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Cel l Cy c l e 47S ept ember2015|Buda pes t , Hung a r y Da nubi usHea l t hS paRes or tMa r g i t s z i g e t Or g a ni z er s Bél aNov á k F r a nkUhl ma nn This meeting was funded by the European Molecular Biology Organization within the EMBO Workshop programme. Sponsors: Cover image kindly provided by Mark Petronczki 16th European Cell Cycle Workshop and EMBO Workshop on the Cell Cycle 4 – 7 September 2015 Danubius Health Spa Resort Margitsziget Budapest, Hungary Organizers: Béla Novák and Frank Uhlmann EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Programme overview Friday, 4 September 2015 Saturday, 5 September 2015 Sunday, 6 September 2015 Monday, 7 September 2015 09:00 09:00 - 10:15 Meiosis 10:15 Star Auditorium 09:00 - 10:15 Cell Cycle and Development Star Auditorium 09:00 - 10:15 Mitotic Exit and Cytokinesis Star Auditorium 10:15 10:15 - 10:45 Coffee break Jasmine corridor 10:15 - 10:45 Coffee break Jasmine corridor 10:15 - 10:45 Coffee break Jasmine corridor 10:45 10:45 - 12:00 Meiosis 12:00 Star Auditorium 10:45 - 12:00 Cell Cycle and Development Star Auditorium 10:45 - 12:00 Mitotic Exit and Cytokinesis Star Auditorium 12:00 12:00 - 13:00 Lunch 12:00 - 13:00 Lunch 12:00 - 12:15 Closing remarks Star Auditorium Platán Restaurant Platán Restaurant 13:00 - 15:00 Poster session Jasmine rooms 13:00 - 15:00 Poster Session Jasmine rooms 15:00 - 16:15 S Phase Star Auditorium 15:00 - 16:15 Mitosis Star Auditorium 16:15 - 16:45 Coffee break Jasmine corridor 16:15 - 16:45 Coffee break Jasmine corridor 16:45 - 18:00 S Phase 16:45 - 18:00 Mitosis Star Auditorium Star Auditorium Free time Free time 19:00 - 22:00 19:00 - 22:00 Boat tour and dinner on the Danube Gala dinner 10:45 13:00 15:00 15:00 16:15 16:15 16:45 15:00 - 17:00 Arrival and Refreshment Jasmine corridor 16:45 17:00 17:00 - 18:00 Keynote lecture 18:00 Star Auditorium 18:00 18:00 - 18:30 Coffee break 18:30 Jasmine corridor 19:00 19:00 20:00 20:00 22:00 18:30 - 20:00 Cell Size Control Star Auditorium 20:00 - 22:00 Dinner and Drinks Platán Restaurant Széchenyi Terrace of Grand Hotel Boat Sirona ii 12:15 - 13:15 Lunch Platán Restaurant EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Programme Programme Friday, 4 September 2015 Arrival and refreshment - 15:00 - 17:00 Keynote lecture: Eric Wieschaus - 17:00 - 18:00 Jasmin corridor Star Auditorium Chair: Patrick O'Farrell (UCSF, San Francisco, United States) 17:00 Cell Cycle Control and the Initiation of Transcription in Drosophila Embryos Shelby Blythe, Stefano Di Talia, Amir Momen Roknabadi, Eric Wieschaus Molecular Biology Department, Howard Hughes Medical Institute, Princeton University Coffee break - 18:00 - 18:30 Jasmin corridor Cell Size Control - 18:30 - 20:00 Chair: 18:30 Star Auditorium Bruce Edgar (University of Heidelberg, Germany) Cell size homeostasis in mammalian cells Clotilde Cadart, Sylvain Monnier, Matthieu Piel Systems Biology of Cell Division and Cell Polarity, UMR 144 Institut Curie/CNRS 18:45 Dilution of the cell cycle inhibitor Whi5 controls budding yeast cell size Kurt Schmoller, Jonathan Turner, Mardo Kõivomägi, Jan Skotheim Department of Biology, Stanford University, Stanford 19:00 Molecular competition and cell size control Eva Parisi, Galal Yahya, David Moreno, Carme Gallego, Attila Csikasz-Nagy, Marti Aldea Molecular Biology Institute of Barcelona, CSIC, Barcelona, Catalonia, Spain 19:15 Regulation of cell size homeostasis in plants Rossana Henriques, Csaba Papdi, Beátrix Horváth, Aladár Pettkó-Szandtner, Zoltán Magyar, Laszlo Bögre Royal Holloway, University of London, School of Biological Sciences 19:30 Mitotic control of the nuclear membrane Snezhana Oliferenko, Maria Makarova, et al. King's College, London Welcome Reception and Dinner - 20:00 - 22:00 iii Platán Restaurant EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Programme Saturday, 5 September 2015 Meiosis - 09:00 – 12:00 Chair: 09:00 Star Auditorium Angelika Amon (Koch Institute/HHMI/MIT, Cambridge, USA) The final cut: control of chromosome segregation in meiosis II Orlando Arguello-Miranda, Ievgeniia Zagoriy, Valentina Mengoli, Wolfgang Zachariae Laboratory of Chromosome Biology, Max Planck Institute of Biochemistry, Germany 09:30 To activate, or not to activate cyclin B-Cdk1: that is the question of the oocyte at G2/M-phase border Daisaku Hiraoka, Takeo Kishimoto Science & Education Center, Ochanomizu University, Tokyo, Japan 9:45 Chromosome orientation in meiosis and mitosis Yoshinori Watanabe University of Tokyo, Japan Coffee break - 10:15 - 10:45 10:45 Jasmin corridor Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes. Zuzana Holubcova, Martyn Blayney, Kay Elder, Melina Schuh MRC Laboratory of Molecular Biology, Cambridge, UK 11:15 How oocytes try to get it right: spindle checkpoint control in meiosis Antoine Vallot, Ioanna Leontiou, Warif El Yakoubi, Damien Cladière, Eulalie Buffin, Katja Wassmann Institut de Biologie Paris Seine, Paris, France 11:30 Challenges of segregating chromosomes in oocytes Martin Anger, et al. 11:45 How is bivalent cohesion maintained in oocytes arrested for months in prophase I? Sabrina Burkhardt, Mate Borsos, Jonathan Godwin, Takayuki Hirota, Mitinori Saitou, Paula Cohen, Kikue Tachibana-Konwalski Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna Lunch - 12:00 - 13:00 Platán Restaurant Poster session 1 - 13:00 - 15:00 Jasmine rooms iv EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Programme Saturday, 5 September 2015 S Phase - 15:00 - 18:00 Star Auditorium Chair: Frank Uhlmann (The Francis Crick Institute, London, United Kingdom) 15:00 When DNA replication does not finish on time Magda Magiera, Julie Petit, Nicolas Talarek, Elisabeth Gueydon, Etienne Schwob Institute of Molecular Genetics, CNRS UMR5535 & University Montpellier, France 15:30 Visualizing developmentally programmed endoreplication in mammals and drug-induced cell cycle modulation using ubiquitin oscillators Atsushi Miyawaki Riken, Japan 16:00 Developmental activation of the DNA damage checkpoint Chames Kermi, Susana Prieto, Siem van der Laan, Nikolay Tsanov, Benedicte Recolin, Domenico Maiorano Institute of Human Genetics. CNRS-UPR1142. Montpellier. France Coffee break - 16:15 - 16:45 16:45 Jasmine corridor DNA replication control during the Mid-Blastula Transition in Xenopus laevis Philip Zegerman, Clara Collart, Jim Smith Gurdon Institute, University of Cambridge, UK 17:15 A chromatin perspective of cell cycle progression in whole developing organs Crisanto Gutierrez Centro de Biologia Molecular Severo Ochoa, CSIC-UAM 17:45 Entangling the function of the large number of plant CDKs - The plant specific CYCLIN-DEPENDENT KINASE B1-CYCLIN B1 complex mediates homologous recombination repair in Arabidopsis Annika Weimer, Sascha Biedermann, Hirofumi Harashima, Peter Dörner, Naoki Takahashi, Masaaki Umeda, Arp Schnittger University of Hamburg, Department of Developmental Biology, Germany Boat tour and dinner on the Danube - 19:00 - 22:00 v Boat Sirona EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Programme Sunday, 6 September 2015 Cell Cycle and Development - 09:00 - 12:00 Star Auditorium Chair: Snezhana Oliferenko (King's College London, United Kingdom) 09:00 Growth-dependent G1/S control in Drosophila endocycles Bruce Edgar, Norman Zielke, Jinyi Xiang, Xueyang Yu, Jonathan Bohlen German Cancer Research Center (DKFZ), Heidelberg, Germany 09:30 Emergence of heterochromatin and prolongation of S phase in Drosophila embryos. Patrick H O'Farrell, Kai Yuan, Antony W Shermoen Dept. of Biochem. and Biophys. UCSF, San Francisco, CA, US 10:00 S-phase waves of Cdk1 activity synchronize mitosis in the early Drosophila embryo Victoria Deneke, Anna Melbinger, Massimo Vergassola, Stefano Di Talia Department of Cell Biology, Duke University Medical Center, Durham, USA Coffee break - 10:15 - 10:45 10:45 Jasmin corridor From clocks to dominoes: cell cycle remodeling during hES cell differentiation Silvia Santos MRC-Imperial College, London, UK 11:15 Growth Regulation by the Hippo Pathway Nicolas Tapon The Francis Crick Institute, London, UK 11:45 Cytoskeletal Dynamics during Inter-nuclear Spacing in the Drosophila Syncytial Embryo Ojas Deshpande, Jorge de-Carvalho, Ivo Telley Instituto Gulbenkian de Ciência, Oeiras, Portugal Lunch - 12:00 - 13:00 Platán Restaurant Poster session II - 13:00 - 15:00 Jasmin rooms vi EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Programme Sunday, 6 September 2015 Mitosis - 15:00 - 18:00 Star Auditorium Chair: David Morgan (University of California, San Francisco, United States) 15:00 How is cohesin positioned in mammalian genomes? Jan-Michael Peters 15:30 Pds5 proteins are key regulators of cohesin dynamics Ana Losada, Miguel Ruiz-Torres, et al. Chromosome Dynamics Group, Spanish National Cancer Research Centre, Madrid, Spain 16:00 Mitotic chromosomes acquire mechanical independence through Ki-67 Sara Cuylen, Claudia Blaukopf, Thomas Müller-Reichert, Beate Neumann, Ina Poser, Jan Ellenberg, Anthony A. Hyman, Daniel W. Gerlich Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Coffee break - 16:15 - 16:45 16:45 Jasmin corridor Effects of aneuploidy on fitness and tumorigenesis Angelika Amon, et al. Koch Institute/HHMI/MIT, Cambridge, USA 17:15 Integration of microtubule-kinetochore attachment formation and spindle checkpoint signalling by phosphatase complexes Ulrike Gruneberg University of Oxford, UK 17:45 Functions of Greatwall-PP2A/B55 pathway in the mammalian cell cycle Mónica Alvarez-Fernández, María Sanz-Flores, Belén Sanz-Castillo, Marcos Malumbres Cell Division and Cancer Group, CNIO, Madrid, Spain Gala dinner - 19:00 - 22:00 Széchenyi Terrace of Grand Hotel vii EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Programme Monday, 7 September 2015 Mitotic Exit and Cytokinesis - 09:00 - 12:00 Star Auditorium Chair: Silvia Santos (MRC-Imperial College, London, United Kingdom) 09:00 Mechanisms of mitotic regulation by the APC/C Dan Lu, Juliet Girard, Arda Mizrak, David Morgan University of California, San Francisco 09:30 Deciphering the role of the GWL/ARPP19-ENSA/PP2A pathway in the control of the cell cycle Anna Castro, et al. CRBM-CNRS, Montpellier, France 10:00 PP2A-B55 is an NLS-directed Cdk-counteracting phosphatase coordinating mitotic spindle reorganisation with nuclear envelope reformation and cytokinesis. Michael Cundell, Ricardo Nunes Bastos, Elena Poser, Shabaz Mohammed, Francis Barr University of Oxford, Department of Biochemistry Coffee break - 10:15 - 10:45 10:45 Jasmin corridor Spindle pole body specification in budding yeast Jette Lengefeld, Manuel Hotz, Yves Barral Institute of Biochemistry, Department of Biology, ETH Zürich, Switzerland 11:15 A novel protein network stabilizes the yeast septin ring upon mechanical stress Laura Merlini, Maria Angeles Juanes, Alessio Bolognesi, Yves Barral, Simonetta Piatti Centre de Recherche en Biochimie Macromoléculaire-CNRS, Montpellier, France 11:45 Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing. Marina Vietri, et al. Department of Molecular Cell Biology, Oslo University Hospital, Norway Closing Remarks - 12:00 - 12:15 12:00 Star Auditorium Closing Remarks Tim Hunt Lunch - 12:15 - 13:15 Platán restaurant viii ABSTRACTS OF ORAL PRESENTATIONS EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Keynote lecture Cell Cycle Control and the Initiation of Transcription in Drosophila Embryos Shelby Blythe1, Stefano Di Talia1, 2, Amir Momen Roknabadi1, Eric Wieschaus1 1 2 Molecular Biology Department, Howard Hughes Medical Institute, Princeton University Department of Cell Biology, Duke University School of Medicine Following fertilization in Drosophila, the embryo undergoes 13 synchronous nuclear mitoses. It then pauses cell cycle progression to partition the nuclei into individual cells and to assign those cells distinct developmental fates. The pause in cell cycle is analogous to the midblastula transition observed in many organisms and is associated with the degradation of a subset of maternally supplied mRNAs and large-scale transcriptional activation of the genome. In my talk, I will describe experiments that investigate the remodeling of the rapid maternally controlled cell cycle into the extended cell cycle characteristic of later stages, the role of the DNA replication checkpoint in this process, and the relationship between this cell cycle transition and the restructuring of chromatin associated with the transcriptional activation. Using interphase duration as an assay for checkpoint activity, we have shown that activation of the checkpoint depends not on total DNA content, but instead on the dosage of DNA that is transcriptionally engaged. We have extended these observations using ChIP-seq to reconstruct the genome-wide distribution of RNA Polymerase II (Pol II). We find that large-scale de novo recruitment of poised Pol II to thousands of sites coincides with activation of the MBT checkpoint, and that the checkpoint itself is not required for this initial recruitment. Instead we see that loci occupied by Pol II at the MBT recruit a sensor of replication stress generally thought to be upstream of checkpoint activation (Rp-A70). Reducing Pol II recruitment in zelda mutants reduces recruitment of Rp-A70 and partially rescues the lethality associated with checkpoint mutants. These results suggest a model where checkpoint activation and timing of the MBT is triggered by the large-scale zygotic genome activation. The cell cycle pause at cycle 14 is associated with a downregulation of the Drosophila homologues of the cdc25 phosphatase, String and Twine. Re-initiation of cell division occurs in a defined temporal sequence at gastulation and requires renewed zygotic transcription of cdc25-String. I will describe genetic screens designed to identify the global and region specific transacting elements that time the re-initiation of these divisions. 1 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Size Control Cell size homeostasis in mammalian cells Clotilde Cadart1, 2, Sylvain Monnier1, Matthieu Piel1 1 2 Systems Biology of Cell Division and Cell Polarity, UMR 144 Institut Curie/CNRS, 26 rue d’Ulm 75248 Paris Cedex 05 France Univ Paris-Sud The way mammalian cells coordinate cell growth and cell cycle progression in order to correct for potential mistakes at division and maintain cell size homeostasis is not yet known. Although often hypothesized, the requirement of a size checkpoint similar to that in S. Pombe has not been confirmed experimentally. To monitor cell growth over several cell cycles we developed two volume measurement methods. The first method is based on fluorescence-exclusion and enables precise growth rate measurement throughout the cell cycle. The second method uses confinement inside micro-channels and enables inducing asymmetric divisions to assess potential size-correction mechanisms during the following cell cycle. The combination of these two methods allowed characterizing the homeostasis process in different cell types. We show that, in HeLa, MDCK and HT29 cells, there is no apparent cell size checkpoint and that cells instead maintain size homeostasis through a constant volume increase at each cell division cycle, independent of their volume at birth. This process, similar to what was recently observed in bacteria (1) enables the progressive reduction of errors in volume segregation. In one other cell type, Raji cells, the homeostasis mechanism appears less efficient and the amount of volume gained is partly dependent on initial size, with big cells gaining more volume than smaller ones. In HeLa, MDCK and HT29 cells, the central mechanism allowing homeostasis seems to rely on the fact that cells with smaller growth rate (which is often the case of smaller cells) have longer cell division cycle timing and thus eventually grow as much than cells with larger growth rates. In Raji cells, the same inverse correlation between volume at birth and cell cycle length is found but it is not sufficient to allow small cells to gain the same amount of volume than big cells. We are now combining volume measurement with complementary techniques to assess cell dry mass (quantitative phase) and cell cycle phase. This allows us to measure all the key parameters of cell growth. These measures are then performed on cells grown in various conditions such as confinement, osmotic shocks, rapamycin treatment or nutrient depletion to perturb cell growth or cyclin inhibitors to perturb cell cycle progression. With this approach we will be able to understand the coupling between initial cell size, growth rate and cell cycle duration that leads to size homeostasis in cultured cells. We also aim at testing the robustness of this homeostasis mechanism in various environments and identify conditions in which size is not maintained. Reference: (1) Campos, M., Surovtsev, I. V., Kato, S., Paintdakhi, A., Beltran, B., Ebmeier, S. E., & Jacobs-Wagner, C. (2014). A Constant Size Extension Drives Bacterial Cell Size Homeostasis. Cell, 159(6), 1433–1446. doi:10.1016/j.cell.2014.11.022 2 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Size Control Dilution of the cell cycle inhibitor Whi5 controls budding yeast cell size Kurt Schmoller, Jonathan Turner, Mardo Kõivomägi, Jan Skotheim 1Department of Biology, Stanford University, Stanford, CA 94305, USA Cell size fundamentally affects all biosynthetic processes by determining the scale of organelles and influencing surface transport. Although extensive studies have identified many mutations affecting cell size, the molecular mechanisms underlying size control have remained elusive. In budding yeast, size control occurs in G1 phase prior to Start, the point of irreversible commitment to cell division. It was previously thought that activity of the G1 cyclin Cln3 increased with cell size to trigger Start by initiating the inhibition of the transcriptional inhibitor Whi5. Our work overturns this model. While Cln3 concentration does modulate the rate at which cells pass Start, its synthesis increases in proportion to cell size so that its total concentration is constant during pre-Start G1. Rather than increasing Cln3 activity, we identify decreasing Whi5 activity — due to the dilution of Whi5 by cell growth — as a molecular mechanism through which cell size controls proliferation. Whi5 is synthesized in S/G2/M phases of the cell cycle in a largely size-independent manner. This results in smaller daughter cells being born with higher Whi5 concentrations that extend their pre-Start G1 phase. Thus, at its most fundamental level, budding yeast size control results from the differential scaling of Cln3 and Whi5 synthesis rates with cell size. More generally, our work shows that differential size-dependency of protein synthesis can provide an elegant mechanism to coordinate cellular functions with growth. 3 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Size Control Molecular competition and cell size control Eva Parisi1, Galal Yahya1, David Moreno1, Carme Gallego1, Attila Csikasz-Nagy2, Marti Aldea1 1 2 Molecular Biology Institute of Barcelona, CSIC, Barcelona, Catalonia, Spain Institute for Mathematical and Molecular Biomedicine, King's College London, London, UK Coordination of cell growth and DNA replication ensures size adaptation and homeostasis. Cells are able to adapt their size to growth rate both at population and single-cell levels, which suggests that growth is intimately linked to the molecular mechanisms that govern the cell cycle. Budding yeast cells, as most eukaryotic cells, exert this coordination essentially during G1, where a critical size must be attained before cells trigger Start. The most upstream activator of cell cycle entry in budding yeast is Cln3, a cyclin that critically depends on molecular chaperones to accumulate in the nucleus in late G1. On the other hand, chaperones are massively involved in key growth processes, and we have investigated the possibility that coordination between proliferation and growth relies on the competition for limiting stocks of shared chaperones. As deduced from mathematical modeling, a molecular competition device would be able to (1) accumulate Cln3 in the nucleus in a non-linear manner during G1, and (2) trigger Start at a cell size that is proportional to growth rate. We have used different experimental approaches to test predictions of the model, and found that availability of chaperones is negatively correlated with growth rate. More important, chaperone availability increases during G1 as cells grow, thus emerging as a new strong candidate for the sizer mechanism. Our data support the notion that competition for molecular chaperones would provide Start with an upstream switch-like mechanism, and adjust the critical size to the individual growth potential. 4 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Size Control Regulation of cell size homeostasis in plants Rossana Henriques1, Csaba Papdi2, Beátrix Horváth2, Aladár Pettkó-Szandtner3, Zoltán Magyar3, Laszlo Bogre2 1 Center for Research in Agricultural Genomics (CRAG), Consortium CSIC-IRTA-UAB-UB, Parc de Recerca UAB, Edifici CRAG, Campus UAB, Bellaterra (Cerdanyola del Vallés), 08193 Barcelona, Spain. 2 Royal Holloway, University of London, School of Biological Sciences, Egham Hill, Egham, TW20 0EX, UK 3 Institute of Plant Biology, Biological Research Centre, Temesvári kru. 62., POB 521, H-6701, Szeged, Hungary How cell growth and proliferation are coupled in meristematic plant cells is not well understood yet it is pivotal to determine organ growth. Cell growth, which relies on protein translation, constrains cell proliferation. The hypothesis is that translational control of specific mRNAs provides important regulatory links to couple cell growth and cell proliferation. Key pieces of evidence in plants are that silencing or overexpression of translation/ribosome associated protein EBP1 constrains or promote cell proliferation, respectively. We propose that translation and cell proliferation are further integrated with each other by growth regulatory signalling pathways; TOR and S6K. Emerging data indicate that these pathways are known to affect translation as well as the commitment to DNA replication and mitosis. We have shown that the Arabidopsis S6 Kinase1 inhibits cell proliferation through the RBR1-E2FB complex. S6K1 interacts with RBR1 via its N-terminal RBR1 binding motif, promotes its nuclear localization and consequent RBR1-dependent repression of cell cycle genes through E2FB. S6K1 and E2FB are in a mutually antagonistic relationship both in their protein abundance and in their activity. S6K1 activity to phosphorylate RBR1 is increased when E2FB is silenced. Conversely, transcriptional activation of cell cycle genes by E2FB is elevated when S6K1 is silenced. The same antagonism holds for S6K1 and E2FB protein abundance; S6K1 is stabilised when E2FB is silenced, while E2FB is stabilised when S6K1 is silenced. The double inhibitory regulatory connection between S6K1 and E2FB might constitute a regulatory switch to determine whether cells divide or grow. 5 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Size Control Mitotic control of the nuclear membrane Snezhana Oliferenko, Maria Makarova, et al. King's College London Eukaryotes remodel the nucleus during mitosis using a variety of mechanisms that differ in the timing and the extent of nuclear envelope (NE) breakdown. We probe the principles enabling this functional diversity by exploiting the natural divergence in NE management strategies between the related fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus. Using these two systems, we have shown that the mitotic control of the nuclear surface area may determine the choice between the nuclear envelope breakdown and a fully closed division. I will present our recent work on the mechanistic basis of this variability and argue that comparative cell biology studies using two fission yeast species could provide unique insights into physiology and evolution of mitosis. 6 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Meiosis The final cut: control of chromosome segregation in meiosis II Orlando Arguello-Miranda, Ievgeniia Zagoriy, Valentina Mengoli, Wolfgang Zachariae Laboratory of Chromosome Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany The generation of haploid gametes from diploid germ cells during meiosis depends on DNA replication being followed by two rounds of chromosome segregation, called meiosis I and –II. Whereas the mechanisms governing the segregation of homologous chromosomes in meiosis I have been studied extensively, comparatively little is known about the control of chromatid segregation in meiosis II. This is in part due to the perception of meiosis II as a “mitosis-like division”. However, meiosis II differs from mitosis in fundamental aspects: in contrast to mitosis, meiosis II is preceded not by an S-phase but by an M-phase (meiosis I) and it is not followed by another S-phase but by a differentiation program that generates gametes. Furthermore, meiosis II chromosomes are unique because they consist of recombined chromatids held together solely at their centromeres by cohesin that has been protected from cleavage by the separase protease in meiosis I. Cleavage of cohesin at entry into anaphase I requires phosphorylation of its meiosis-specific Rec8 subunit. At centromeres, this phosphorylation is prevented by the phosphatase PP2A, which is recruited to kinetochores by the shugoshin protein. It is currently unclear whether cleavage of centromeric cohesin in meiosis II requires phosphorylation of Rec8 and whether this depends on the inactivation or removal from kinetochores of PP2A. A detailed understanding of meiosis II has been hampered by technical challenges. Experimental manipulation of meiosis II has to be performed without perturbing meiosis I, which requires conditional methods for protein inactivation that work in the short interval between meiosis I and –II. We use budding yeast to investigate how centromeric cohesin is cleaved at the onset of anaphase II and how this event is coordinated with exit from meiosis II and spore formation, the yeast equivalent of gametogenesis. 7 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Meiosis To activate, or not to activate cyclin B-Cdk1: that is the question of the oocyte at G2/M-phase border Daisaku Hiraoka, Takeo Kishimoto Science & Education Center, Ochanomizu University, Tokyo 112-8610, Japan Some cells, particularly non-mammalian oocytes at the G2/M-phase border lack the checkpoint control. Nevertheless, they properly respond only to physiologically relevant extracellular stimuli, implying a mechanism that prevents unexpected cell cycle transition in response to noise stimuli. However, it remains unclear how these cells cancel noise stimuli. Here we investigated how noise stimuli are processed using a simple, highly synchronized binary response system, the starfish oocyte, in which the maturation-inducing hormone 1-methyladenine (1-MeAde) causes meiotic G2/M-phase transition via the Gβγ-PI3K-Akt pathway with no requirement of new protein synthesis; Akt directly phosphorylates both Cdc25 and Myt1 to reverse the balance of their activities, resulting in activation of cyclin B-Cdk1. We found that a subthreshold noise stimulus by 1-MeAde can initiate cyclin B-Cdk1 activation. However, cyclin B-Cdk1-dependent negative feedback (Cdk-NF) causes dephosphorylation of Akt substrates, thereby canceling noise signaling and preventing full activation of cyclin B-Cdk1. By contrast, after suprathreshold stimuli, Gβγ activates an additional, novel pathway that is distinct from the PI3K-Akt pathway but promotes the PI3K-Akt-dependent phosphorylation, thereby overriding Cdk-NF and fully activating cyclin B-Cdk1. These findings demonstrate that noise canceling is a self-regulatory decision process mediated by the final effector of the signaling pathway. Reference Kishimoto, T. (2015). Entry into mitosis: a solution to the decades-long enigma of MPF (review). Chromosoma, 124, DOI 10.1007/s00412-015-0508-y, in press. 8 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Meiosis Chromosome orientation in meiosis and mitosis Yoshinori Watanabe Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo During mitosis, replicated chromosomes (sister chromatids) become attached at the kinetochore by spindle microtubules emanating from opposite poles and segregate equationally. In the first division of meiosis, however, sister chromatids become attached from the same pole and co-segregate, while homologous chromosomes connected by chiasmata segregate to opposite poles. Recent our studies together with others have elucidated the molecular mechanisms determining chromosome orientation, and consequently segregation, in meiosis. Comparative studies of meiosis and mitosis lead to the general principle that kinetochore geometry and tension exerted by microtubules synergestically generate chromosome orientation. Disorder of this regulation may lead to birth defects and tumorigenesis in humans. 9 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Meiosis Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes. Zuzana Holubcova1, Martyn Blayney2, Kay Elder2, Melina Schuh1 1 2 MRC Laboratory of Molecular Biology Bourn Hall Clinic Aneuploidy in human eggs is the leading cause of pregnancy loss and several genetic disorders such as Down syndrome. Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg's progenitor cell. The basis for particularly error-prone chromosome segregation in human oocytes is not known. We analyzed meiosis in more than 100 live human oocytes and identified an error-prone chromosome-mediated spindle assembly mechanism as a major contributor to chromosome segregation defects. Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. Instead, spindle assembly was mediated by chromosomes and the small guanosine triphosphatase Ran in a process requiring ~16 hours. This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs. 10 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Meiosis How oocytes try to get it right: spindle checkpoint control in meiosis Antoine Vallot1, 2, Ioanna Leontiou1, 2, Warif El Yakoubi1, 2, Damien Cladière1, 2, Eulalie Buffin1, 2, Katja Wassmann1, 2 1 2 Paris Sorbonne Universities, Institut de Biologie Paris Seine (IBPS), Paris, 75005 France CNRS UMR7622 Developmental Biology Lab, Paris, 75005 France In meiosis I, homologous chromosomes consisting of two sister chromatids each are segregated to opposite poles of the bipolar spindle. This requires the orientation of sister kinetochores to the same pole through monopolar attachment to the meiotic spindle. Because chromosomes are held together by chiasmata, tension-generating attachments can be established. Correct attachment of chromosomes to the bipolar spindle is verified by the spindle assembly checkpoint (SAC), as in mitosis. In mitosis, the checkpoint not only verifies whether all sister kinetochores are attached, but also whether those attachments are under tension. Tension is established not only between sister kinetochores, but also in the kinetochore itself ("intra-kinetochore tension"). In the absence of tension, an error-correction mechanism is activated. Missing tension allows the kinase Aurora B to phosphorylate substrates that destabilize kinetochore-microtubule attachments. Tension-dependent relocalization of Aurora B and recruitment of phosphatases to the kinetochore counterbalance Aurora B substrate phosphorylation, leading to the stabilization of correctly attached kinetochores. In meiosis I, the tension applied on bivalents does not lead to tension between mono-oriented sister kinetochores, and it has been suggested that missing inter-kinetochore tension (now between pairs of sister kinetochores) of wrongly attached chromosomes is not recognized by the meiotic SAC. Stabilization of attachments which is dependent on BubR1-dependent PP2A localization to kinetochores in mitosis, requires BubR1 function independent of kinetochore localization in meiosis (Touati et al, 2015). BubR1 levels decrease with age in oocytes, therefore it is important to clarify whether the meiotic SAC can recognize unstable attachments that are not under tension.To clarify whether the meiotic SAC is sensitive to missing tension in mouse oocyte meiosis I, we analyzed oocytes with shorter spindles and reduced tension, and localization of different checkpoint proteins at tension-less kinetochores, as well as checkpoint response in oocytes without a functional SAC. Our data show that the SAC is activated in meiosis when kinetochores are properly attached, but not under tension. SAC activation leads to a metaphase delay and prevents timely Securin degradation and Polar body extrusion. Bub1 but not Mad2 is recruited to tensionless kinetochores. Inactivation of the SAC abolishes checkpoint response to loss of tension, showing that even though attachments are monopolar, the meiotic SAC can distinguish between kinetochores that are attached, and kinetochores that are attached and under tension. Touati, S.A., Buffin, E., Cladiere, D., Hached, K., Rachez, C., van Deursen, J.M., and Wassmann, K. (2015). Nat Commun 6, 6946. 11 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Meiosis Challenges of segregating chromosomes in oocytes Martin Anger, et al. Department of Histology and Embryology Faculty of Medicine, Masaryk University Kamenice 3, building A1 62500 Brno, Czech Republic & Central European Institute of Technology - CEITEC Veterinary Research Institute Hudcova 70 Brno 621 00 Czech Republic Mammalian oocytes and embryos are frequently affected by aneuploidy arising during chromosome segregation. The reason why chromosome segregation errors are more frequent in germ cells and embryos in comparison to somatic cells is unknown. We believe that the problem lies in less stringent chromosome segregation control mechanisms operating in these cells. In our study we focused on a functional analysis of a surveillance checkpoint mechanism called Spindle Assembly Checkpoint (SAC) in live oocytes. Using micromanipulation and live cell confocal microscopy, we have tested whether the SAC is capable to prevent metaphase to anaphase transition in oocytes in which the chromosomes are not congressed properly or in which single chromatids are present in meiosis I. Similar approach was used for monitoring the activity of SAC on individual kinetochores and for correlation of this activity with chromosome movements, spindle formation, onset of Anaphase Promoting Complex (APC) activation and polar body extrusion (PBE) at various time points during the first meiotic division. Our results show important and unexpected differences in SAC function in oocytes compared to somatic cells. In contrast to the somatic cells, oocytes were unable to mount functional SAC response and to prevent anaphase onset in presence of unaligned chromosomes or single chromatids. Our results indicate that the checkpoint mechanisms involved in monitoring chromosome segregation, are in oocytes unable to detect or respond to errors in this process, which might explain a high incidence of aneuploidy in these cells. Project support: This work was supported by Czech science foundation projects P502/12/2201 and 15-04844S and by MEYS projects ED1.1.00/02.0068—CEITEC and LH 13072—Kontakt II. 12 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Meiosis How is bivalent cohesion maintained in oocytes arrested for months in prophase I? Sabrina Burkhardt1, Mate Borsos1, Jonathan Godwin2, Takayuki Hirota3, Mitinori Saitou3, Paula Cohen4, Kikue Tachibana-Konwalski1 1 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences University of Oxford 3 Kyoto University 4 Cornell University 2 It is presumed that sister chromatid cohesion is established during DNA replication in embryonic oocytes and maintained during a prolonged prophase I arrest until its destruction at the meiotic divisions in adult female mammals. Rec8-containing cohesin complexes, which may contain Smc1β, maintain bivalent cohesion that is essential for chromosome segregation. Unlike Rec8, Smc1β is nonessential for cohesion establishment but its absence causes premature cohesion loss and infertility. Conditional deletion of Smc1β using Gdf9-iCre thought to be active after birth does not affect fertility, suggesting its transcription is not required for cohesion maintenance. Whether new cohesive structures strengthen meiotic cohesion in oocytes arrested for months or decades at the dictyate stage of meiosis I remains unknown. Since both DNA synthesis-dependent and independent mechanisms can generate cohesive structures entrapping sister chromatids, cohesion might be maintained with or without building additional structures in arrested oocytes. To distinguish between these, the ability of Rec8 activated at different meiotic stages to generate functional cohesion is tested by TEV protease cleavage and live-cell imaging. Genetic activation of Rec8 using Cre recombinase under the Gdf9 or Spo11 promoter results in functional cohesion, likely due to cohesin loading during DNA replication. In contrast, timely controlled Rec8 activation in arrested oocytes before growth using tamoxifen-inducible Dppa3-Mer-Cre-Mer consistently does not rescue cohesion despite new cohesin transcription. We conclude that cohesion is maintained after birth without building new Rec8-cohesive structures in arrested mouse oocytes. Our results imply that women’s fertility depends on the longevity of cohesin complexes that become cohesive on chromosomes in oocytes in utero. 13 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations S phase When DNA replication does not finish on time Magda Magiera, Julie Petit, Nicolas Talarek, Elisabeth Gueydon, Etienne Schwob Institute of Molecular Genetics, CNRS UMR5535 & University Montpellier, France It was long assumed that cells would not enter mitosis until chromosome replication is completed. This concept stems almost entirely from studies in which the progression of replication forks was affected by chemical or enzymatic impediments. It is now well established that, in such case, long stretches of single-stranded DNA are generated, which activate the Mec1/ATR checkpoint pathways that delay mitosis until the problem is fixed. We devised two strategies to address what happens when S phase is still under progress, without fork perturbation, when cells are programmed to enter mitosis. In the first strategy we postponed S phase to a later point in the cell cycle, so that its completion temporally overlaps with mitosis. This indicated that physiological DNA replication can delay mitosis through activation of both Mec1/ATR and Mad2/SAC checkpoint mechanisms, thus preserving genome integrity. Interestingly Mec1 and Mad2 become together essential in cells where S phase and mitosis are triggered by the same set of cyclin-Cdk, revealing an evolutionary driving force for checkpoint emergence. In the second strategy we extended S phase by lowering the number of active origins. Such cells enter mitosis with under-replicated DNA, undetected by checkpoints, and become highly genetically unstable. The DNA damage response (DDR) is activated only when cells are allowed to progress past the G2/M transition, irrespective of the amount of unreplicated DNA, indicating that there is no checkpoint monitoring the completion of S phase. We will discuss the mechanism by which DNA replication is completed during mitosis and how this leads to genome instability. Altogether our work underscores the key role of origin licensing and replication fork density in the maintenance of genome integrity. It challenges the dogma that cells cannot enter mitosis before S phase is finished and provides a mechanism for the breakage of late-replicating fragile sites. The mechanisms we uncovered in yeast offer a conceptual framework to explain the high chromosomal instability of cancer cells with altered replication programmes. 14 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations S phase Visualizing developmentally programmed endoreplication in mammals and drug-induced cell cycle modulation using ubiquitin oscillators Atsushi Miyawaki RIKEN Brain Science Institute RIKEN Center for Advanced Photonics We used transgenic mice expressing Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator) probes that report the activity of APCCdh1 and SCFSkp2, to visualize spatio-temporal dynamics of altered cell-cycle progression. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APCCdh1 and SCFSkp2 in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs is varied with the polyploidy level. We also obtained threedimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. We also used population and time-lapse imaging analyses of cultured cancer cells expressing Fucci, to find great diversity in the cell-cycle alterations induced by anticancer drugs. Drug-induced cell cycle modulation varied not only between different cell types or following treatment with different drugs, but also between cells treated with different concentrations of the same drug or following drug addition during different phases of the cell cycle. By combining cytometry analysis with the Fucci probe, we have developed a novel assay that fully integrates the complexity of cell cycle regulation into drug discovery screens. This assay system will represent a powerful drug-discovery tool for the development of the next generation of anti-cancer therapies. 15 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations S phase Developmental activation of the DNA damage checkpoint Chames Kermi, Susana Prieto, Siem van der Laan, Nikolay Tsanov, Benedicte Recolin, Domenico Maiorano Institute of Human Genetics. CNRS-UPR1142. Montpellier. FRANCE The cell cycles of early embryos are very rapid due to contraction of gap phases and reduced S phase length. In this highly contracted cell cycle the DNA damage checkpoints that restrain proliferation in the presence of DNA damage are inefficient and may constitute an adaptation to rapid proliferation. We have previously shown that in mouse embryonic stem cells inhibition of the G1/S checkpoint is untimely linked to maintenance of pluripotency through developmental regulation of protein phosphatase Cdc25A stability. We have also shown that cell cycle remodelling is essential for the execution of a viable differentiation programme (van der Laan et al., 2013). Here we have investigated the molecular grounds of checkpoint inhibition in the early embryonic cleavages of the vertebrate Xenopus laevis. Previous studies have suggested that checkpoint activation is triggered by the increase of the nucleus-to-cytoplasmic (N/C) ratio of the embryo due to absence of cell growth, which leads to titration of maternal-limiting factors, whose identity has been so far mysterious. We report that the Rad18 ubiquitin ligase, a master regulator of DNA damage tolerance that promotes monoubiquitylation of the replication factor PCNA (mUb), is a critical maternal checkpoint silencing factor in Xenopus embryos. We have observed high levels of Rad18 and PCNAmUb in Xenopus embryos strongly decreasing prior to MBT, at a developmental stage that coincides with acquisition of the competence to sense DNA damage. Using Xenopus cell-free extracts, we also show that Rad18 and not PCNA is depleted from egg cytoplasm and titrated on chromatin by increasing the N/C ratio. Moreover, we show that inactivation of Rad18 function in vitro and in vivo, leads to DNA damagedependent checkpoint activation, monitored by Chk1 phosphorylation upon UV irradiation. The molecular mechanism involves inhibition of replication fork uncoupling, a critical determinant of checkpoint activation. Strikingly, we have observed that this embryonic-like regulation can be reactivated in somatic mammalian cells by ectopic Rad18 expression. Finally we report high Rad18 expression in cancer stem cells and show that it is relevant to resistance to DNA damaging agents. Altogether these data propose Rad18 as a critical checkpoint-inhibiting factor and put forward Rad18 as novel target in sensitizing cancer stem cells to DNA damaging agents. Reference van der Laan S, Tsanov N, Crozet C, Maiorano D. High Dub3 expression in mouse ESCs couples the G1/S checkpoint to pluripotency. Mol Cell. 2013 Nov 7;52(3):366-79. 16 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations S phase DNA replication control during the Mid-Blastula Transition in Xenopus laevis Philip Zegerman1, Clara Collart1, 2, Jim Smith2 1 2 Gurdon Institute, University of Cambridge, UK MRC National Institute for Medical research, London, UK Early embryonic divisions from flies to amphibia are extremely rapid, but at a point called the midblastula transition (MBT), multiple events are coordinated including the remodelling of the cell cycle, the onset of bulk zygotic transcription and the activation of checkpoints. How these events are coordinated is not known. We have shown in Xenopus laevis that four DNA replication factors are limiting for replication initiation in vivo in MBT stage embryos and are required for the developmental activation of the kinase Chk1. While Chk1 activation is essential for gastrulation in Xenopus, we show that this is independent of the roles of Chk1 in controlling cell cycle checkpoints. Another kinase, DDK, is also regulated during embryogenesis. Cdc7-Drf1 is the predominant form of DDK in early embryos, but Drf1 is replaced by the paralogue Dbf4 at around the time of gastrulation. We show that Drf1 down-regulation is Chk1dependent at the MBT in Xenopus. This is important because knockdown of Drf1 rescues the gastrulation defect caused by inhibition of Chk1. This suggests that we have uncovered a new essential role for Chk1-dependent control of DDK in metazoa. The mechanisms and physiological consequences of the developmental regulation of replication kinases will be discussed. 17 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations S phase A chromatin perspective of cell cycle progression in whole developing organs Crisanto Gutierrez Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Nicolás Cabrera 1, 28049 Madrid, Spain Cellular homeostasis during organogenesis depends on the adequate balance between different cell populations. In the case of plants organogenesis is postembryonic and leads to the formation of new organs in a continuous manner during the entire life of the organism. Consequently, every growing organ requires the activity of stem cells, the proliferation of their derivatives, the decrease in cell division competence and the initiation of differentiation. The latter is frequently associated with the switch to the endocycle in many cells. We are interested in understanding the crosstalk between changes in chromatin organization and function, and cell proliferation control in the various cell populations within a developing organ. In the growing Arabidopsis root cell proliferation and differentiation are integrated radially and longitudinally by gene expression patterns guided by patterning genes. We have found that after chromatin replication in the S-phase an extensive chromatin reprogramming is at the basis of cell population dynamics through the root compartments. Thus, the histone H3.1/H3.3 balance, which is important for gene expression, defines a boundary within the proliferation compartment, where a low ratio identifies cells in their last cycle before exit to differentiation. The spatial pattern of this reprogramming process coincides with repression of genes at this proliferation boundary. Furthermore, we have identified key patterning transcription factors that, together with other transcription factors, regulate the coordinate expression of H3 and their chaperone genes. Interestingly, a high H3.1/H3.3 ratio and H3.1 eviction pattern is reproduced also in the endocycling cells before differentiation, revealing a common principle of chromatin reprogramming in proliferating and endoreplicating cells. The use of appropriate tools for in vivo monitoring of cell cycle progression together with highresolution gene expression data of individual cell types and screens for upstream regulators of cell cycle are being instrumental in the identification of cellular factors that contribute to the maintenance of an adequate balance of cell populations during organogenesis. 18 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations S phase Entangling the function of the large number of plant CDKs - The plant specific CYCLIN-DEPENDENT KINASE B1-CYCLIN B1 complex mediates homologous recombination repair in Arabidopsis Annika Weimer1, Sascha Biedermann1, Hirofumi Harashima1, Peter Dörner2, Naoki Takahashi3, Masaaki Umeda3, Arp Schnittger1, 4 1 Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS - UPR2357, Université de Strasbourg, 12, Rue du Général Zimmer, 67084 Strasbourg Cedex, France. 2 University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, Scotland, UK 3 Graduate School of Biological Sciences; Nara Institute of Science and Technology; Takayama; Ikoma, Nara, Japan; JST; CRESTl 8916-5 Takayama; Ikoma, Nara, Japan. 4 University of Hamburg, Biozentrum Klein Flottbek, Department of Developmental Biology, Ohnhorststr. 18 - 22609 Hamburg, Germany A common response to DNA damage is the inhibition of cell division by inactivation of cyclindependent kinases (CDKs). Puzzlingly, recent evidence has revealed that CDK activity is required for DNA repair pathways, especially for homologous recombination repair (HR), resulting in the conundrum how mitotic arrest and effective repair can be reconciled. Here we show that the group of so far ill-characterized plant specific B1-type CDKs (CDKB1) are major regulators of DNA damage response in plants. We identify the group of B1-type cyclins (CYCB1) as the specific partners of B1type CDKs in HR and reveal that RADIATION SENSITIVE 51 (RAD51), a core component of HR, is a substrate of CDKB1-CYCB1 complexes. Conversely, mutants in CDKB1 and all four CYCB1 genes fail to recruit RAD51 to DNA after damage. CYCB1;1 is specifically activated under DNA damage condition and we show that this activation is directly controlled by SUPRESSOR OF GRAMMA RADIATION 1 (SOG1), a plant specific transcription factor that acts similarly to p53 in animals. Thus, while the main mitotic cell cycle activity is blocked under DNA damage conditions, CDKB1-CYCB1 complexes are specifically activated to mediate HR. 19 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Cycle and Development Growth-dependent G1/S control in Drosophila endocycles Bruce Edgar, Norman Zielke, Jinyi Xiang, Xueyang Yu, Jonathan Bohlen German Cancer Research Center (DKFZ)-Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH) Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany Endocycles are variant cell cycles comprised of DNA Synthesis (S)- and Gap (G)-phases but lacking mitosis. Such cycles facilitate post-mitotic growth in many animal and plant cells. DNA replication in endocycling Drosophila cells is triggered by Cyclin E/Cdk2 activity, but this kinase must be inactivated during each G-phase to allow pre-Replication Complexe (preRC) assembly for the next S-phase. Using genetic tests in parallel with computational modeling, we recently demonstrated that Drosophila’s endocycles are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4Cdt2 ubiquitin ligase, and this in turn mediates E2F1 destruction. We propose that it is the transient loss of E2F1 during S-phases that creates the window of low Cdk activity required for preRC formation. In support of this model, overexpressed E2F1 accelerates endocycling, whereas a stabilized variant of E2F1 blocks endocycling by de-regulating target genes including CycE, Cdk1 and mitotic Cyclins. Since rates of E2F1 accumulation can control the frequency of endocycle S phases, understanding how E2F1 expression is affected by cell growth and growth factor signaling is key to understanding the control of cell cycle progression in these cells. In Drosophila salivary glands, we find that altering cell growth by changing nutrition or TOR signaling impacts E2F1 translation, and that this connection makes endocycle progression growth-dependent. Assays in S2 cells show that the 5’ UTR of E2F1 mRNA is important in mediating its translational control, likely in response to TOR and other growth-regulatory signals. In midgut enteroblasts, endocycling rates are also determined by E2F1 expression levels, but in this case a critical input is EGFR/MAPK signaling. Many of the regulatory interactions essential to the growthand translation-dependent G1/S cell cycle oscillator we describe are conserved, suggesting that elements of this mechanism may act in most growth-dependent cell cycles in animal and plant cells. 20 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Cycle and Development Emergence of heterochromatin and prolongation of S phase in Drosophila embryos. Patrick H O'Farrell, Kai Yuan, Antony W Shermoen Dept. of Biochem. and Biophys. UCSF, San Francisco, CA, US Emergence of heterochromatin and prolongation of S phase in Drosophila embryos. Kai Yuan, Antony W. Shermoen and Patrick H. O’Farrell The first event in the slowing of the cell cycle at the midblastual transition in Drosophila embryos is not introduction of a gap phase but the prolongation of S phase. This prolongation is due to a change in the replication from simultaneous initiation of replication all sequences to a sequential process where each of a number of satellite sequences exhibits a stereotyped delay of initiation of replication. Without the resulting extension of S phase in cycle 14, gastrulation is disturbed by a premature mitosis. The onset of late replication is the result of downregulation of cyclin:Cdk1, which, if active during interphase, drives early replication of otherwise late replicating sequences. The late replicating satellite sequences are normally heterochromatic, but lack molecular hallmarks of heterochromatin during the early cell cycles. We examined the onset of heterochromatin formation and its coordination with replication using TALE-lights to tag the highly repetitive sequence that characterizes each block of satellite sequence, and visualizing replication by the transient recruitment of GFP-PCNA while following the onset of HP1-RFP binding. There were several surprising and interesting outcomes. - The initial delay in replication of the satellite sequence in cycle 14 s did not depend on prior recruitment of HP1, which followed the slightly delayed replication of the 359 repeat and did not occur at all during cycle 14 on the 1.686 satellite. - The HP1 recruitment to 359 occurred only after the completion of its replication implying that the timing of this recruitment is due to some other signal. - Mutations introduced into HP1 showed that the early recruitment of HP1 to the 359 sequence is independent of the chromo domain, but depends on C-terminal extension that includes a protein interaction domain. Other foci of HP1 recruitment appeared to depend on the chromo domain indicating diversity in the interactions that initiate heterochromatin formation. - Absence of the 359 sequence in the mother resulted in failure to properly recruit HP1 to the paternal 359 sequence showing that a sequence specific maternal signal promotes embryonic establishment of heterochromatin on this satellite. - In the absence of HP1 binding, replication timing of a satellite was advanced in cycle 15, and reciprocally, it was delayed by artificial recruitment of HP1 using a TALE fusion, showing that HP1 binding impacts replication timing. 21 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Cycle and Development S-phase waves of Cdk1 activity synchronize mitosis in the early Drosophila embryo Victoria Deneke1, Anna Melbinger2, Massimo Vergassola2, Stefano Di Talia1 1 2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, United States Department of Physics, University of California San Diego, La Jolla, CA 92093, United States Since the classical work of Fisher and Kolmogorov in 1930s, chemical waves have been proposed as a mechanism to coordinate events in biological systems. Chemical waves have the potential of transferring information across large spatial scales very rapidly. However, the importance of such waves during embryonic development remains unclear. In the earliest phases of development of most metazoans, mitoses are organized across spatial scales of about 1mm. It has been proposed that trigger waves of Cdk1 activity might play a role in coordinating mitosis in embryos. However, the physical properties of these waves and the molecular mechanisms regulating them remain unclear. Using quantitative live imaging of a FRET biosensor for the activity of Cdk1, we provide a direct visualization of traveling waves of Cdk1 activity in Drosophila embryos. We demonstrate that the speed of the mitotic waves is not regulated by the activity of Cdk1 during mitosis, but by the activity of Cdk1 during S-phase. Consistently, the progressive slowdown of the mitotic waves observed during development is regulated by the activity of the DNA replication checkpoint through the Chk1/Wee1 pathway. Mathematical modeling of Cdk1 and Chk1 activities and ablation experiments indicate that a trigger wave in S-phase is followed by a phase wave that regulates entry into and exit from mitosis. Theoretical arguments argue that the scaling of the speed with Cdk1 activity display universal signatures of the dynamic of a bistable system, in which waves are initiated by stochastic fluctuations. Our findings demonstrate how the physical properties of chemical waves can be modulated by the dynamics of signaling pathways to coordinate cellular events during development. 22 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Cycle and Development From clocks to dominoes: cell cycle remodeling during hES cell differentiation Silvia Santos MRC-Imperial College London During cell decision-making signal transduction networks dynamically change in time and space in response to cues, and thereby trigger different cellular states. The decision to divide is one of the most fundamental cellular responses and the evolutionarily conserved networks that control cell division adapt and remodel in a variety of biological contexts - during development and homeostasis, infections and malignancy, in response to drugs and stresses. A striking example of this versatility occurs during development where the same core regulators drive structurally different divisions. Divisions in the embryo are clock-like, fast, short and synchronous with no checkpoints or gap phases. With time, these divisions become longer and asynchronous. The resulting somatic like cycles have checkpoint control and gap phases, and the initiation of events is dependent on completion of early events, just like a falling domino. The question, thus, arises on how do the same cell cycle regulators self-organize and remodel in time and space to generate structurally different cell division cycles? In our lab we use human embryonic stem cells as a model for the embryonic cell cycle and monitor the activities, concentrations and spatial distribution of key cell cycle regulators in single cells, during ES cell differentiation. In my talk I will be discussing how combining single cell imaging and omics approaches with mathematical modelling is allowing us to shed light into how cell cycle networks remodel in time and space during embryonic to somatic transition. 23 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Cycle and Development Growth Regulation by the Hippo Pathway Nicolas Tapon Francis Crick Institute Lincoln's Inn Fields Laboratory 44 Lincoln's Inn Fields London WC2A 3LY Genetic screens in Drosophila melanogaster have identified the Hippo (Hpo) pathway as one of the major signalling pathways required for tissue size control. The Hpo pathway controls tissue and organ size by both inhibiting cell proliferation and promoting apoptosis. Subsequent studies in mammals have shown that this growth control function is conserved and that Hpo signalling is dysregulated in many types of cancer. At the core of the Hippo pathway lies a kinase cascade comprising the Ste20-related kinase Hpo and the Dbf2-related kinase Warts (Wts). Upon Hpo activation, the downstream kinase Wts phosphorylates and inhibits the pro-growth transcriptional co-activator Yorkie (Yki). Hpo signalling has been proposed to sense various local cues relating to cell density (contact inhibition of growth and mechanical tissue properties) or patterning (morphogen gradients), and translate these cues into a growth arrest signal once an individual tissue has reached its appropriate size. I will discuss our latest findings on the relationship between tissue growth and mechanics, as well as the control of the Hpo pathway by nutritional signals. 24 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Cell Cycle and Development Cytoskeletal Dynamics during Inter-nuclear Spacing in the Drosophila Syncytial Embryo Ojas Deshpande, Jorge de-Carvalho, Ivo Telley Instituto Gulbenkian de Ciência, Oeiras, Portugal The positioning of the nucleus, the central organelle of the cell, is an active and regulated process crucially linked to cell cycle, differentiation, migration, and polarity. Alterations in positioning have been correlated with cell and tissue function deficiency and genetic or chemical manipulation of nuclear position is embryonic lethal. Nuclear positioning plays a key role during early embryonic developmental stages in insects, such as Drosophila, where the nuclei divide without cytokinesis and spread evenly throughout the large syncytial embryo cell. Little mechanistic insight is presently available due to the high opacity caused by yolk within eggs and early embryos, which has restricted the microscopic study of nuclear positioning in pre-blastoderm embryos to chemically fixed samples only. We use a novel technique whereby we create cytoplasmic explants from pre-blastoderm stage embryos of Drosophila that reconstitute nuclear divisions. Using time-lapse microscopy and image processing we quantify the cytoskeletal dynamics and nuclear positioning and dissect the forcegenerating structures that lead to nuclear movement. It is intriguing that the daughter nuclei originating from adjacent mitotic divisions remain separated, resulting in a uniform nuclear arrangement in the absence of compartmentalisation. Astral microtubules play a key role in separation of daughter nuclei during mitosis. We hypothesise that microtubule-mediated repulsion maintains the minimum distance between the neighbouring nuclei, creating nuclear domains. Using transgenic fly lines and high-resolution imaging we are investigating the localisation and function of candidate microtubule interacting proteins (Feo) and molecular motors (Klp3a, Klp61f) that are known to generate stable microtubule overlaps. We show that microtubule overlaps between asters from neighbouring nuclei do occur and Klp61F localises along the aster microtubules. We are now testing if these candidate proteins are preferentially enriched at these overlaps. 25 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitosis How is cohesin positioned in mammalian genomes? Jan-Michael Peters Research Institute of Molecular Pathology (IMP), Vienna During S-phase newly synthesized “sister” DNA molecules become physically connected. This sister chromatid cohesion resists the pulling forces of the mitotic spindle and thereby enables the biorientation and subsequent symmetrical segregation of chromosomes. Cohesion is mediated by ring-shaped cohesin complexes, which are thought to entrap sister DNA molecules topologically. In mammalian cells, cohesin is loaded onto DNA at the end of mitosis by the NIPBL-MAU2 (Scc2-Scc4) complex, becomes acetylated during S-phase, and is stably “locked” on DNA during S- and G2-phase by sororin. Sororin stabilizes cohesin on DNA by inhibiting Wapl, which can otherwise release cohesin from DNA again. In addition to mediating cohesion, cohesin also has important roles in organizing higher-order chromatin structures and in gene regulation. Cohesin performs the latter functions in both proliferating and post-mitotic cells and mediates at least some of these together with the sequencespecific DNA-binding protein CTCF, which co-localizes with cohesin at many genomic sites. Cohesin is required for long-range chromatin interactions, which can either support or suppress enhancerpromoter interactions, and is thought to contribute to the folding of chromosomes into topologically associated domains (TADs). Whereas cohesin might be able to mediate cohesion by connecting any region of sister chromatids, it is important that cohesin associates with specific binding sites in the genome to be able to contribute to higher-order chromatin structure and gene regulation. We therefore analyzed how cohesin is positioned in the mouse and human genome. Our results indicate that cohesin is initially recruited to DNA at NIPBL-MAU2 binding sites, which are distinct from cohesin’s final binding sites. Wapl enables reversible interactions of cohesin with these sites to allow the passage of large protein complexes that move along DNA processively, such as RNA polymerases. To obtain mechanistic insight into how cohesin might translocate from loading to CTCF sites we have begun to reconstitute these processes from purified components and visualized in real time how cohesin interacts with DNA. 26 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitosis Pds5 proteins are key regulators of cohesin dynamics Ana Losada1, Miguel Ruiz-Torres1, et al. 1 Chromosome Dynamics Group, Spanish National Cancer Research Centre, Madrid, Spain Bioinformatics Unit, Spanish National Cancer Research Centre, Madrid, Spain 3 Research Institute of Molecular Pathology (IMP), Vienna, Austria. 2 Cohesin is a ring-shaped complex that entraps the sister chromatids from the time they emerge from the replication fork until their separation in anaphase, thereby ensuring both faithful chromosome segregation and accurate DNA repair by homologous recombination. Cohesin may also promote long-range DNA looping which contributes to transcriptional regulation and organization of DNA replication factories. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21 and either SA1 or SA2. Cohesion factors Pds5, Wapl and Sororin bind cohesin and modulate its interaction with chromatin. There are two versions of Pds5 in vertebrates, Pds5A and Pds5B, and in order to address their functional specificity, we generated single and double knock out alleles for Pds5A and Pds5B. Analyses of Mouse Embryonic Fibroblasts (MEFs) carrying these alleles showed that simultaneous depletion of both Pds5 proteins negatively affects Wapl and Sororin binding to chromatin, therefore altering cohesin dynamics and preventing cohesion establishment. To better characterize the role of Pds5 proteins on cohesin’s association to chomatin, we have now performed Fluorescence Recovery After Photobleaching (FRAP) in quiescent cells. Depletion of both Pds5 proteins clearly reduces the mobility of cohesin, similar to the effect previously observed in MEFs lacking Wapl. Moreover, upon serum stimulation, these cells have trouble to reenter the cell cycle and start DNA replication. Consistent with this defect, we have observed that establishment of the pre-replication complex (preRC) on chromatin is delayed. This delay is likely a consequence of impaired synthesis and accumulation of preRC components including MCMs, Cdc6 and Orc1, rather than a recruitment failure. These results underscore the importance of Pds5 in regulating cohesin dynamics and further suggest that this dynamic behaviour is important for transcriptional regulation. 27 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitosis Mitotic chromosomes acquire mechanical independence through Ki-67 Sara Cuylen1, Claudia Blaukopf1, Thomas Müller-Reichert2, Beate Neumann3, Ina Poser4, Jan Ellenberg3, Anthony A. Hyman4, Daniel Gerlich1 1 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria. Experimental Center, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany 3 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany 4 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 2 Eukaryotic genomes are partitioned into chromosomes, which during cell division form spatially separate compact bodies to move one copy of the replicated genome to each daughter cell. How mitotic chromosomes acquire the biophysical properties enabling their independent motility is poorly understood. Here, we report that the chromosome surface is cell cycle-regulated. Chromosomes have low adhesion towards each other during early mitotic stages, whereas they cluster during mitotic exit to shape one mass of chromatin prior to nuclear envelope reassembly. We identified Ki-67 as a critical factor required to maintain mitotic chromosomes spatially separate, which is necessary for their independent motility and timely segregation. Our study elucidates a biomechanical role of the mitotic chromosome periphery and uncovers an important function of Ki67, a widely used cancer prognostic marker. 28 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitosis Effects of aneuploidy on fitness and tumorigenesis Angelika Amon, et al. Koch Institute for Integrative Cancer Research Howard Hughes Medical Institute Massachusetts Institute of Technology 76-561 500 Main Street Cambridge MA 02139 Aneuploidy, a karyotype that is not a multiple of the haploid complement, is a hallmark of cancer. 90 percent of all solid human tumors harbor an incorrect karyotype. Thus, determining how aneuploidy arises and how it impacts cellular behavior is critical for our understanding of tumorigenesis. We developed in vitro and in vivo mouse models to study the effects of aneuploidy on cellular fitness and tumorigenesis. Using hematopoietic reconstitution assays we find that aneuploidy leads to decreased fitness of stem cells in vivo. Their ability to to proliferate and reconstitute the hematopoietic system in vivo is significantly reduced leading to macrocytopenia and bone marrow failure. Our studies further indicate that aneuploidy also reduces the fitness of immortalized and transformed cells. Thus, aneuploidy has a significant impact on fitness in all contexts. The implications of these findings for tumorigenesis and ways in which aneuploidy could promote tumorigenesis despite its anti-proliferative effects will be discussed. 29 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitosis Integration of microtubule-kinetochore attachment formation and spindle checkpoint signalling by phosphatase complexes Ulrike Gruneberg University of Oxford Faithful chromosome segregation in eukaryotic cells requires the formation and maintenance of stable microtubule-kinetochore attachments. The spindle assembly checkpoint (SAC) is an important regulatory mechanism ensuring the presence of correctly attached chromosomes before anaphase onset. Both timely activation as well as inactivation of the SAC are key prerequisites for the successful orchestration of chromosome segregation. The Mps1 kinase plays a crucial role in triggering and maintaining SAC signalling by phosphorylating the Knl1 kinetochore protein, thus forming a binding platform for the key SAC proteins Bub1, Bub3 and BubR1. Prompt silencing of the SAC upon bipolar attachment thus requires a phosphatase activity opposing Mps1, initiating the loss of Bub1, Bub3 and BubR1 from the kinetochore. In yeast, PP1 has been shown to fulfil this function. We have taken an unbiased approach to identify the phosphatase activity controlling Knl1 phosphorylation and BubR1 and Bub1 kinetochore localisation in spindle checkpoint arrested mammalian cells, and have recently identified BubR1-tethered PP2A-B56 as a key phosphatase opposing Mps1 activity towards Knl1. Consequently, knock down of this phosphatase complex resulted in retention of phospho-Knl1 as well as Bub1 and BubR1 kinetochore localisation in the absence of Mps1 activity. We suggest that PP2A-B56 triggers anaphase onset by eliciting the loss of BubR1 and Bub1 from kinetochores when Mps1 activity drops at the metaphase to anaphase transition. Our latest results indicate that the same pool of PP2A-B56 also regulates Mps1 activity itself and may control the residence time of Mps1 at the kinetochore, thus contributing to the integration of microtubule-kinetochore attachment formation and spindle checkpoint signaling. 30 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitosis Functions of Greatwall-PP2A/B55 pathway in the mammalian cell cycle Mónica Alvarez-Fernández, María Sanz-Flores, Belén Sanz-Castillo, Marcos Malumbres Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain Protein phosphorylation is an essential mechanism to control progression throughout the different phases of the cell cycle. During the last years, the Greatwall-PP2A axis has emerged as a new pathway implicated in control of mitosis. Greatwall kinase, also known as Mastl (microtubuleassociated serine/threonine kinase-like protein) in mammals, specifically inhibits PP2A complexes containing the B55 family of regulatory subunits, which in mammals is composed of 4 different isoforms (PPP2R2A-D). Inhibition of PP2A-B55 complexes by Greatwall occurs in a kinase-dependent manner through the phosphorylation of two small proteins, α-endosulfine and Arpp19, the only two Greatwall substrates identified to date. We previously generated the first genetic model of Greatwall in mammals. Constitutive ablation of Mastl in mice resulted in embryonic lethality, indicating that Greatwall activity is essential for embryonic development. By using a Mastl conditional knockout, we also found that cells lacking Greatwall display a mitotic collapse after nuclear envelope breakdown, characterized by condensation defects and prometaphase arrest, accompanied by chromosome segregation defects and a global reduction in the phosphorylation of Cdk substrates. Interestingly, these defects were partially rescued by concomitant depletion of PP2A B55 regulatory subunits. In order to explore the physiological relevance of PP2A/B55 complexes in mammals, we have now generated knockout mouse models for two of those B55 regulatory subunits, PPP2R2A (B55 alpha) and PPP2R2D (B55 delta), which have been reported as the ubiquitous and cell-cycle related ones. Our preliminary data indicates that, whereas B55 delta knockout mice are viable and do no present obvious phenotypes, deletion of PPP2R2A (B55 alpha) compromises mouse survival. Phenotypes both at the organism and cellular levels are currently being characterized. Interestingly, our recent findings indicate that, beyond mitosis, Greatwall kinase is also important for other cell cycle phases. Moreover, Greatwall is overexpressed in several human tumors and its depletion leads to impaired proliferation of human tumor cell lines, although its relevance to cancer is mostly unknown. On the other hand, PP2A is a well-known tumor suppressor, and its reactivation has been proposed as a therapeutic strategy for cancer treatment. Therefore, we propose here that, in addition to its antimitotic role, inhibition of Greatwall may also have therapeutic benefits through the reactivation of PP2A-dependent pathways. 31 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitotic Exit and Cytokinesis Mechanisms of mitotic regulation by the APC/C Dan Lu, Juliet Girard, Arda Mizrak, David Morgan University of California, San Francisco The dramatic and beautiful events of chromosome segregation are governed by a multi-subunit ubiquitin ligase called the anaphase-promoting complex or cyclosome (APC/C), which catalyzes the ubiquitination and destruction of securin, cyclins, and other important regulatory proteins. The APC/C is activated at specific cell-cycle stages by association with an activator subunit, Cdc20 or Cdh1, which provides binding sites for specific substrate sequence motifs, or degrons. Like other members of the RING family of E3s, the APC/C catalyzes direct ubiquitin transfer from an E2ubiquitin conjugate (E2-Ub) to lysine residues on the protein substrate. We are using experimental and computational methods to dissect the mechanisms underlying the ordered degradation of APC/C substrates. Our single-cell analyses of GFP-tagged APC/C substrates in budding yeast suggest that the timing of substrate degradation is determined by changes in substrate degrons and other mechanisms that govern substrate interaction with the APC/C. Biochemical experiments show that degrons might not simply enhance substrate affinity for the APC/C but also enhance the enzyme’s catalytic function: for example, mutation of a specific degron motif in the S cyclin, Clb5, reduces maximal catalytic rate with that substrate. We are now using computational modeling to develop a more complete understanding of how changes in substrate affinity and catalytic rate can provide the precisely ordered degradation of different APC/CCdc20 substrates. These studies reveal that following APC/C activation, the multi-step ubiquitination process provides an adjustable delay in the onset of substrate degradation. Small variations in substrate affinity and/or catalytic rate can have a major impact on the timing of degradation onset, and robust degradation timing can be achieved over a broad range of parameters. When two substrates share the same pool of APC/CCdc20, their relative enzyme affinities and rates of turnover can influence the partitioning of APC/CCdc20 among substrates, and under some conditions competition between substrates can occur. However, increased expression of the early APC/CCdc20 substrate Clb5 does not delay the degradation of the later substrate securin, arguing against a role for competition in establishing securin degradation timing. These studies provide a conceptual framework for understanding the factors that determine the timing of substrate modification in the cell cycle. 32 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitotic Exit and Cytokinesis Deciphering the role of the GWL/ARPP19-ENSA/PP2A pathway in the control of the cell cycle Anna Castro, et al. CRBM-CNRS Greatwall has recently been identified as an essential kinase to promote mitotic entry and to maintain the mitotic state. Studies in the xenopus egg extracts subsequently characterised the two first substrates of Gwl, Arpp19-ENSA whose phosphorylation promote PP2A-B55 binding and inhibition, and mitotic entry. We are now studying the the role of Arpp19 and ENSA on the control of the cell cycle in mammalian cells and the function of Gwl in the control of normal and oncogenic cell cycle. 33 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitotic Exit and Cytokinesis PP2A-B55 is an NLS-directed Cdk-counteracting phosphatase coordinating mitotic spindle reorganisation with nuclear envelope reformation and cytokinesis. Michael Cundell, Ricardo Nunes Bastos, Elena Poser, Shabaz Mohammed, Francis Barr University of Oxford, Department of Biochemistry Prior to mitosis, many spindle assembly factors (SAFs) and cytokinesis-promoting factors (CYFs) are targeted to the nucleus by polybasic nuclear localisation signals (NLS) leading to their effective sequestration. In metazoan mitosis Cdk1:CycB mediated phosphorylation drives dissolution of the nuclear envelope, activates liberated SAFs and inhibits CYFs, thereby simultaneously promoting mitotic spindle formation whilst preventing precocious cytokinesis. Cdk1:CycB activity reinforces the mitotic state by inhibiting PP2A-B55, one of the major Cdk-counteracting phosphatases, by the Greatwall/ENSA pathway. Previously we have shown that regulation of PP2A-B55 by this pathway during mitotic exit is crucial to explain how anaphase spindle formation and cytokinesis are initiated later than sister chromatid separation. However, it has remained unclear how PP2A-B55 selects specific substrates during mitotic exit, and if there are any general principles governing its action. Using whole cell temporal profiling of protein phosphorylation we have identified 310 phosphosites on 211 proteins that are specifically targeted by PP2A-B55 during anaphase and regulated by the Greatwall/ENSA pathway. This work uncovers a general principle explaining substrate selection by PP2A-B55. Analysis of specific substrates in vitro using purified components and in vivo shows that PP2A-B55 requires a polybasic patch or NLS adjacent to the target phosphosite. This suggests that the NLS is not only used for interphase nuclear sequestration, but also during mitotic exit to ensure the order of cell-cycle events through timely and targeted dephosphorylation of a distinct pool of substrates by PP2A-B55. PP2A-B55 inactivates SAFs such as TPX2, and activates CYFs including the anaphase spindle protein PRC1 and RhoGEF ECT2 at the metaphase to anaphase transition. As a consequence, PP2A-B55 coordinates remodelling of the metaphase spindle with anaphase central spindle formation and cytokinesis. Moreover, PP2A-B55 dephosphorylates numerous components of the nuclear pore complex and is necessary for timely nuclear envelope reformation. Removal of the Greatwall/ENSA pathway therefore results in entrapment of the partly segregated chromatin and central spindle within the confines of the nuclear envelope, as well as precocious anaphase spindle formation and cytokinesis. Our results demonstrate that PP2A-B55 provides a means to coordinate the temporal regulation of multiple proteins required for metazoan mitosis, by integrating nuclear sequestration in G2, with regulation by Cdk1 in M-phase and dephosphorylation during mitotic exit. 34 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitotic Exit and Cytokinesis Spindle pole body specification in budding yeast Jette Lengefeld, Manuel Hotz, Yves Barral Institute of Biochemistry, Department of Biology, ETH Zürich, 8093 Zurich, Switzerland A number of cell types, including stem cells and budding yeast, segregate their centrosomes (or spindle pole bodies – SPBs – in yeast) in a non-random manner at mitosis. For example, Drosophila neuroblasts retain the young centrosome upon cell division while their differentiating daughters inherit the old one. Likewise, budding yeast mother cells retain the new SPB and segregate the old SPB to the bud. How these cells know which of the two centrosome/SPB is the old vs the young one, and how they use this information to orient spindle poles appropriately along the division axis is unknown. It is also unclear whether this process is a secondary consequence of other events, or the result of dedicated pathways. We have started to dissect the molecular events specifying the relative identity of the two SPBs of budding yeast through the cell cycle. These studies indicate that different mechanisms are involved, depending on whether the old SPB of a considered cell was already the old SPB in the previous cycle (established old SPB) or new (newly old SPB). In the second case, phosphorylation of yeast centriolin by the wee1 kinase prior to SPB duplication is required to instruct the “newly old” SPB to adopt the “old” identity. SPBs that have already went through a full cell cycle as old SPB no-longer need instruction by wee1 but require activity of the NEK-related kinase, Kin3 to maintain the old identity. Remarkably, we find that in both cases these pathways act at least in part by promoting the recruitment of the bipartite GAP for the GTPase Tem1 to the old SPB in early metaphase. Furthermore, ablation of the target modification sites did not cause any other major effect than randomizing SPB inheritance, and particularly did not affect spindle alignment with the division axis. Together, these data indicate that SPB specification and spindle orientation is a dedicated process, the selective advantage of which remains to be determined. Our data provide means to investigate what are the specific effects of SPB mis-orientation for cellular physiology. 35 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitotic Exit and Cytokinesis A novel protein network stabilizes the yeast septin ring upon mechanical stress Laura Merlini1, Maria Angeles Juanes1, Alessio Bolognesi2, Yves Barral2, Simonetta Piatti1 1 2 Centre de Recherche en Biochimie Macromoléculaire-CNRS, Montpellier (France) Institute of Biochemistry, ETH Zurich (Switzerland) Septins are conserved GTP-binding proteins that play a critical role in cytokinesis, cell polarity and membrane remodelling. In higher eukaryotes septin dysfunctions have been linked to tumorigenesis and neurodegenerative diseases. In many cell types septins assemble into filaments and rings at the neck of cellular appendages and/or at the cleavage furrow to help compartmentalize the plasma membrane and support cytokinesis. How septin ring assembly is coordinated with membrane remodelling and controlled by mechanical stress at these sites is however unclear. Through a genetic screen we uncovered an unanticipated link between the conserved Rho1 GTPase (i.e. the yeast counterpart of metazoan RhoA) and its effector protein kinase C (Pkc1) with septin ring stability in yeast. Both Rho1 and Pkc1 stabilize the septin ring, at least partly through phosphorylation of the membrane-associated F-BAR protein Syp1, which colocalizes asymmetrically with the septin ring at the bud neck. Syp1 is displaced from the bud neck upon Pkc1-dependent phosphorylation at two serines, thereby impacting the rigidity of the new-forming septin ring. Our data suggest that Syp1 may affect septin dynamics through the glucan synthase Fks1, which remodels the cell wall. We propose that Rho1 and Pkc1 coordinate septin ring assembly with membrane and cell wall remodelling partly by controlling Syp1 residence at the bud neck. 36 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Oral presentations Mitotic Exit and Cytokinesis Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing. Marina Vietri, et al. Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, N-0379 Oslo, Norway. Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway. At the onset of metazoan cell division the nuclear envelope breaks down to enable capture of chromosomes by the microtubule-containing spindle apparatus. During anaphase, when chromosomes have separated, the nuclear envelope is reassembled around the forming daughter nuclei. How the nuclear envelope is sealed, and how this is coordinated with spindle disassembly, is largely unknown. Here we show that endosomal sorting complex required for transport (ESCRT)-III, previously found to promote membrane constriction and sealing during receptor sorting, virus budding, cytokinesis and plasma membrane repair, is transiently recruited to the reassembling nuclear envelope during late anaphase. ESCRT-III and its regulatory AAA ATPase VPS4 are specifically recruited by the ESCRT-III-like protein CHMP7 to sites where the reforming nuclear envelope engulfs spindle microtubules. Subsequent association of another ESCRT-III-like protein, IST1, directly recruits the AAA ATPase spastin to sever microtubules. Disrupting spastin function impairs spindle disassembly and results in extended localization of ESCRT-III at the nuclear envelope. Interference with ESCRT-III function in anaphase is accompanied by delayed microtubule disassembly, compromised nuclear integrity and the appearance of DNA damage foci in subsequent interphase. We propose that ESCRT-III, VPS4 and spastin cooperate to coordinate nuclear envelope sealing and spindle disassembly at nuclear envelope-microtubule intersection sites during mitotic exit to ensure nuclear integrity and genome safeguarding, with a striking mechanistic parallel to cytokinetic abscission. 37 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) POSTER ABSTRACTS Poster presentations EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 1 Deciphering the function of the CPC at the equatorial cortex in anaphase Ingrid Adriaans, Bas Ponsioen, Susanne Lens Department of Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands A divergent chromosome content, also known as aneuploidy, is a common feature of solid tumors. Aneuploidy is the consequence of impaired fidelity of chromosome segregation in anaphase called Chromosomal INstability (CIN). Tetraploidization is one of the causes of CIN and can arise due to cell fusion, mitotic slippage or cytokinesis failure. In the following multipolar mitosis, a tetraploid cell has an increased incidence of mis-segregating chromosomes1,2. The Chromosomal Passenger Complex (CPC), consisting of INCENP, Survivin, Borealin and Aurora B kinase, guards chromosomal stability through regulation of several important processes during cell division, one of these being the proper execution of cytokinesis, thus preventing tetraploidization3.While present at the inner centromere during prometaphase and metaphase, the CPC relocates to the central spindle and the equatorial cortex during anaphase3. Global inhibition of Aurora B kinase activity just before anaphase onset was shown to prevent cleavage furrow ingression, whereas kinase inhibition in early anaphase resulted in regression of an initially fully ingressed cleavage furrow4. How and which (i.e. central spindle or cortical) pool of the CPC controls the onset of cytokinesis is not known. To investigate the function of the CPC at the equatorial membrane we generated a membrane-localized FRET-based sensor of Aurora B kinase activity5. Several membrane localization domains were tested and we found that fusion of the FRET-based sensor to a TUBBY domain which binds PtdIns(4,5)P26, resulted in uniform localization at the cell membrane during all stages of mitosis, including anaphase. Although Aurora B is active during mitosis, we did not detect phosphorylation of the membrane-localized sensor in early mitosis. Phosphorylation of the sensor at the cell membrane started in anaphase before visible membrane ingression, and was most prominent in the equatorial furrow. Similar to FRET-based Aurora B sensors that were targeted to chromatin, kinetochores or centromeres5,7, phosphorylation of the membrane localized-sensor also rapidly diminished after inhibition of Aurora B. Importantly, phosphorylation of the membrane-localized sensor was dependent on the presence of Mklp2, the mitotic kinesin-like protein that promotes the relocation of Aurora B from centromeres to the central spindle and equatorial cortex in anaphase. These initial results demonstrate that the TUBBY-FRET-based Aurora B sensor is a specific tool for detecting and measuring quantitative changes in Aurora B-dependent phosphorylation at the cell membrane. It will allow us to elucidate how this local activity is regulated and how it is associated with initiation of cytokinesis. References: 1. N.J. Ganem et al., Nature 460, 278-282 (2009) 2. Silkwort et al., PLOS one 4, e6564 (2009) 3. Van der Horst et al., Chromosoma 123, 25-42 (2014) 4. Guse et al., Current biology 15, 778-86 (2005) 5. Fuller et al., Nature 453, 1132-6 (2008) 6. Szentpetery et al., BMC Cell Biology, 10:67 (2009) 7. Liu et al., Science 323, 1350-3 (2009) 38 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 2 Temporal control of mitosis: the many roles of positive feedback Ana Rita Araujo, Rahuman Sheriff, Silvia DM Santos MRC-Clinical Sciences Centre, Imperial College London, London, UK The cell cycle is characterized by a sequence of events by which a cell grows, replicates its genetic information and its components and gives rise to two identical daughter cells. Even though the molecular machinery that drives cell division cycles is the same in all the tissues, within the same organism, the cell cycle length varies amongst different cells. Preliminary quantification of the length of cell cycle phases in single cells by live-cell imaging showed high variability in the dynamics of cell cycle phases. An exception of this was seen in mitosis, where different cells seem to keep a constant length of time to complete this phase. Surprisingly, there is no correlation between cell cycle length and mitosis duration. In other words, it does not matter if a cell runs through the cell cycle fast or slow, once it reaches mitosis it will complete mitosis in a short and synchronous manner. We and others have shown that positive feedback regulation is crucial to keep mitotic events synchronized (Holt et al 2008, Santos et al 2012). We hypothesized that positive feedback might be the molecular mechanism that keeps the time of mitosis constant across different cell lines with variable cell cycle lengths. To test this hypothesis we combined live cell imaging and computational modelling and showed that when positive feedback is perturbed the switch-like activation of Cdk1 is compromised, leading to a much more variable and sluggish completion of mitosis. Importantly, perturbing positive feedback gave rise to a correlation between the length of the cell cycle and duration of mitosis. This work shows that positive feedback, a recurrent motif in cell cycle control, may also be important to keep mitosis short, synchronous and temporally insulated from earlier cell cycle events. We hypothesized that positive feedback may be a recurrent strategy to help keep events modular in other biological systems. 39 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 3 Meiosis II: a unique form of cell division Orlando Argüello-Miranda, Ievgeniia Zagoriy, Valentina Mengoli, Wolfgang Zachariae Laboratory of Chromosome Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. Meiosis, the basis for sexual reproduction, consists one round of chromosome replication followed by two rounds of chromosome segregation, called meiosis I and –II. The second meiotic division presents several features that set it apart from all other forms of cell division. (1) Unlike mitosis, it is neither preceded nor followed by DNA replication. Instead, it occurs directly after the M phase of meiosis I and prior to a differentiation process that creates the gametes. (2) Meiosis II segregates chromatids that have undergone recombination and are held together by cohesin located solely around their centromeres. While meiosis II is frequently called a mitosis-like division, it remains unclear to what extend principles of mitotic chromosome segregation and cell cycle control apply to meiosis II. The analysis of meiosis II has been hindered by the difficulty of manipulating meiosis IIspecific processes without affecting meiosis I. To address this problem, we have developed a protocol that generated meiotic cultures of budding yeast that progress through meiosis II in a highly synchronous manner. We find that meiosis II includes unique mechanisms that coordinate the removal of centromeric cohesin with exit from M phase and spore formation, the yeast equivalent of gametogenesis. 40 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 4 Regulation of the G1-to-S transcriptional wave in an unperturbed cell cycle Isabel Alves-Rodrigues, Angel Guerra-Moreno, Maribel Marquina, Elena Hidalgo, Jose Ayte Oxidative Stress and Cell Cycle, Universitat Pompeu Fabra, Barcelona, 08003, Spain. Inactivation of the Retinoblastoma protein (pRB) leads to unregulated cell cycle progression promoting uncontrolled cell growth, genomic instability and aneuploidy, hallmarks of tumor progression. pRB tumor suppressor activity is achieved through binding and regulating the E2F family of transcription factors. The fission yeast MBF complex, which is the functional homolog of mammalian E2F/pRB, drives the G1-to-S wave of transcription controlling the expression of genes that are directly or indirectly required for DNA synthesis. MBF has been found bound to its target promoters throughout the cell cycle, implicating that regulation of MBF activity is not achieved by simply modulating its DNA-binding activity. We have recently shown that the MBF complex is regulated by both the DNA replication checkpoint and the DNA damage checkpoint, but in opposite directions: while the former activates MBF-dependent transcription, the latter down-regulates MBF activity by directly phosphorylating the core subunit of the MBF complex, Cdc10 (Gomez-Escoda et al, 2011; Ivanova et al 2013). To characterize the regulation of MBF in an unperturbed cell cycle, we have developed a fluorescence-based reporter strain that can measure MBF activity in vivo. Using reverse genetics to introduce this reporter system in a fission yeast knock-out (KO) collection that includes over 3000 non-essential mutants, we have performed a double High Throughput Screening (HTS) on live cells by i) FACS on 96-well plates and ii) Cell Sorting of individual mutants from a pooled population of the barcoded KO strains. With this double screening, we have been able to identify individual regulators of MBF. Among the isolated genes, we will discuss the roles of the COP9/Signalosome and the histone deacetylase (HDAC) Hst4 in the regulation of MBF activity. Gómez-Escoda, B., Ivanova, T., Calvo, I.A., Alves-Rodrigues, I., Hidalgo, E. and Ayté, J. (2011) Yox1 links MBF-dependent transcription to completion of DNA synthesis. EMBO Reports 12:84-89. Ivanova, T., Alves-Rodrigues, I., Gómez-Escoda, B., Dutta, C., DeCaprio, J.A., Rhind, N., Hidalgo, E. and Ayté, J. (2013) The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor. Mol. Biol. Cell 24:3350-3357 41 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 5 Releasing the brake - the functional role of phosphatases in spindle assembly checkpoint silencing. Debora Bade1, Vincent Groenewold1, 2, Antoinette Teixeira1, Geert Kops1, 2, 3 1 Molecular Cancer Research, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands. Department of Medical Oncology, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands. 3 Cancer Genomics Netherlands, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands. 2 The spindle assembly checkpoint (SAC) plays an important role as proof-reading machinery during cell division: it delays anaphase onset until all chromosomes are attached in a bipolar manner and consequently directly protects the cell from aneuploidy. Although it is fairly established how the SAC is initiated and maintained, the actions leading to its silencing and subsequently to mitotic exit are poorly understood. Specifically the fact that many proteins change their phosphorylation state upon SAC silencing has not been addressed systematically yet. In this project, I assess the differential phosphorylation sate of the SAC proteome of mitotic cells either displaying unattached kinetochores or bi-oriented chromosomes. I thereby specifically concentrate on kinetochore complexes such as the KMN, MCC, APC/C, RZZ, and SKA. Furthermore, I am currently attributing the (de)phosphorylation event(s) to specific kinases, such as MPS1, or phosphatases, such as PP2A and PP1. In detail, I am using APEX and BioID purification experiments to confirm suspected and to identify new substrates of SAC phosphatases/kinases. Next, I am addressing the functional contributions of selected phosporylation/dephosphorylation events to SAC activation/silencing. In conclusion, this project aims to draw a comprehensive picture of the SAC phosphoregulation. 42 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 6 A dynamical framework for the all-or-none G1/S transition in individual human cells: a role for Emi1 in the irreversible transition Alexis Barr2, Tongli Zhang2, Chris Bakal1, Bela Novak2 1 2 Institute of Cancer Research Department of Biochemistry, University of Oxford Whilst the irreversible and all-or-none nature of the G1/S transition is well understood in yeast, we lack an understanding of the systems-level properties that control this transition in mammalian cells. Here we use quantitative single cell imaging to track the expression of key G1/S regulators in human cells to parametrise a new model of the G1/S transition. We show that a proteolytic double negative feedback loop between Cdk2:Cyclin and the Cdk inhibitor, p27Kip1, drives a switch-like S-phase entry. Furthermore, our model predicts that Emi1 levels are critical in maintaining an irreversible G1/S transition, which we validate using Emi1 knockdown and live imaging of G1/S reporters. Thus we provide a dynamical framework to understand the irreversible G1/S transition in human cells. 43 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 7 A 14-3-3 phospho docking protein stabilises bipolarity of the acentrosomal meiotic spindle Robin Beaven, Hiro Ohkura Wellcome Trust Centre for Cell Biology, The University of Edinburgh, UK To faithfully segregate chromosomes, spindles need to maintain their bipolar organisation. Centrosomes are key to this function during mitosis, but it is unclear how bipolar spindles are formed and stably maintained in female meiosis, where centrosomes are absent. Using Drosophila oocytes we identified a new function for 14-3-3epsilon in this context. The 14-3-3s are phospho-S/T docking proteins which bind and regulate a multitude of interactors, so acting at the core of diverse signalling pathways. We found that loss of 14-3-3epsilon caused female sterility and frequent tripolar spindles. Live imaging showed that bipolar spindles could form but were highly unstable. Mass spectrometry was then used to identify binding partners of 14-3-3epsilon in oocytes. They included the kinesin Ncd, loss of which is also known to result in highly unstable meiotic spindles. We had previously shown that the pole localisation of the microtubule regulator Msps (XMAP215) is lost in ncd mutants, and depletion of Msps results in tripolar spindles similar to 14-3-3epsilon depletion. We hypothesise that 14-3-3epsilon could regulate Ncd’s ability to transport Msps to the spindle poles, and indeed find Msps localisation to be disrupted in 14-3-3epsilon’s absence. We have also identified a predicted 14-3-3 binding site and neighbouring Aurora phosphorylation site in the tail of Ncd, and hypothesise that Aurora kinases act to regulate 14-3-3’s binding to Ncd. These molecular insights are shedding light on a mechanism which the meiotic spindle utilises to overcome its inherent instability, resulting from its lack of centrosomes. 44 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 8 Annexin A2 is required for the early steps of cytokinesis Christelle Benaud, Gaelle Le Dez, Svetlana Mironov, David Reboutier, Claude Prigent Université de Rennes I, Institut de Génétique et Développement, Centre National de la Recherche Scientifique, UMR 6290, Rennes, France Cytokinesis requires the formation of an actomyosin contractile ring between the two sets of sister chromatids. Annexin A2 is a calcium and phospholipid binding protein implicated in cortical actin remodeling. We report that Annexin A2 accumulates at the equatorial cortex at the onset of cytokinesis and depletion of Annexin A2 results in cytokinetic failure, due to a defective cleavage furrow assembly. In absence of Annexin A2, the small GTPase RhoA that regulates cortical cytoskeletal rearrangement fails to form a compact ring at the equatorial plane. Furthermore, Annexin A2 is required for cortical localization of the RhoGEF Ect2 and to maintain the association between the equatorial cortex and the central spindle. Our results demonstrate that Annexin A2 is necessary in the early phase of cytokinesis. We propose that annexin A2 participates in the central spindle-equatorial plasma membrane communication. 45 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 9 Cdk1-mediated regulation of the protein phosphatase scaffold RepoMan Junbin Qian, Jin Huang, Sofie De Munter, Bart Lesage, Monique Buellens, Mathieu Bollen Laboratory of Biosignaling & Therapeutics, Department of Cellular and Molecular Medicine, University of Leuven, Belgium RepoMan is a mitotic interactor of protein phosphatases PP1 and PP2A-B56. During prometaphase PP2A-B56 promotes the chromosome targeting of RepoMan, whereas RepoMan-associated PP1 opposes the centromeric targeting of Aurora B through dephosphorylation of Histone H3 at Thr3. Here, we show that the Cdk1-mediated phosphorylation of RepoMan reduces the binding of PP1 but enhances the recruitment of PP2A-B56. Upon inactivation of Cdk1 at the beginning of anaphase PP2A-B56 dissociates from RepoMan and the binding of PP1 is increased, culminating in the bulk recruitment of RepoMan to the chromosomes. This phosphatase switch serves to make the chromosome-binding of RepoMan Cdk1-independent and irreversible. We also show that the reduced PP1-RepoMan interaction during prometaphase prevents the precocious dephosphorylation of Aurora B and Importin beta which are substrates of RepoMan-associated PP1 in anaphase. Hence, the Cdk1-mediated phosphorylation of RepoMan contributes to the ordered dephosphorylation of mitotic-exit substrates. 46 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 10 The Drosophila protein Matrimony is a functional homolog of kinetochore proteins Spo13, Moa1, and Meikin Amanda Bonner1, R. Scott Hawley1, 2 1 2 Stowers Institute for Medical Research Department of Molecular and Integrative Physiology, University of Kansas Medical Center During meiosis homologous chromosomes must first pair and then segregate from each other in a reductional division before going through a second equational, mitosis-like division in which sisters separate to create haploid gametes. Proper segregation of homologs during the first meiotic division depends on both the formation of chiasmata between homologs and the mono-orientation of sister kinetochores towards the same pole. Meiosis-specific kinetochore proteins have been characterized in budding and fission yeasts, and, very recently, in mammals, namely Spo13, Moa1, and Meikin, respectively. While these proteins share no sequence homology, all three are expressed specifically during meiosis I, co-precipitate with Polo-like kinases, and aid in the protection of cohesion between sister chromatids at the centromere. Here we provide evidence that the Drosophila protein Matrimony (Mtrm) plays a similar role during female meiosis. Mtrm, which was originally identified genetically as a gene required for the proper segregation of achiasmate chromosomes, has also been shown to be a strong interactor of Polo kinase. Also, despite the overall lack in sequence homology among these four proteins, Mtrm contains an LxExxxN degron motif that was originally shown in Spo13 to be required for both proteins to be recognized by the Anaphase Promoting Complex (APC). There is also a shared region of homology between Mtrm and Meikin that contains a canonical Polobox binding motif. We demonstrate here that, as is the case for mutants in Spo13, Moa1, and Meikin, mutations in Mtrm cause precocious separation of sister chromatids and chromosome missegregation defects, particularly when recombination between homologs is absent. These findings help explain the curious segregational defects we see in a mtrm-/+ heterozygote, where homologs that are connected by a chiasmata disjoin as expected, while achiasmate homologs seemingly segregate at random. We also show that Mtrm can inhibit cell cycle progression when expressed during mitosis, as has also been previously shown with Spo13. On the basis of these data, we suggest that Mtrm joins a very newly described family of meiosis-specific kinetochore proteins that are important for regulating Polo-like kinase’s activities during the first meiotic division. 47 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 11 Degradation of Ndd1 by APC/CCdh1 generates a feed forward loop that times mitotic protein accumulation Michael Brandeis1, Julia Sajman1, Drora Zenvirth1, Mor Nitzan2 1 The Department of Genetics, The Alexander Silberman Institute of Life Sciences,The Hebrew University of Jerusalem, Jerusalem 9190401, Israel 2 The Racah Institute of Physics, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel Ndd1 activates the Mcm1–Fkh2 transcription factor to transcribe mitotic regulators. The anaphasepromoting complex/cyclosome activated by Cdh1 (APC/C-Cdh1) mediates the degradation of proteins throughout G1. Here we show that the APC/C-Cdh1 ubiquitinates Ndd1 and mediates its degradation, and that APC/C-Cdh1 activity suppresses accumulation of Ndd1 targets. We confirm putative Ndd1 targets and identify novel ones, many of them APC/C-Cdh1 substrates. The APC/CCdh1 thus regulates these proteins in a dual manner—both pretranscriptionally and posttranslationally, forming a multi-layered feedforward loop (FFL). We predict by mathematical modelling and verify experimentally that this FFL introduces a lag between APC/C-Cdh1 inactivation at the end of G1 and accumulation of genes transcribed by Ndd1 in G2. This regulation generates two classes of APC/C-Cdh1 substrates, early ones that accumulate in S and late ones that accumulate in G2. Our results show how the dual state APC/C-Cdh1 activity is converted into multiple outputs by interactions between its substrates. 48 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 12 The double role of Cdc20 in the Spindle Assembly Checkpoint Andrea Ciliberto1, Elena Chiroli1, Paolo Bonaiuti1, Fridolin Gross2 1 2 IFOM, Via Adamello 16, Milan Institute for Theoretical Biology, Charite and Humboldt University, Invalidenstrasse 43, Berlin The activation of the Anaphase Promoting Complex or Cyclosome (APC/C) drives cells from metaphase to anaphase. APC/C ubiquitinates two key substrates – mitotic cyclins and securin – whose degradation allows the progression of the cell cycle oscillator and the separation of sister chromatids. To recognize its substrates, APC/C needs to be bound to its cofactor Cdc20. Cdc20, however, is not simply an activator of APC/C, as it can also inhibit APC/C. More precisely, in the presence of unattached kinetochores Cdc20 is part of a multi-protein complex, the Mitotic Checkpoint Complex or MCC, which inhibits APC/C and arrests cells in metaphase. MCC is the final output of a signaling pathway known as the Spindle Assembly Checkpoint which prevents cells from becoming aneuploid. The double effect of Cdc20 on APC/C prompted opposite interpretations regarding its role during a mitotic arrest. Cdc20 degradation has been proposed to be required for timely APC/C activation (i.e., Cdc20 as an inhibitor) but also for proper checkpoint maintenance (i.e., Cdc20 as an activator). Here, we use a combination of mathematical models and experiments to show that, during an arrest induced by the SAC, Cdc20 is primarily an inhibitor of APC/C. It can only switch to an activator when its synthesis becomes uncoupled from degradation. 49 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 13 Mitotic catastrophe: insights from synthetic yeast Claude Gérard1, John Tyson2, Damien Coudreuse3, Bela Novak1 1 Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK Department of Biological Sciences, Virginia Tech, Blacksburg, USA 3 Institute of Genetics and Development, CNRS UMR 6290, Rennes, France 2 Proper progression through the cell cycle is fundamental for cellular life. From the control of mitotic entry to the regulation of the onset of DNA replication, the cyclin-dependent kinase (CDK) family is the primary node of the circuit driving the eukaryotic cell division cycle. Modulation of CDK activity relies on a host of inputs, highlighting the complexity of this critical process. However, discerning essential controls from secondary regulation is a challenge that may limit our understanding of the core engine behind cell cycle progression. Building on past studies demonstrating that a single cyclin is sufficient for driving this process in fission yeast, we generate and analyze basic synthetic systems providing controlled CDK/Cdc2 activity levels and bypassing a large part of the endogenous regulatory network. Interestingly, the simplicity of the synthetic yeasts we have built makes them particularly well-adapted for mathematical modeling and theoretical dissection of the organization of cell cycle control. A combination of modeling and experimental approaches using fission yeast cells operating with minimal cell cycle networks indeed allowed us to propose a novel mechanism for the origin of mitotic catastrophe, a lethal result of major deregulation and overlap of cell cycle phases. Our studies reveal a cumulative effect of deregulated G1/S cyclin-dependent CDK activity on mitotic control in the absence of the Wee1/Cdc25 feedback loop. This work highlights the importance of coupling classical genetics with synthetic biology and mathematical modeling for understanding normal and pathological cell cycle events. 50 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 14 Single-cell monitoring of cell cycle progression in whole developing organs. Bénédicte Desvoyes, María Delgado-Barea, Sofía Otero, María Isabel Lopez, Crisanto Gutierrez Centro de Biología Molecular Severo Ochoa (CSIC-UAM), calle Nicolas Cabrera 1, 28049 Madrid, Spain. Plant organ development is mainly a postembryonic process that occurs in the adult in a continuous manner. Consequently, a strict balance between cell proliferation and differentiation is required. In addition, many plant cells undergo one or more endoreplication cycles as part of their normal differentiation program. Therefore, growing plant organs consist of populations of dividing, endoreplicating and differentiated cells all derived from a few stem cells. The transitions between the different cell pools are integrated and respond to developmental and environmental cues. In fact, cell cycle control influences greatly developmental programs, e.g. increased or decreased proliferation rates affect organ growth and shape. To get insight into the coordination of the different processes that influence organ development it is important to be able to identify cells progressing in vivo through the cell cycle in a non-invasive manner. We have used fluorescently-labeled proteins that unambiguously identify cells in G1, S/G2 and G2 and generated plants that express them under their own promoters in a single plant. (1) As a G1 marker, we used a CFP-labeled prereplication complex protein that is loaded in late M/early G1 and rapidly degraded soon after S-phase initiation in a proteasome-dependent manner. (2) S-phase cells are followed by the incorporation of a canonical histone H3.1-mRFP, which is maintained through mitosis in actively dividing cells and excluded late in G2 in cells undergoing their last mitotic cycle. (3) Finally, G2 cells are labeled with CYCB1;1-GFP, with a maximum in late G2 and degraded by the APC in anaphase. These markers are also useful to follow cells undergoing endoreplication cycles. The combination of these three markers in a single organism allows monitoring all the cell cycle phases and constitutes an important tool to study cell cycle regulation in the context of a growing organism during normal growth or in response to internal and external signals. 51 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 15 Modeling the dynamics of spindle assembly checkpoint signaling during mitotic exit progression Amalie Dick1, Lukas Hutter2, Béla Novák2, Daniel Gerlich1 1 2 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Oxford Centre for Integrative Systems Biology, Department of Biochemistry, University of Oxford, Oxford, UK The spindle assembly checkpoint contributes to faithful chromosome segregation by delaying anaphase onset until all chromosomes have attached their sister kinetochores to opposing spindle poles. Satisfaction of the spindle assembly checkpoint leads to rapid APC/C activation, which promotes mitotic exit by targeting cyclin B1 for ubiquitin/proteasome-mediated degradation. Cdk1 inactivation by cyclin B1 degradation in turn contributes to permanent spindle assembly checkpoint silencing, which has been proposed as a mechanism to warrant unidirectional progression through mitotic exit. Here, we performed quantitative live cell imaging and mathematical modeling to better understand the kinetics of APC/C activation and the regulation of spindle assembly checkpoint signaling at the metaphase-to-anaphase transition. By acutely detaching chromosomes from the spindle using laser microsurgery or nocodazole, we found that the spindle assembly checkpoint remained proficient until the very end of metaphase, whereas it was unresponsive already during very early stages of anaphase. To investigate how the spindle assembly checkpoint rapidly switches from a responsive to a non-responsive state, we established live cell assays probing for the effect of partial Cdk1 inactivation. The quantitative data were integrated into a mathematical model that describes the kinetics of the regulatory system in terms of its key components. Our model recapitulates key features of spindle assembly checkpoint signaling from prometaphase until mitotic exit. This helps us to understand how the steady states of the system determine its response to lack of attachment. Our analysis shows how thresholds for inactivation and re-activation of spindle assembly checkpoint signaling arise at the systems-level, and establish a “point of no return” at the metaphase-to-anaphase transition as Cdk1-activity drops. 52 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 16 Roberts Syndrome Esco2-E450del Mutant Partially Rescues Cohesion at Pericentric Regions of Esco2 Deficient MEFs. Peter Ditte, Gregor Eichele Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Although most of known ESCO2 mutations in Roberts Syndrome (RBS) lead to nonsense-mediated decay or to severely truncated proteins, there are three mutations located in acetyltransferase domain that stand out because they do lead to a protein of normal size. Among them p.E453del corresponds to in-frame deletion of glutamic acid at position 453 and despite the fact that it causes RBS, the protein possesses full auto-acetylation activity. No cytogenetic or in vivo biochemical data is available since only one case of stillborn fetus with E453del mutation was reported. Using BAC transgenomics we prepared MEF cell line expressing murine Esco2-E450del in Esco2 deficient background. The expression profile of Esco2-E450del is similar to wild type protein with peak of expression in mid S-phase. The level of Smc3 acetylation in Esco2-E450del MEFs is decreased during S-phase compared to wild type Esco2 and comparable to Esco2 null cells. The data shows that Esco2-E450del is unable to effectively acetylate Smc3 in vivo. As a consequence of impaired sister chromatid cohesion at pericentric regions, mitotic chromosomes of Esco2 deficient MEFs typically show railroad track appearance, what can be evaluated by distance between centromeres. In Esco2E450del MEFs, the average distance between centromeres is clearly shorter than in Esco2 null cells albeit still significantly longer than in Wt cells, suggesting that Esco2-E450del MEFs partially establish sister chromatid cohesion at centromeres. Altogether, our data provide the first biochemical evidence that E450del mutation has severe impact on ability of Esco2 to acetylate Smc3 in vivo, yet showing milder cohesion defects at centromeric regions of Esco2 deficient MEFs. Furthermore, the case of Esco2-E450del indicates that at least some RBS mutations may not be null. 53 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 17 Rab35 GTPase couples lumen formation to cell division during polarity establishment in 3D renal cysts Kerstin Klinkert, Arnaud Echard Membrane Traffic and Cell Division Lab Institut Pasteur - CNRS UMR3691 25-28 rue du Docteur Roux, 75015, Paris, France Most of the human cancers arise from epithelial cells and the loss of epithelial apico-basal polarity is a hallmark in tumor progression. It has been shown that the initiation of epithelial polarity is tightly connected to cell division and polarized membrane traffic, but how this is coordinated at a molecular level remains unknown. We addressed this question in renal MDCK cells, which develop 3D polarized cysts with an open apical lumen from a single cell when cultured in Matrigel. Unpolarized renal MDCK cells establish an apical membrane already at the first cell-cell interface during the two-cell stage, and this is coupled to cytokinesis by a unknown mechanism. Unexpectedly, we found that the Rab35 GTPase directly interacts with the cytoplasmic tail of the apical transmembrane protein Podocalyxin (PODXL, aka GP135), a classical apical marker essential for epithelial polarity and lumen formation. Interestingly, Rab35 was enriched at the future apical membrane during the first cell division before PODXL or any other tested apical proteins could be detected. Depletion of Rab35 or replacement of endogenous PODXL by a mutant unable to interact with Rab35, prevented PODXL and other apical proteins to become enriched at the future apical membrane during the first cell division. Consequently, a complete inversion of apico-basal polarity was observed in these conditions. Furthermore, the experimental delocalization of Rab35 to mitochondrial membranes induced a striking accumulation of vesicles containing PODXL, Crb3, Cdc42 and aPKC around the mitochondria, leading to a loss of apico-basal polarity. We propose that Rab35 acts as a direct tether for transcytosed PODXL-containing vesicles at the future apical membrane, and thereby plays a crucial role in triggering the establishment of apical polarity. In addition, this work provides a molecular mechanism for coupling the site of cytokinesis to the initiation of polarity and lumen localization in 3D structures. 54 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 18 Kinase-Phosphatase interplay regulates Kar9 function in spindle positioning Ana-Maria Farcas, Mathias Bayer, Yves Barral Institute of Biochemistry, ETH Zurich Faithful alignment of the mitotic spindle with the polarity axis of the cell drives asymmetric division of polarized cells. In constitutively asymmetric dividing cells, such as budding yeast, this process is also essential in order to ensure that each daughter inherits a full copy of the genome. In Saccharomyces cerevisiae the protein Kar9 mediates the alignment of the spindle with the motherbud axis of the metaphase cell. Kar9 localizes to aster microtubules in an asymmetric fashion, almost exclusively to the plus-end of microtubules emanating from the old spindle pole body (SPB, the yeast equivalent of the centrosome). Interaction of Kar9 with microtubule plus-ends depends on its interaction with the EB1 homolog Bim1. Furthermore, Kar9 interacts with the type V myosin Myo2, which walks along actin cables emanating from the bud cortex, carrying Kar9 and microtubule tips towards the bud. Thereby, the Bim1-Kar9-Myo2 pathway drives both the orientation and alignment of the spindle in mitotic yeast cells. Accordingly, symmetric localization of Kar9 to both asters causes both SPBs to orient towards the bud and impairs spindle alignment with the mother-bud axis. Therefore, the mechanisms ensuring Kar9 asymmetry are crucial for proper spindle positioning. Here, we show that the protein phosphatase 1 (Glc7 in budding yeast), interacts with, and controls Kar9 function, most likely by counteracting Clb4/Cdk1- and Dbf2-mediated phosphorylation of Kar9. By using a series of Kar9 mutants (e.g. phospho-mimicking, phospho-ablating, phosphatase-docking and chimeras), we aim to understand the mechanism by which Clb4/Cdc28, Dbf2 and Glc7 regulate Kar9 distribution and function. 55 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 19 The phosphatase regulator NIPP1 is essential for the proliferation and differentiation of male germ cells Monica Ferreira1, 2, Shannah Boens1, Kathelijne Szeker1, Aleyde Van Eynde1, Margarida Fardilha2, Mathieu Bollen1 1 Laboratory of Biosignaling & Therapeutics, Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium. 2 Institute for Research in Biomedicine-iBiMED, Health Sciences Department, University of Aveiro, Aveiro, Portugal. NIPP1, for Nuclear Inhibitor of Protein Phosphatase 1 (PP1), regulates transcription and pre-mRNA splicing by targeting PP1 to specific nuclear substrates. The deletion of NIPP1 in mice is early embryonic lethal. Studies on cultured cells suggest a role for NIPP1 in cell proliferation and differentiation. To examine the role of NIPP1 in adult germline cell proliferation and differentiation, we generated an inducible NIPP1 knockout (KO) mouse model. We observed that the Crerecombinase induced deletion of NIPP1 in four-weeks-old testis causes a progressive loss of germ cells within 8 weeks, culminating in a Sertoli cell-only phenotype. At earlier time points, the mitotic germ cells (spermatogonia) failed to progress beyond early spermatogenic events due to proliferation defects and cell cycle dysregulation, resulting in an increased rate of apoptosis. Several recombination-related proteins were downregulated after the deletion of NIPP1 and the incidence of unrepaired DNA breaks was decreased, as detected by reduced γH2AX foci on the axes of autosomal chromosomes in spermatocytes during meiotic prophase I. Consistent with this finding, radiation-induced γH2AX foci formation was compromised in the NIPP1 KO testis. Altogether our results show that NIPP1 is essential for both male germline stem-cell proliferation and the differentiation of spermatocytes. 56 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 20 How budding yeast control extracellular matrix remodelling during cytokinesis? Magdalena Foltman1, 2, Iago Molist1, 2, Alberto Sanchez-Diaz1, 2 1 Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria, CSIC, Albert Einstein 22, 39011, Santander, Spain 2 Departamento de Biología Molecular, Universidad de Cantabria, Facultad de Medicina, Cardenal Herrera Oria s/n, 39011, Santander, Spain During cytokinesis cells coordinate contraction of the actomyosin ring with the ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as important players during cytokinesis. In budding yeast the chitin synthase Chs2 makes the primary septum, a special layer of ECM that is essential for cell division. To try to understand how yeast cells coordinate actomyosin ring contraction, plasma membrane ingression and remodelling of the extracellular matrix we have used budding yeast Chs2 and Inn1 proteins to isolate ‘ingression progression complexes’ (IPCs) that contain key actomyosin rings components from yeast cells undergoing cytokinesis synchronously. We have identified, together with Chs2 and Inn1, actomyosin ring components Cyk3, myosin type II, the IQGAP protein Iqg1 and Hof1. We propose that IPCs are central to the mechanism by which cells coordinate cytokinesis. We have found that the catalytic domain of Chs2 interacts directly with the C2 domain of Inn1 and the transglutaminase-like domain of Cyk3. We have now data indicating that Inn1, Chs2 and Cyk3 form a stable complex. We found that chitin synthase Chs2 is activated by C2 domain of Inn1, as well as the transglutaminase-like domain of Cyk3. Finally we have exploited an experimental system that allowed us to determine a previously unknown role for the C-terminus of Inn1 and to understand how Inn1 and Cyk3 finely regulate Chs2 activity in vivo. 57 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 21 Aurora A mutant neuroblasts are defective for cyclin B degradation in the absence of the Spindle Assembly Checkpoint. Régis GIET Université of Rennes 1-CNRS Control of the balance between stem cell renewal, differentiation and accurate chromosome segregation are essential to ensure tissue homeostasis. Drosophila mutants for sas-4 and aurA display brain tumors with extra Neuroblasts (NBs), triggered by loss of cell identity. These mutants also show defective mitotic spindle assembly and consequent mitotic delay caused by the activation of the Spindle Assembly Checkpoint (SAC). In an attempt to determine if generating aneuploidy would compromise NB proliferation, we inactivated the SAC by removing the SAC protein Mad2 from aurA and sas-4 mutants. Removal of the SAC in the sas-4 mutant impaired NB proliferation and chromosome segregation. By contrast, removing the SAC in the aurA mutant did not prevent brain overgrowth, tumor formation, and chromosome segregation. Monitoring Mad2 and cyclin B dynamics in live aurA neuroblasts revealed that chromosome alignment and SAC satisfaction were not coupled to timed cyclin B degradation. We therefore show for the first time the existence of an Aurora A-dependent mechanism that promotes timed and efficient cyclin B degradation. As a consequence, aurA mutants NBs are delayed in mitosis, and perform accurate chromosome segregation even in the absence of the spindle assembly checkpoint. 58 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 22 The kinetochore-to-cytoplasm ratio determines the strength of the spindle assembly checkpoint during early embryonic divisions of C. elegans Matilde Galli, David Morgan UCSF, Department of Physiology The spindle assembly checkpoint (SAC) delays mitotic progression when chromosomes are not properly attached to microtubules of the mitotic spindle. Interestingly, cells exhibit a large variation in the extent to which they delay mitotic progression upon activation of the SAC. In C. elegans, previous studies have shown that early embryonic divisions are only moderately delayed upon disruption of their mitotic spindle. To determine whether this is due to an intrinsically weak checkpoint in C. elegans, or whether the SAC becomes stronger during later developmental stages, we systematically assayed the mitotic delay induced by microtubule disruption at different stages of development. To disrupt microtubules during larval stages, when cells are impermeable to microtubule drugs, we inducibly expressed DarpinD1, a designed ankyrin repeat protein selected to bind tubulin and promote microtubule depolymerization. Interestingly, in contrast to the weak SAC in early embryos, activation of the SAC during larval intestinal divisions delays mitosis for several hours. To understand when this strong SAC arrest arises during development, we measured the SAC at different stages of embryonic development. Strikingly, we found a gradual increase in the arrest time, ranging from the reported 2-2.5-fold delay in 2-cell embryos to a 10-fold delay at the 50-cell stage. We hypothesized that the gradual increase in strength of the SAC could be explained by a decrease in cellular size in older embryos. Specifically, the strength of the SAC could depend on the ratio between the amount of unattached kinetochores, where the checkpoint signal is generated, and the amount of cytoplasm. To test whether cell size, and not a developmental timer, determines the strength of the checkpoint, we depleted ani-2 to induce a spread in embryo sizes and then assayed the mitotic delay in differently-sized two-cell embryos. Here again, smaller cells displayed a longer mitotic delay, arguing against a developmental timer controlling the strength of the checkpoint. Furthermore, we find that triploid embryos, which have an increased amount of kinetochores, exhibit a stronger mitotic delay upon disruption of the mitotic spindle. Together, these results demonstrate that the kinetochore to cytoplasm ratio determines the extent to which embryonic cells delay mitosis upon activation of the SAC. Our work provides new insights into why cells exhibit large variations in their ability to delay mitotic progression upon disruption of the mitotic spindle. 59 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 23 PP2ACdc55 opposes Cdk substrate phosphorylation in budding yeast interphase Molly Godfrey, Frank Uhlmann Chromosome Segregation Lab, Francis Crick Institute Lincoln's Inn Fields Laboratory In the quantitative model for the cell cycle, rising levels of Cyclin-dependent-kinase (Cdk) activity determine the timing of cell cycle events and progression through the different cell cycle stages. Recently, the importance the importance of Cdk-opposing phosphatase activity in this quantitative cell cycle model has come to light. This is particularly true when it comes to mitotic exit in S. cerevisiae, where the ratio of Cdk/Cdc14 phosphatase activity determines the orderly progression through mitotic exit in a quantitative manner. Similarly, we postulate that Cdk-opposing phosphatase activity in interphase could be an important element in this quantitative model for cell cycle progression. This activity could be required for ordered phosphorylation of Cdk phosphoproteins during interphase and preventing too early cell cycle transitions. Evidence from Xenopus as well as yeast suggests that PP2ACdc55 is a good candidate for such a phosphatase. We have shown that in the absence of PP2ACdc55, global interphase Cdk phosphorylation levels are elevated, independently of PP2A’s role in regulating Cdk tyrosine phosphorylation. We have also identified specific Cdk targets whose phosphorylation is advanced and increased in this case. This indicates that PP2ACdc55 may be directly opposing Cdk phosphorylation events throughout interphase, from G1 until the onset of mitosis. PP2ACdc55 might be part of a mechanism that sets Cdk thresholds for cell cycle progression. 60 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 24 Pausing the cell cycle at the mid-blastula transition in Drosophila embryos Jörg Großhans, Boyang Liu, Hung-wei Sung, Franziska Winkler Institute for Developmental Biochemistry, Medical School, University of Göttingen During early embryonic development in Drosophila, the mode of the cell cycle switches from fast nuclear cycles to a mode with an extended G2 phase. This switch in cell cycle mode is an essential part of a developmental switch, namely the mid-blastula transition. Concommitantly with the G2 pause, zygotic gene expression is activated, maternal mRNA are degraded and cellularisation starts. To reveal the functional relationship between these processes and to determine the molecular mechanism of the robust cell cycle pause, we isolated and characterised mutants with more or fewer cell cycles prior to the switch. We previously reported that premature activation of zygotic transcription is sufficient to trigger the complete developmental switch. By analysing a novel mutation in the protein phosphatase V (PP-V) which leads to an additional nuclear division, we find that PP-V required for timely pause of the cell cycle. Furthermore, a novel mutation in the the gene encoding the enyzme serine hydroxymethyl transferase, leads to a precocious pause after 12 instead of 13 nuclear divisions. We will present our unpublished analyses and findings how PP-V and SHMT are involved in cell cycle control during MBT. 61 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 25 Identification of novel regulators of mitotic chromosome architecture Christoph Schiklenk, Jin Wang, Carlo Klein, Christian Haering Cell Biology and Biophysics Unit, EMBL Heidelberg Chromosome condensation is an essential prerequisite for successful chromosome segregation during mitosis and meiosis. Even though some proteins that are essential for the formation of mitotic chromosomes, including condensin complexes and topoisomerase II, have been recently identified, the complete repertoire of molecular machines that organise mitotic chromosomes has remained elusive. Using a newly developed high-throughput assay that quantitatively measures chromosome condensation dynamics in live fission yeast cells, we screened for yet undiscovered factors involved in chromosome condensation. I will present novel candidate proteins that we identified in this screen as regulators of mitotic chromatin architecture. One of these proteins encodes a large zincfinger protein named Zas1. Zas1 is essential for cell division and localizes to chromosomes during interphase and during cell divisions, consistent with a direct role in regulating chromosome architecture. I will discuss in detail the function of Zas1 and its role in mitotic chromosome condensation. 62 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 26 Exploring the recruitment and regulation of Mad1 during mitosis Daniel Hayward, Ulrike Gruneberg Sir William Dunn School of Pathology, University of Oxford The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying mitotic progression until all kinetochores are attached to microtubules, therefore avoiding aneuploidy which can lead to cell death or cancer. This is achieved by kinetochores unattached to microtubules generating the mitotic checkpoint complex (MCC), which in turn inhibits the anaphasepromoting complex (APC/C) and maintains sister chromatid cohesion. Mad1 (mitotic arrest deficient 1) is an essential SAC protein, recruiting Mad2 which is an indispensable member of the MCC. Both Mad1 and Mad2 are the first SAC proteins lost at the kinetochore upon microtubule-kinetochore attachment. Depletion of Mad1 in cells results in a defective mitotic checkpoint whilst artificial tethering of Mad1 to the kinetochore results in mitotic arrested cells with the SAC permanently on. Although the importance of Mad1 has been extensively demonstrated, the nature of its regulation and to what it binds at the kinetochore are poorly understood. Here we explore how Mad1 binds to the kinetochore in Human cells and discover how mitotic kinases and phosphatases effect its localisation. We expand upon previous work and show that both MPS1 and Aurora B kinases are required for initial Mad1 localisation to kinetochores. Both kinases are also required for maintenance of Mad1 at kinetochores in the presence of microtubules, but are dispensable when microtubules are depolymerised. We further explore the action of these kinases and their substrates. In addition to previous work suggesting the importance of Bub1 and the RZZ complex in Mad1 localisation we also identify the Ndc80 complex as vital in establishing Mad1 at the kinetochore. We can now present a clearer model of Mad1 regulation at the kinetochore. 63 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 27 Protein phosphatase 1 is required for inactivation of Greatwall during mitotic exit in Xenopus embryos Andreas Heim1, 2, Anja Konietzny1, Thomas U. Mayer1, 2 1 2 Department of Molecular Genetics, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany Entry into and exit from M-phase are triggered by changes in the phosphorylation state of multiple cell cycle regulators. In Xenopus laevis oocytes and embryos, the M-phase promoting activity of cyclin-dependent kinase 1 (Cdk1)/cyclin-B is actively counteracted by protein phosphatase 2A in complex with a regulatory B55 subunit (PP2A-B55). Thus, to facilitate faithful and irreversible entry into M-phase, Cdk1/cyclin-B and PP2A-B55 must be regulated in a manner such that their activities are mutually exclusive. At mitotic entry, Cdk1/cyclin-B-activated Greatwall (Gwl) kinase phosphorylates the small heat stable proteins Arpp19/Ensa which enables them to tightly bind to and inhibit PP2A-B55. While it is well established that both Gwl and Arpp19/Ensa have to be dephosphorylated during mitotic exit to facilitate PP2A-B55 re-activation, the precise underlying mechanism remains elusive. Here, we demonstrate that protein phosphatase 1 (PP1) is essential for Gwl inactivation at mitotic exit. PP1 dephosphorylates Gwl at autophosphorylation sites resulting in Gwl inactivation. Subsequently, PP2A-B55 can completely dephosphorylate Gwl to reset it to the interphasic state, which is important to prevent untimely Gwl reactivation. Thus, our work identifies PP1 as the sought-after phosphatase that breaks the Gwl-Arpp19/Ensa module of PP2A-B55 inhibition and thereby provides a molecular explanation for the essential function of PP1 during exit from mitosis in Xenopus early embryos. 64 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 28 Novel stage-specific fluorescent indicators for mapping Ca2+ signals during mitosis in mammalian cells Nordine Helassa1, 2, Lee Haynes2, Robert D Burgoyne2, Katalin Torok1 1 2 St George’s University of London, Institute of Cardiovascular and Cell Sciences, London, UK University of Liverpool, Institute of Translational Medicine, Department of Cellular and Molecular Physiology, Liverpool, UK The division of cells by mitosis is crucial for normal development, growth and tissue renewal and its tight regulation is essential to prevent abnormal cell proliferation. Understanding the mechanistic details and regulation of mitosis is important to mammalian cell biology since irregularities or failure of this process can lead to aneuploidy, genetic instability, premature aging and malignant potential. Ca2+ signalling is fundamental to multiple key physiological processes ranging from fertilisation to cell death. In spite of the evidence indicating a role for Ca2+ signalling during progression of the cell cycle and, in particular, during mitosis, there have been conflicting reports that have hindered progress in this field. Our hypothesis is that Ca2+ signals are important for critical stages of cell division in mammalian cells but that such events are spatio-temporally restricted to such an extent as to have evaded detection in previous studies employing conventional cytosolic Ca2+ dyes. In order to address these issues experimentally we are taking a novel approach of using targeted, genetically encoded, calcium probes (GCaMPs) to monitor Ca2+ signalling during mitosis. GCaMPs are based on a circularly permutated EGFP molecule (cpEGFP) flanked at the N and C termini by the smooth muscle myosin light chain kinase derived RS20 peptide and calmodulin (CaM), respectively. Upon Ca2+ binding, the formation of a tight complex between RS20 and CaM, stabilises the deprotonated form of cpEGFP inducing a fluorescence enhancement. However, the high affinity and slow Ca2+-response kinetics of the current GCaMPs does not make them suitable for measuring localised, short lived mitotic Ca2+ transients. To optimize GCaMP6 for mapping mitotic Ca2+ signals, we decreased the binding affinity of the Ca2+.CaM.RS20 complex and accelerated the Ca2+-response kinetics by point mutations using rationale design. Newly engineered GCaMP6 proteins were characterised in terms of dynamic range, Ca2+ affinity and association and dissociation kinetics. Dissociation constants (Kd) for Ca2+ obtained from the equilibrium Ca2+ binding experiments ranged from 0.1 to 2.6 microM with Fmax/Fmin of ~ 20, making these variants suitable for tracking Ca2+ influx in multiple scenarios. In stopped-flow kinetics experiments, we showed that the variant GCaMP6f RS1 EF4 had half times (t1/2) for Ca2+ rise and decay 5-fold and 2-fold faster than GCaMP6f at 37°C, respectively (t1/2for rise of 3.3 ms and t1/2 for decay 15.7 ms). The improved GCaMP6 variants were used to generate a library of mitosis stagespecific Ca2+ sensors by fusing to proteins relevant for mitosis that have specific localisations during the process (e.g. RhoA, Arf6, pericentrin, actin, tubulin, lamp1). We demonstrated by spinning-disk confocal microscopy that the targeted Ca2+ probes express and localize properly which provide useful tools to accurately map Ca2+-signalling activity with high temporal and spatial resolution throughout mitosis. Funded by the Wellcome Trust (094385/Z/10/Z) to KT and Leverhulme Trust (RPG-2014-194) to LH and RB. 65 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 29 Possible novel mechanism of degradation for the human new cyclin I Sara Hernández-Ortega, Samuel Bru, Natalia Ricco, Javier Jiménez, Josep Clotet Grup de Noves Ciclines, Departament de Ciències Bàsiques, Universitat Internacional de Catalunya, Josep Trueta s/n, 08195, Sant Cugat del Vallès. Cell cycle is controlled by CDKs along with their correspondent partner cyclins in each phase of the cycle. Some years ago, our group described in S. cerevisiae that Pcl1, a not essential cyclin, have special relevance when the environmental conditions are not favorable. Furthermore, we also described that this cyclin was degraded by Dma1 instead of the canonical E3 ligases for cell cycle cyclins, Grr1 nor Cdc4, and that this E3 ligase acts recognizing a sequence that we named DDD (Hernández-Ortega et al., JBC, 2013). Our group is now interested in describing the possible homologous of this cyclin in human. We are currently investigating if cyclin I, a small human new cyclin that appears to be acting with CDK5, the Pho85 homolog, is being phosphorylated by this CDK. In addition, cyclin I has a peak of expression in G1/S as Pcl1 (Nagano et al., Cell cycle, 2013). We made an alignment between these two cyclins and we saw that cyclin I has conserved the DDD sequence so we are trying to find if the mechanism of degradation is also conserved by the RING human E3 ligases, Chfr or Rnf8. This possible conserved mechanism of regulation could help to elucidate the specific roles of the redundant CDK/cyclins complexes along the cell cycle or their function in different stages of development or tissues. 66 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 30 A Consensus Motif for PP2A-B56 Emil Peter Thrane Hertz1, Thomas Kruse1, Jón Otti Sigurdsson1, Norman Davey2, Jesper Velgaard Olsen1, Jakob Nilsson1 1 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark 2 UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland Reversible protein phosphorylation is central to biochemical processes in all living organisms. During mitosis, kinases and phosphatases ensure fidelity in chromosome alignment and segregation. While the kinases working during mitosis have been extensively studied the characterization of the opposing phosphatases remains rudimentary. Protein Phosphatase 2A (PP2A) in complex with B56 regulatory subunits is emerging as an essential regulator of various mitotic and meiotic events. How PP2A-B56 is regulated and recruited to binding partners remains unknown. We have now for the first time discovered a short consensus motif in proteins binding to PP2A-B56 allowing us to predict binding partners and precisely address the role of these interactions. Furthermore, we have identified a fully conserved binding pocket on B56 subunits that mediate the interaction with the consensus motif. We will present our progress in understanding the molecular determinants of the consensus motif that dictates interaction with PP2A-B56 and the function of this important phosphatase. 67 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 31 De novo formation of centrosome-like structures and their roles in the progression of the first mitosis and diploid nucleus formation in parthenogenetic embryos of Drosophila ananassae Kazuyuki Hirai1, Haruka Suzuki2, Yohei Minakuchi3, Atsushi Toyoda3, Muneo Matsuda1 1 Kyorin University School of Medicine International Christian University 3 National Institute of Genetics 2 Parthenogenesis, development of an unfertilized egg into a new individual, has been known to occur naturally in a wide range of animal taxa. Genetic variants that allowed this unusual reproductive strategy have arisen independently in evolutionary histories of sexual reproduction of each species. Parthenogenetic Drosophila may serve as a useful experimental model for studying the adaptation of basic nature in cytological as well as genetic respects for reproductive strategies. We have recently established parthenogenetic strains of Drosophila ananassae and its closely related species. Most of the unfertilized eggs laid by parthenogenetic mothers show developmental arrests and die as haploid embryos, but others can develop into diploid females with a low but significant probability (~8%). We now report the essential mechanisms by which the following problems are solved: Mitotic spindle assembly around the female pronucleus without paternally-derived centrosomes and restoration of diploidy. Our genetic and cytological experiments confirmed that parthenogenetic eggs finished meiosis normally, giving rise to haploid gametes. We found that, in unfertilized eggs of both sexual and parthenogenetic strains of the species, an acentrosomal metaphase spindle is assembled around the chromosomes of the female pronucleus, after completion of S phase. It is striking that eggs laid by parthenogenetic females assembled centrosome-like structures de novo, which were scattered in the anterior part of the egg cytoplasm. These microtubule organizing centers contain pericentriolar material recruitment factors Asterless and Centrosomin. We observed some eggs with such centrosome-like structures located at one or both of the poles of the bipolar spindle assembled around the female pronucleus. Interestingly, anaphase or telophase figures were observed only in the presence of the centrosome-like structures at spindle poles, while no mitotic stages beyond metaphase were observed in the absence of centrosome-like structures at spindle poles in unfertilized eggs of sexual and parthenogenetics strains. Our data suggest a role of the centrosome-like structures in stimulating the metaphaseanaphase transition by binding to the pole of the acentrosomal spindle around the female pronucleus. The first mitosis produces two adjacent daughter nuclei, one associated with a centrosome-like structure and the other without it. Two metaphase spindles are assembled around the cleavage nuclei, but two poles from the separate spindles are often connected by a single centrosome-like structure. We found that the first diploid nucleus is formed by fusion of two haploid chromosome sets at telophase of the second nuclear cycle, resembling the gonomeric division in sexual development. Among three nuclei produced by the second division, the diploid nucleus with a centrosome-like structure might have the ability to proliferate preferentially. The function of centrosomes essential for the initiation of sexual and parthenogenetic development will be discussed. 68 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 32 Maintenance of genome integrity in Arabidopsis root meristem is regulated by the RETINOBLASTOMA-RELATED protein Beatrix Horvath1, 2, Hana Kourova3, Szilvia Nagy4, Zoltan Magyar5, Gabino SanchezPerez2, Csaba Papdi1, Edit Nemeth1, Tamas Meszaros4, Pavla Binarova3, Laszlo Bogre1, Ben Scheres2, 6 1 School of Biological Sciences, Centre for Systems and Synthetic Biology, Royal Holloway, University of London, Egham Hill, Egham, TW20 0EX, UK 2 Department of Molecular Genetics, Utrecht University, 3584 CH Utrecht, The Netherlands 3 Institute of Microbiology AS CS, Laboratory of Functional Cytology, v.v.i.,Víde?ská 1083 Prague 4 , 14 200, Czech Republic 4 Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, H-1094, Budapest, Hungary 5 Institute of Plant Biology, Biological Research Centre, Temesvári krt. 62, POB 521, H-6701, Szeged, Hungary 6 Department of Plant Sciences, Wageningen University Research Centre, 6708 PB Wageningen, The Netherlands Meristematic cells are prone to DNA damage, but are equipped with an array of response pathways to protect genome integrity, including the activation of repair mechanisms, the delay or arrest of cell cycle, the induction of differentiation or cell death. The RETINOBLASTOMA-RELATED is the major regulator for cell proliferation, and its role during DNA damage response in plant is hitherto unknown. Using micro-array analysis, Chromatin –Immunoprecipitation and genetic studies we show that RETINOBLASTOMA-RELATED, a central regulator for cell proliferation, in conjunction with the E2FA and E2FB transcription factors, directly represses the expression of DNA damage response genes; AtBRCA1, AtPARP2, and the newly characterised putative RING finger domain containing E3 ligase, which we named LIFERING. AtBRCA1, a known DNA repair mediator, when mutated becomes hypersensitive to genotoxin-induced cell death. LRN acts against the DNA damage-induced cell differentiation, while AtPARP2 functions to restrain cell proliferation to impose quiescence. RBR silencing leads on one hand to cell death, which relies on the function of the three target genes, and on the other hand to overproliferation which depends on the opposing functions of AtPARP2 and LIFERING. We conclude that RBR provides an input for mitogenic signals to regulate distinct DNA damage responses. The balanced action of the RBR target genes, AtBRCA1, AtPARP2 and LIFERING collectively determines the outcome of DNA damage responses. 69 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 33 Cell length growth patterns in fission yeast reveal a novel size control mechanism operating in late G2 phase Anna Horváth, Ákos Sveiczer Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science Individual cell length growth patterns were analysed of several fission yeast strains, wild type and some cell cycle mutants by fitting different functions to the data. During the growing period, most cells’ growth patterns could be best described by a bilinear function (two linear segments separated by a breakpoint, RCP2); however, linear patterns also occurred in several cases, but exponential ones only very rarely. The bilinear patterns were separated into two growing parts by RCP2, and the position of size control was examined by regression analyses of appropriate growth parameters in both segments. The existence of known size controls in late G1, in mid G2, and in late G2 during the fission yeast cell cycle was verified. However, in spite of the former general view, we now discovered that late G2 size control is a general characteristic third event in the cycle, when cell size is monitored. The level of the critical late G2 size to be reached in an individual fission yeast cell is influenced by its growth rate, similarly to budding yeast, which suggests an evolutionary conserved mechanism. 70 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 34 Modelling the role of systems-level feedbacks in the detection of premature sister chromatid separation by the spindle assembly checkpoint Lukas Hutter1, Mihailo Mirkovic2, Raquel Oliveira2, Bela Novak1 1 2 Department of Biochemistry, University of Oxford Instituto Gulbenkian de Ciencia, Lisbon The Spindle Assembly Checkpoint (SAC) delays anaphase onset in response to chromosomes that are erroneously attached to the mitotic spindle. Satisfaction of the SAC through biorientation of chromosomes crucially depends on sister chromatid cohesion. However, recent findings suggest that the SAC fails to robustly prevent mitotic progression in the absence of cohesin. Here, we present a detailed theoretical study of the dynamics of the SAC in Drosophila Neuroblasts that were challenged with precocious loss of cohesin. In this setup, full sister chromatid separation does not elicit a robust checkpoint response and they abnormally exit mitosis with high segregation errors after a short mitotic delay. By adopting a stochastic modelling approach to explain quantitative live-cell imaging experiments performed on these cells, we are able to highlight the role of multiple feedback loops in the mitotic signalling networks: our analysis shows how the coupling of SAC-signalling and error correction mechanisms can promote the observed gradual accumulation of stable end-on attachments as the signalling capacity of the SAC declines, and how transient attachments in turn drive the decline in SAC signalling capacity. Thus, our results provide a framework to understand how cohesion defects may escape SAC surveillance and therefore give rise to aneuploid cells. 71 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 35 A dynamic description of progression through meiosis Katarzyna Jonak, Wolfgang Zachariae Laboratory of Chromosome Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany Mitotic and meiotic divisions are controlled by the activities of Cdk1-cyclin B and APC/C-Cdc20. However, these types of division differ in fundamental aspects. The events of the mitotic cell cycle are controlled by a regulatory system that re-creates the starting-conditions for a new cycle. By contrast, meiosis is a linear sequence consisting of a single S phase followed by two consecutive M phases and a differentiation program dedicated to the generation of gametes or spores. It is unclear how the mitotic cell cycle control mechanism is modified to regulate meiosis and, in particular, how meiotic cells irreversibly exit from M phase after exactly two divisions. Thus, we combine mathematical modelling with biological experiments in budding yeast to study the dynamics of key meiotic regulators and to understand how these regulators form a network that generates two, and only two, waves of Cdk1 and APC/C-Cdc20 activity. Recently, we have developed a model for the irreversible transition from prophase I into metaphase I in response to the completion of meiotic recombination (Okaz et al., 2012). To generate a model for the entire meiosis, we have supplemented the prophase-to-metaphase I model with a Cdk1-APC/C-Cdc20 oscillator taken from mitotic cell cycle models (Chen et al., 2004; Tyson and Novak, 2008) and a hypothetical terminator mechanism that stops the oscillator at the exit from meiosis II. Currently, we are investigating the mechanisms that govern exit from meiosis II in order to replace the hypothetical terminator with its biological counterpart. Chen, K., Calzone, L., Csikasz-Nagy, A., Cross, F.R., Novak, B., and Tyson, J.J. (2004). Mol. Biol. Cell 15(8), 3841-3862. Okaz, E., Arguello-Miranda, O., Bogdanowa, A., Vinod, P.K., Lipp, J.J., Markova, Z., Zagoriy, I., Novak, B., and Zachariae, W. (2012). Cell 151(3), 603-618. Tyson, J.J., and Novak, B. (2008). Current Biology 18(17), R759-R768. 72 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 36 Assaying bistability and investigating its major determinants in the G2/M switch system Stephy Joseph Genome damage and Stability centre University of Sussex Department of biochemistry University of Oxford Wellcome Trust Centre for Cell Biology University of Edinburgh Models of the Cdk1 activation feedback loops that ensure robust and irreversible transition between G2 and M-phase suggest that this system behaves like a bistable switch. This model predicts that cells can only be in a G2, or M-state but that intermediate states are not allowed implying a system property termed hysteresis. If the G2/M transition displays hysteresis, Cdk1 activity thresholds should differ between mitotic entry and exit. One would also expect a timelag in Cdk1 activation inversely proportional to the input signal strength at levels close to the threshold. Both predictions have been confirmed experimentally in cellfree Xenopus egg extracts. However, it remains to be shown that the G2/M transition is bistable in intact mammalian cells. Moreover, we do not know how the various components of the switch system contribute to bistability and how loss of this property would affect mitotic progression. We have developed assays to investigate the dynamic properties of the Cdk1 activation switch in cdk1 analogue sensitive DT40 and HeLa cells that allowed us to verify bistability of the G2/M transition in these cellular model systems. We found that modulating both the negative and positive feedback loops in the Cdk1 activation reaction shifts the thresholds up and down, but does not abolish hysteresis. We are currently investigating how nuclear translocation of cyclin B and cytoplasmic translocation of Greatwall kinase may affect the systems behaviour and are hypothesising that these events may be the key determinants of hysteresis of the Cdk1 activation switch. Overall, these data will be used to build an accurate and experimentally verified mathematical model of the G2/M transition in mammalian cells. 73 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 37 Downregulation of PP2ACdc55 at anaphase onset by Zds1 and separase SORAYA JÁTIVA, et al. BELLVITGE INSTITUTE OF BIOMEDICAL RESEARCH (IDIBELL) Exit from mitosis and completion of cytokinesis require the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. In budding yeast, Cdc14 phosphatase is a key mitotic regulator that is activated in anaphase to counteract Cdk activity. In metaphase, Cdc14 is kept inactive in the nucleolus sequestered by its inhibitor Net1. At anaphase onset, downregulation of PP2ACdc55 phosphatase by separase and Zds1 protein promotes Net1 phosphorylation and consequently, Cdc14 release from the nucleolus. The mechanism by which Zds1 and separase impinge on PP2ACdc55 activity remains to be elucidated. Previous results show that Zds1 exert its biological function as PP2ACdc55 regulator, by controlling the subcellular localisation of the PP2A regulatory subunit Cdc55. Our previous results suggest that the activity of PP2ACdc55 cannot be modulated solely through regulation of its localization, and that an additional regulatory step may be required to control PP2ACdc55 activity during mitotic exit. Here we show that Cdc55 regulatory subunit is phosphorylated during anaphase upon PP2ACdc55 downregulation. Our results suggest that PP2ACdc55 activity is modulated throughout Cdc55 posttranslational modifications in a separase and Zds1-dependent manner. 74 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 38 A comprehensive study of NRF2-controlled cell cycle arrest during oxidative stress Orsolya Kapuy1, Anita Kurucz1, Tamás Korcsmáros2, 3, Gábor Bánhegyi1 1 Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary The Genome Analysis Centre, Norwich, UK 3 Institute of Food Research, Norwich, UK 2 Oxidative stress is one of the main environmental factors that can influence cellular homeostasis. Treatment with oxidative agents (such as H2O2, tBHP) results in activation of several signal transduction pathways of that stress response mechanism; meanwhile the ongoing cell division cycle has to be blocked. NRF2 (nuclear factor erythroid 2-related factor 2) has a key role to enable cell adaptation to oxidative stress via transcriptionally controlling more than 2000, mainly cytoprotective, genes. However, details about its effect on cell cycle regulation have not been explored yet. Our goal is to reveal a crosstalk between NRF2 and the key elements of the cell cycle regulatory network. We approach our scientific questions from a so called systems biological perspective by using both experimental and theoretical methods. It was shown recently that Cyclin D, the main inducer of G1/S transition, got immediately downregulated via PERK pathway in response to oxidative stress resulting in cell cycle arrest. We suggest that PERK kinase inhibits the key cyclin molecule throughout NRF2 induction by using various methods. We are planning to investigate whether this connection between NRF2 and Cyclin D is direct and/or indirect. We assume that the indirect regulatory connection between NRF2 and Cyclin D is generated by CDK inhibitors (for example p15, p16, p21, p27). We claim that a positive feedback loop between NRF2 and CDK inhibitors assures a rapid cell cycle arrest even at low level of oxidative stress. Performing a systematic analysis we explore the role of each above mentioned CDK inhibitor in down-regulating Cyclin D with respect to oxidative agent. Our experimental results presumably inter-correlated with our theoretically generated model. With computational simulations of the underlying control network various mutant phenotypes might be also tested. Our results suggest that the role of NRF2 during oxidative stress is even more complex as it was known until now. NRF2 not only induces the survival process and arrests cell division cycle, but later it is able to suppress the stress response by transcriptionally down-regulating the mechanism. We claim that the generated feedback loops (both positive and negative ones) between NRF2 and its targets guarantee the precise response mechanism with respect to oxidative agents. At low level of stress NRF2 quickly induces the cyto-protective process, meanwhile mTOR (mammalian targets of rapamycin), another key sensor of cellular homeostasis, gets inhibited. However at high level of stress the cyto-protective process has only a transient induction followed by mTOR re-activation. 75 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 39 The role of Cyclin B3 in Mammalian Spermatogenesis Mehmet Erman Karasu1, 2, Andrew C Koff1, 2, Scott Keeney1, 2, 3 1 Molecular Biology Program, Memorial Sloan Kettering Cancer Center Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center 3 Howard Hughes Medical Institute 2 Important components of the cell cycle machinery include cyclins and their catalytic kinase partners, cyclin-dependent kinases (Cdks). It has been shown in budding yeast that temporal regulation of cyclin-Cdk activity is critical for orchestrating key events during meiosis, as in mitosis. Such regulated events include initiation of homologous recombination and segregation of homologous chromosomes. Similar to budding yeast, in mouse male meiosis (spermatogenesis), cyclins show a sequential expression pattern. In mouse, two cyclins – Cyclin A1 and Cyclin B3 – are meiosis-specific and display interesting expression patterns. Cyclin A1 is expressed in spermatocytes from late pachytene to diplotene, and disruption of Cyclin A1 causes arrest in meiotic prophase and infertility in male mice. Cyclin B3 is expressed in both spermatocytes and oocytes from leptotene to zygotene, when most DSB formation occurs, suggesting that Cyclin B3 may regulate events during early meiosis, such as initiation of meiotic recombination or pairing of homologs. Moreover, in transgenic mice in which expression of Cyclin B3 was prolonged until the end of meiosis, spermatogenesis was disrupted and sperm count was reduced. Additionally, disruption of Cyclin B3 in flies resulted in infertility in females. Based on these findings, Cyclin B3 has long been thought to be a prime candidate for a key cell cycle regulator of early meiotic events. To investigate the role of Cyclin B3 in meiosis, we generated Cyclin B3 knock-out (KO) animals by CRISPR-Cas9 genome editing. We obtained in-frame deletions, out-of-frame deletions, substitutions and insertions at the Cyclin B3 locus. Analysis of Cyclin B3 KO animals indicate that Cyclin B3 protein is not present, and mRNA transcript levels are reduced 6-10 fold. Surprisingly, however, Cyclin B3 KO mice are fertile and testis size and weight are comparable to wild-type littermates. The detailed phenotype of Cyclin B3 KO mice will be presented. 76 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 40 HOW DOES CDC14 ORDER EVENTS DURING MITOTIC EXIT IN SACCHAROMYCES CEREVISIAE? Meghna Kataria, Frank Uhlmann The Francis Crick Institute - Lincoln's Inn Fields Accurate progress through the cell division cycle necessitates robust order and timing of various cytological events, the molecular basis for which is incompletely understood. The quantitative model of cell cycle progression proposes that this order is dictated by increasing kinase activity as cells traverse through the various stages. Lately, the historically overlooked contribution of the concomitant phosphatases has come into focus – a process well studied in mitotic exit in budding yeast. In this organism, Cdc14 is the major phosphatase which reverses cyclin-dependent kinase (Cdk)-mediated phosphorylation events upon its activation at anaphase, and is required for cells to exit mitosis. Ordered dephosphorylation of proteins dictates the sequence of events during mitotic exit and cytokinesis. This order is largely imposed by differing catalytic efficiencies of Cdc14 towards its substrates. This has spurred the idea that preferred substrates might possess high affinity for the phosphatase. In this study, we seek a molecular understanding of mitotic exit by probing the biochemical characteristics of Cdc14 – sequence features within substrates that might underlie differential affinities. We probe this by characterizing its interactions with substrates that are dephosphorylated very early into anaphase, such as Fin1. Preliminary data indicates that Cdc14 binds to Cdk1 phosphorylation sites, in addition to other features that are now being validated. The eventual objective is to alter the dephosphorylation timing of ‘early’ proteins not only in vitro, but also in vivo to study its effects on cell cycle progression. 77 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 41 The condensin component NCAPG2 regulates chromosome segregation Jae Hyeong Kim1, Jaegal Shim1, Seoung Min Bong1, Young-Joo Jang2, Chang-Woo Lee3, Byung Il Lee1,Kyungtae Kim1, et al. 1 Research Institute, National Cancer Center, Goyang, Gyeonggi 410-769, Republic of Korea Laboratory of Cell Cycle and Signal Transduction, Department of Nanobiomedical Science & BK21 PLUS Research Center for Regenerative Medicine, Dankook University, Cheonan, Chungnam 330-714, Republic of Korea 3 Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746, Republic of Korea 2 The early event of microtubule–kinetochore attachment is a critical stage for precise chromosome segregation. Here we report that NCAPG2, which is a component of the condensin II complex, mediates chromosome segregation through microtubule–kinetochore attachment by recruiting PLK1 to prometaphase kinetochores. NCAPG2 colocalises with PLK1 at prometaphase kinetochores and directly interacts with the polo-box domain (PBD) of PLK1 via its highly conserved C-terminal phospho-Thr (1007VLS-pT-L1011) containing region and conserved crystal structure with other PBD binding phospho-peptide. In both humans and in vivo model system, when NCAPG2 is depleted, the attachment of the spindle to the kinetochore is loosened and misoriented, caused by the disruption of PLK1 localisation to the kinetochore and by the decreased phosphorylation of its kinetochore substrate, BubR1. These findings suggest that NCAPG2 plays an important role in regulating proper chromosome segregation through a functional interaction with PLK1 during mitosis. 78 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 42 Interaction of MAP kinase MPK6 with γ-tubulin and microtubule plus-end protein EB1c and regulatory function of MAP kinases in mitotic chromosome segregation in acentrosomal plant cells Lucie Kohoutová1, Hana Kourová1, Szilvia K. Nagy2, Jindřich Volc1, Petr Halada1, Tamás Mészáros2, Irute Meskiene3, László Bögre4, Pavla Binarová1 1 Institute of Microbiology AS CR, v. v. i., Videnska 1083, 142 20 Prague 4, Czech Republic Semmelweis University, Department of Medical Chemistry, Molecular Biology, Budapest, Hungary 3 Max F. Perutz Laboratories, University of Vienna, Vienna, Austria 4 Royal Holloway, University of London, School of Biological Sciences, Egham, United Kingdom 2 Mitogen-activated protein (MAP) kinases pathways play role in stress response and developmental signalling in eukaryotes and modulate cell division through cytoskeleton. Knowledge of protein targets of MAP kinase signalling among microtubular proteins is limited and uncovering of new substrates and interactors of MAP kinases can help understanding of the cell cycle regulation. We found an interaction of MPK6 and γ-tubulin in cytoplasmic extract and with microtubules in Arabidopsis. Signal of active MAP kinases detected by p-ERK antibody was present together with γtubulin on the specific subset of mitotic microtubules. We further found that microtubule plus-end binding protein EB1c interacts with MPK6. We showed that EB1c is phosphorylated by MKK4activated MPK6 in vitro while phosphorylation was not shown for EB1a and γ-tubulin. Moreover, we confirmed phosphorylation of EB1c in vivo in Arabidopsis and the phosphorylation was reduced after treatment with specific MEK1/2 inhibitor U0126. Active MAP kinases and EB1c-GFP protein localized with mitotic spindles with a slight enrichment in vicinity of the kinetochores and on shortening kinetochore fibres. Arabidopsis plants with reduced activity of MPK3, MPK4, and MPK6 kinases due to overexpression of MAP kinase phosphatase AP2C3 showed mitotic and cytokinetic defects and misaligned spindles and phragmoplasts. We found that congression and segregation of chromosomes was impaired in AP2C3 plants and similar defects of spindle assembly checkpoint were observed when activity of MAP kinases was inhibited by U0126. KO mutants mpk6-2 showed misaligned spindles only in response to stress. Our data showed that γ-tubulin, a protein involved in microtubule nucleation and organization, and microtubule plus-end protein EB1c with function in the cell division are two novel partners for MPK6 signalling. Furthermore, our data suggest synergistic and partially overlapping functions for MPK3, MPK4, and MPK6 kinases in regulation of mitosis in plant cells. Supported by Grant from Grant Agency of the Czech Republic P501-15-1657S 79 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 43 Functional analysis of the spindle checkpoint in plants - are we really all MAD? Shinichiro Komaki, Arp Schnittger University of Hamburg Cell division accuracy is required for genomic stability and proper organ development. The spindle assembly checkpoint (SAC) plays a key role for the fidelity of cell division by preventing anaphase onset until all chromosomes are correctly attached to the spindle. The core proteins of the SAC are highly conserved from yeast to plants, including MAD1, MAD2, MAD3/BUBR1, BUB1, BUB3 and MPS1. In yeast and mammalian cells, lack of the SAC activity causes missegregation of chromosomes, resulting in aneuploidy. In plants, homologs of the core SAC components were identified but surprisingly, all but one mutant in the core SAC genes are viable raising the question whether the SAC or its regulators have a conserved function in the eukaryotic kingdom. To investigate the requirement of the SAC activity in plants, mutants for the central SAC components of Arabidopsis were also grown on media containing a microtubule-destabilizing drug oryzalin. Oryzalin reduced root growth of mad1, mad2, bubr1, mad3.2 and mps1, suggesting that at least some of the molecular functions of the SAC components are also conserved in Arabidopsis. Examination of the expression patterns of the core SAC components during plant development using different genomic fusions of the respective regulators to beta-glucuronidase (GUS) showed that most of the Arabidopsis SAC genes have complex regulatory elements, most likely present in their intron and/or 3’ UTR region that may serve to integrate developmental with environmental signals. Based on the GUS reporter lines, we have then constructed protein reporters fused to GFP. Live imaging revealed that MAD1-GFP and MAD3.2-GFP localized to unattached kinetochores, which is the typical localization pattern of the SAC reported previously. However, the other SAC components showed diverse localization patterns. In particular, BUB3.1-GFP and BUB3.2-GFP concentrated at the midzone of the plant-specific cytokinetic phragmoplast, suggesting a function of the SAC in cytokinesis. Further analyses on the role of the SAC in mitosis and during cytokinesis are currently under way. 80 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 44 CDK1 structures reveal conserved and unique features of the essential cell cycle CDK. Svitlana Korolchuk2, Nicholas Brown1, Mathew Martin2, Will Stanley2, Martin Noble2, Jane Endicott2 1 2 MRC National Institute for Medical Research Newcastle University CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. We have determined the crystal structures of complexes of CDK1–Cks1 and CDK1–cyclin B–Cks2. These structures reveal a number of important aspects of CDK1 structure and function. They confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodeled by cyclin binding. In the context of previously reported CDK-cyclin structures, the structure of CDK1–cyclin B increases the observed diversity with which the CDK responds to cyclin binding. Relative to CDK2– cyclin A, CDK1–cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation, and shows differences in the substrate sequence features that determine activity. CDK1–cyclin B demonstrates a relatively relaxed specificity for residues adjacent to the site of phosphorylation, a phenomenon that correlates with this CDK–cyclin pair appearing to have a less rigidly constrained activation segment. The CDK1–cyclin B structures also reveal potential novel protein interaction sites that might regulate CDK1 activity. In addition to a conserved surface on the CDK1 C-terminal fold, there is a cleft between the two subunits not seen in the structure of CDK2–cyclin A but apparent in CDK9–cyclin T, where it is exploited by the human immunodeficiency virus 1 Tat. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We have also determined the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors. We are currently aiming to extend our observations to characterise CDK1 activation by cyclin A and other activators. 81 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 45 Role of the DmUsp5 in coupling ubiquitin homeostasis to development and apoptosis Levente Kovács1, Olga Nagy1, Margit Pál1, Octavian Popescu2, Andor Udvardy1, Péter Deák1 1 2 Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary Interdisciplinary Institute of Bio-Nano-Sciences, Molecular Biology Center, Cluj-Napoca, Romania Ubiquitin is a small regulatory protein which is used for posttranslational modification of proteins in a reversible process called ubiquitylation. An enzyme cascade catalyzes the attachment of ubiquitins to the target proteins. This covalent modification facilitates protein-protein interactions, changes enzymatic activities, or targets proteins to proteasomal degradation. This dynamic process is implicated in many basic cellular functions including cell cycle and cell death. For all these functions, the ubiquitylation requires a free mono-ubiquitin pool which is maintained most prominently by ubiquitin recycling. Members of the proteases family called deubiquitylating enzymes or DUBs are implicated in ubiquitin recycling since they can remove ubiquitins from target proteins or process polyubiquitins. Although DUBs seem to be indispensable for the maintenance of ubiquitin homeostasis, their precise physiological importance is still poorly understood. In the genetically well characterized Drosophila model, 46 DUBs have been identified, but the function of most of them is still unknown. One of them is Usp5, an evolutionarily conserved DUB enzyme involved in the disassembly of unanchored polyubiquitin chains. We identified and genetically analyzed the Drosophila orthologue of the human Usp5 (DmUsp5) which turned out to be essential for normal development. A heterologous complementation experiment confirmed functional homology between the DmUsp5 gene and its yeast homologue, Ubp14. Loss of DmUsp5 function results in late lethality that is accompanied by the accumulation of unanchored polyubiquitin chains. It also stabilizes p53 and induces a high incidence of apoptosis in larval brains and imaginal discs. In addition to this, the expression of reaper and hid, but not the grim, proapoptotic genes becomes elevated in DmUsp5 mutants. Most importantly, the expression of another, proteasome-associated DUB, DmUsp14 increased highly in DmUsp5 mutants. It was shown in the unicellular budding yeast that Usp14 orthologue, Ubp6 is expressed and progressively deubiquitylate proteasome-bound substrates at times of ubiquitin depletion. Elevated DmUsp14 expression together with dominant cycloheximide sensitivity indicates that loss of DmUsp5 cause ubiquitin stress in these animals. These observations suggest that the DmUsp5 DUB enzyme plays a critical role in regulating apoptosis and – together with DmUsp14 - moderating ubiquitin homeostasis in Drosophila. This work is supported by HURO/1101/173/2.2., TAMOP-4.2.2.A11/1/KONV-2012-0035 and TAMOP- 4.1.1.C-13-1-KONV grants. 82 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 46 Axial contraction and short-range compaction of chromatin synergistically promote the condensation of mitotic chromosomes. Tom Kruitwagen1, Annina Denoth-Lippuner1, Brian Wilkins2, Heinz Neumann2, Yves Barral1 1 2 Institute of Biochemistry, ETH Zürich, Switzerland Department of Applied Synthetic Biology, University of Göttingen, Germany Prior to their symmetric segregation during mitosis, eukaryotic chromosomes must be extensively folded into units that the spindle can act upon. Despite its importance, the molecular mechanisms underlying chromosome condensation are still unclear. Serine 10 on histone 3 (H3 S10) has been shown to be specifically phosphorylated during mitosis. In most eukaryotes, however, the role and importance of this Aurora B dependent event has remained unclear, since the mutation of this residue from serine to alanine failed to show any clear phenotype. However, we recently showed that the yeast Sir2-related histone deacetylase Hst2 is recruited by H3 S10Ph and to deacetylate histone 4 at lysine 16 (H4 K16), to promote mitosis-specific interactions between neighbouring nucleosomes via H2A and H4. A clearer role was assigned to the condensin complex, whose disruption lead to widespread defects in chromosome condensation in frog egg extracts. However, in other model organisms condensin seemed to be partially dispensable for chromosome condensation. Thus, although a number of factors contribute to chromosome condensation, how they act together to shape mitotic chromosomes remains unclear. Here, we use two microscopic assays to distinguish short-range nucleosome-nucleosome interactions from long-range, axial contraction of mitotic chromosomes. Using these methods, we demonstrate that H3 S10 phosphorylation and the deacetylation of histone H4 tails in early anaphase promote short-range compaction of chromatin through nucleosome-nucleosome interactions, starting from the centromere. During late anaphase, condensin mediates the axial contraction of chromosome arms. These processes are temporally and mechanistically distinct, since mutants disrupting chromatin compaction, such as H3 S10A, have no observable effects on axial contraction, and vice versa. When both pathways are inactivated, synergistic defects on chromosome segregation and cell viability are observed. Interestingly, both pathways rely at least partially on the deacetylase Hst2, the only factor to show both compaction and contraction defects when deleted. 83 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 47 Rif1 is required for resolution of Ultrafine DNA Bridges in anaphase to ensure genomic stability Rutger Hengeveld1, Rudolf de Boer2, Pepijn Schoonen2, Elisabeth de Vries2, Marcel van Vugt2, Susanne Lens1 1 Department of Molecular Cancer Research, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. 2 Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9723 GZ Groningen, The Netherlands. Sister-chromatid disjunction in anaphase requires the resolution of DNA catenanes by topoisomerase II together with PICH (Plk1-interacting checkpoint helicase) and BLM (Bloom’s helicase). We here identify Rif1 as a novel factor involved in the resolution of DNA catenanes that are visible as ultrafine DNA bridges (UFBs) in anaphase to which PICH and BLM localize. Rif1, which during interphase functions downstream of 53BP1 in DNA repair, is recruited to UFBs in a PICHdependent fashion, but independently of 53BP1 or BLM. Similar to PICH and BLM, Rif1 promotes the resolution of UFBs: Its depletion increases the frequency of nucleoplasmic bridges and RPA70positive UFBs in late anaphase. Moreover, in the absence of Rif1, PICH or BLM more nuclear bodies with damaged DNA arise in ensuing G1 cells, when chromosome decatenation is impaired. Our data reveal a thus far unrecognized function for Rif1 in the resolution of UFBs during anaphase to protect genomic integrity. 84 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 48 Molecular ties between the cell cycle and differentiation in embryonic stem cells Victor Li, Marc Kirschner Harvard Medical School Attainment of the differentiated state during the final stages of somatic cell differentiation is closely tied to cell cycle progression. Much less is known about the role of the cell cycle at very early stages of embryonic development. Here, we show that molecular pathways involving the cell cycle can be engineered to strongly affect embryonic stem cell differentiation at early stages in vitro. Strategies based on perturbing these pathways can shorten the rate and simplify the lineage path of ES differentiation. These results make it likely that pathways involving cell proliferation intersect at various points with pathways that regulate cell lineages in embryos and demonstrate that this knowledge can be used profitably to guide the path and effectiveness of cell differentiation of pluripotent cells. 85 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 49 Feedback throughout G2 coordinates production of proteins required for mitosis with mitotic entry Karen Akopyan, Arne Lindqvist Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden During G2 phase a cell is considered to prepare for mitosis, but how such preparation is coordinated with mitotic entry remains poorly understood. We have recently used quantitative immunofluorescence to acquire temporally resolved data through the cell cycle and found that the mitotic kinases Plk1 and Cdk1 are active throughout G2 phase (Akopyan et al, Mol Cell 2014). We find that fitting the quantitative data to an ODE-based model of mitotic entry is most straightforward if Cdk1 activity enhances Cyclin B protein levels. In accordance, we find that Cdk1 inhibition in G2 phase decreases gene-targeted Cyclin B1-YFP levels. Interestingly, during forced stimulation of mitotic entry by Wee1 inhibition, the duration of mitosis inversely correlates to Cyclin B1-YFP levels, suggesting that mid G2 cells lack factors required for mitotic progression. Similarly, a pulse of Cdk1 inhibition in G2 phase prolongs the duration of a forced mitosis, despite full activation of Cyclin B-Cdk1. Our working model is that Cdk1 through a feedback-loop increases mitotic entry network components during G2 phase. Such a feedback could potentially synchronize production of proteins required for a successful mitosis with timing of mitotic entry and may indicate why G2 takes so long in human somatic cells. 86 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 50 Spatial-temporal model for silencing of mitotic spindle assembly checkpoints Jian Liu, et al. National Institutes of Health, Bethesda, MD 20892, USA The spindle assembly checkpoint arrests mitotic progression until each kinetochore secures a stable attachment to the spindle. Despite fluctuating noise, this checkpoint remains robust and remarkably sensitive to even a single unattached kinetochore among many attached kinetochores; moreover, the checkpoint is silenced only after the final kinetochore-spindle attachment. Experimental observations showed that checkpoint components stream from attached kinetochores along microtubules toward spindle poles. Here, we incorporate this streaming behavior into a theoretical model that accounts for the robustness of checkpoint silencing. Poleward streams are integrated at spindle poles, but are diverted by any unattached kinetochore; consequently, accumulation of checkpoint components at spindle poles increases markedly only when every kinetochore is properly attached. This step-change robustly triggers checkpoint silencing after, and only after, the final kinetochore-spindle attachment. Our model offers a unified framework that highlights systems-level coupling between kinetochore-spindle attachment and spindle-pole formation in SAC silencing. It not only explains the remarkable fidelity of chromosome segregation in normal somatic cell mitosis, but also accounts for the frequent aneuploidy rate in cancer cell mitosis and mammalian oocyte meiosis I. 87 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 51 Cell cycle control in MutiCellular Tumor Spheroids Aurélie Gomes1, 2, Odile Mondésert1, 2, Céline Frongia1, 2, Bernard Ducommun1, 2, 3 , Valérie Lobjois1, 2 1 Université de Toulouse, ITAV-USR3505, Toulouse, France CNRS, ITAV-USR3505, Toulouse, France 3 CHU de Toulouse,Toulouse, France 2 Major recent advances have recently been made in the understanding of the molecular pathways regulating the cell cycle machinery and their deregulations in cancer. These results open new avenues for the development of innovative antitumor pharmacological strategies targeting the cell cycle and its checkpoints. MultiCellular Tumor Spheroid (MCTS) are now considered as invaluable models to study cancer cell biology and for the preclinical development of new antiproliferative drugs. To fully exploit the features of MCTS, it is essential to preserve the 3D regionalization level of analysis when investigating the effect of a drug. We will report here a breakthrough in the use of MCTS with new technological developments that can be used to explore the regionalization and the live dynamics of the effects of anticancer drugs in 3D. Cell lines expressing fluorescent cell cycle reporters (i.e. Fucci) and biosensors were engineered and used to study cell cycle dynamics and to characterize the cell cycle parameters regionalization within growing spheroids. We examined the impact of anti-tumor drugs on cell cycle progression and DNA damage Response (DDR) pathway activation induced by chemotherapeutic agents within spheroids using innovative 3D imaging strategy based on 3D light sheet microscopy. This study opens new perspectives for the understanding of cell cycle control and proliferation in 3D multicellular models and for the investigation of the dynamics of the 3D response to novel antiproliferative agents. 88 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 52 Greatwall dephosphorylation and inactivation upon mitotic exit is triggered by PP1 Sheng Ma, Perle Robert, Suzanne Vigneron, Anna Castro, Thierry Lorca CNRS-CRBM Mitotic entry requires cyclin B-Cdk1 activation and PP2A-B55 inhibition. At G2/M the Greatwall kinase (Gwl) is activated and promotes Arpp19/ENSA phosphorylation that once phosphorylated bind and inhibit PP2A-B55. Mitotic exit requires the reactivation of this phosphatase by Gwl inactivation and further dephosphorylation of its substrates. In this study we show that PP1 depletion in Xenopus egg extracts stabilises phosphorylation of Gwl on the activatory site S875 maintaining in this way the full activity of this kinase and blocking meiotic/mitotic exit. In contrast, the inhibition of PP2A-B55 results in Thr194 stabilisation, partial Gwl inactivation and partial meiotic/mitotic exit. Our data also show that if PP2A-B55 is fully reactivated by Arpp19 depletion, this phosphatase is able to rapidly dephosphorylate Gwl in the two activatory sites even in the absence of PP1 suggesting that PP1 would be essential to trigger the Gwl inactivation patwhay. Finally, we show that both phosphatases PP1 and PP2A-B55 can dephosphorylate Gwl on S875 and Thr194 in vitro. These data identify PP1 as the phosphatase first dephosphorylating Gwl and triggering its inactivation upon meiotic/mitotic exit. 89 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 53 Developmental regulation of cell proliferation by E2FB in leaf pavement cells and meristemoids Tünde Leviczky1, Binish Mohammed2, Aladár Pettko-Szandtner1, Beatrix Horváth2, Anita Kovács1, Márta Deli1, Csaba Papdi2, László Bögre2, Zoltán Magyar1 1 2 Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Science, Szeged, Hungary School of Biological Sciences, Royal Holloway University of London, Egham, United Kingdom Leaf growth relies on the number of cells produced before pavement cells exit proliferation and the number of daughter cells generated by asymmetric cell divisions of meristemoids. E2FB is an activator of cell cycle genes in proliferating pavement cells in young developing leaves. In accordance, pavement cells of e2fb mutant prematurely exit cell proliferation while elevated E2FB level delays this exit. During cell cycle exit, RBR represses E2FB activity. We found that E2FB regulates RBR both transcriptionally and on protein level but also controls its phosphorylation level. In accordance CYCD3;1 transcript level was elevated in the ectopic E2FB lines. These feedbacks could provide the underlying mechanism, which controls the switch from cell proliferation to cell cycle exit and differentiation. Therefore elevated E2FB level can alter this switch in cells started to differentiate and they reenter proliferation. In contrast, overexpression of a dominant negative E2FB form, E2FB∆RBR, which is unable to activate cell cycle genes, results in early exit from proliferation and therefore producing smaller leaves. In leaf meristemoids, change in E2F function has totally different outcomes. On one hand, e2fb mutation causes extra divisions of the meristemoids resulted in stomata clusters. On the other hand co-overexpression of the mutant E2FB∆RBR construct with DPA expands the transit amplifying cells produced by meristemoid stem cells. RBR together with E2FB regulates these processes as a repressor. Thus E2FB has dual functions regulated by RBR. (1) RBR represses E2FB activity, dependent on its phosphorylation, regulating cell cycle exit in pavement cells, (2) E2FB forms a repressor complex with RBR to regulate stem cell divisions in meristemoids. 90 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 54 Putting the brakes on Mps1 Kinase. Davinderpreet Mangat, Ulrike Gruneberg University of Oxford Faithful execution of cell division is fundamental for the creation and survival of all organisms. The spindle assembly checkpoint (SAC) is a cell cycle surveillance mechanism that delays mitotic exit in the presence of unattached kinetochores. Both the timely activation and inactivation of the mitotic checkpoint are important for accurate chromosome segregation. The Mps1 kinase, a highly conserved serine/threonine kinase has emerged as a central regulator of the SAC. Mps1 kinase triggers SAC activation by phosphorylating a number of substrates, of which phosphorylation of the scaffold protein Knl1 is best understood. Mps1 mediated phosphorylation of Knl1 provides docking sites for downstream checkpoint proteins including Bub1, Bub3 and BubR1. Inhibiting Mps1 with the specific inhibitor, AZ3146, results in the loss of the Bub and Mad proteins from the kinetochore. Thus, given the critical role Mps1 plays in SAC activation, we hypothesised that the regulation of Mps1 activity may be important for SAC signalling. It has previously been shown that autophosphorylation of the activation loop residue T676 is necessary for efficient SAC signalling. Since the activation of Mps1 is phospho-dependent, we investigated whether this event is negatively regulated by a mitotic phosphatase. Here, we have taken an unbiased siRNA based approach to determine the protein phosphatase responsible for controlling Mps1 activity. Our results suggest that a PP2A-B56 phosphatase complex opposes Mps1 activation by removing a phosphate group from Mps1’s activation loop. Knockdown of the catalytic or regulatory subunits of this phosphatase complex results in the retention of the phospho-Mps1 signal. Moreover, the activation loop of Mps1 is a substrate for PP2A phosphatase complexes in vitro. We suggest that PP2A-B56 promotes mitotic exit by inactivating Mps1 kinase as well as directly inhibiting the Knl1-Bub1 interaction. 91 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 55 Phosphorylation on serine 283 of human CDC25A modulates its stability and activity in G2 and contributes to accelerate entry into mitosis Laurent Mazzolini1, Christine Dozier1, Carine Froment2, Marlene Marcellin2, Odile Schiltz2, Stéphane Manenti1 1 Cancer Research Center of Toulouse, INSERM Unité Mixte de Recherche 1037, CNRS Equipe de Recherche labellisée 5294, Université de Toulouse, 2, avenue Hubert Curien 31037 Toulouse Cedex, France 2 Institute of Pharmacology and Structure Biology, CNRS Unité Mixte de Recherche 5089, Université de Toulouse, 205, route de Narbonne 31077 Toulouse Cedex, France In eukaryotic cells the progression through the cell cycle is tightly regulated in response to intracellular as well as extracellular signals. Central actors of this regulation are constituted by cyclindependent kinases (CDK) which, together with their associated cyclin regulatory subunit, control all cell cycle transitions. The activity of CDK-cyclin complexes themselves is subject to complex regulatory networks involving mostly activating and inhibitory phosphorylation events. The removal of key inhibitory phosphorylations on CDK is performed by the dual specificity phosphatases of the CDC25A family. In mammalian cells three CDC25 isoforms have been identified: CDC25A, CDC25B and CDC25C. While CDC25B and CDC25C activity appears restricted to the regulation of CDK-cyclin complexes involved in mitotic onset, CDC25A regulates both G1/S and G2/M progression. The intracellular function of CDC25A is strongly controlled by phosphorylation events which regulate its stability and activity and are performed by numerous kinases including the CDK themselves. In order to identify new putative regulatory phosphorylations that may impact CDC25A function in the cell, phosphoproteomic studies of CDC25A were undertaken in our team. This led to the identification of new phosphorylation sites among which one, phosphorylation of serine 283, was found to occur in the G2 phase of the cell cycle. This phosphorylation appeared maintained in mitosis and is lost when cells reenter into the next cell cycle. Transient tranfection studies and use of specific kinase inhibitors indicate that this phosphorylation mainly involves CDK-cyclin complexes. Functional studies using cell lines conditionally expressing non-phosphorylable CDC25A mutants showed that this phosphorylation does not impact CDC25A intracellular localization but increases the stability of CDC25A as well as its intracellular activity in G2, contributing to accelerate the G2 to M transition in cells 92 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 56 Asymmetric segregation of a lysine deacetylase links perinuclear organisation and the time of cell cycle entry in budding yeast Manuel Mendoza, et al. Center for Genomic Regulation (CRG) Nuclear organization is associated with cell type-specific patterns of gene expression. However, how differences in nuclear architecture are established during development is not known. Here we report that in asymmetrically dividing budding yeast, the lysine deacetylase Hos3 shapes perinuclear organization in daughter cells to regulate the starting time of the next cell division cycle. Hos3 asymmetrically localizes to the periphery of anaphase daughter nuclei, where it associates with nuclear pores. We find that the G1/S cyclin CLN2 locus is confined to the perinuclear region during G1 in a Hos3-dependent manner, to favor its transcriptional repression. In hos3∆ mutants, the perinuclear anchoring of the CLN2 locus is lost, resulting in premature transition into S phase. Conversely, artificial anchoring of the CLN2 locus to the nuclear periphery delays onset of S phase, even in the absence of Hos3. Thus Hos3 determines the time of cell cycle entry specifically in daughter cells, possibly by regulating the nuclear localisation of the CLN2 locus. These data open the possibility that asymmetric partitioning of lysine deacetylases establishes differences in nuclear architecture to shape developmental decisions during asymmetric division of animal cells. 93 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 57 Identification of novel interaction partners of the Chromosomal Passenger Complex Amanda Meppelink, Sibel Bayrak, Susanne Lens Department of Molecular Cancer Research, ?Center for Molecular Medicine, University Medical Center Utrecht, The Netherlands During mitosis the, in S-phase duplicated genomic contents of a cell needs to be equally distributed over two new daughter cells. To accomplish this the kinetochores of the sister-chromatids have to become attached to microtubules emanating from opposite spindle poles (bipolar attachments) In anaphase the two sisters will then be pulled towards opposing sites of the cell. Chromosome biorientation is essential for genomic stability, and is supported by a correction system that disconnects erroneous kinetochore-microtubule attachments, and that allows stabilization of correct, bipolar attachments. The chromosomal passenger complex (CPC), consisting of INCENP, Survivin, Borealin and Aurora B kinase, is a key component of the error correction machinery. Through phosphorylation of kinetochore substrates that are involved in microtubule binding and in the recruitment of mitotic checkpoint proteins, it destabilizes incorrect kinetochore-microtubule attachments and prevents anaphase onset by keeping the mitotic checkpoint active [1] To further elucidate the role of the CPC during mitosis, we performed large-scale immunoprecipitation experiments in combination with mass spectrometry for the various CPC subunits to identify potential novel interaction partners. Pull-downs were performed in HeLa Kyoto cells expressing bacterial artificial chromosome (BAC) transgenes for Aurora B, INCENP and Borealin [2]. BACs contain large stretches of DNA encoding the complete gene including their endogenous regulatory sequences, resulting in near endogenous expression. All three proteins were C-terminally tagged with a modified version of the localization and affinity purification (LAP) tag [2]. Cells were blocked in mitosis with the Eg5 inhibitor STLC, to mimic a prometaphase-like state. Upon immunoprecipitation (IP) of all three separate CPC components we could identify the remaining three CPC subunits. Next to these we also identified CBX1, CBX3, CBX5, and KIF20A as constant and significant interaction partners, often with a high intensity. We could thus efficiently pull down the entire complex together with various interaction partners. Interestingly, in the IPs of Borealin-LAP we identified SIRT1, a histone deacetylase protein that belongs to the sirtuin family and is known to also deacetylate proteins other than histones [3]. Using Western blot analysis we could validate SIRT1 as a true interactor of Borealin after IP from BorealinLAP cell lines as well as by co-IP of overexpressed SIRT1 and Borealin in HEK293T cells. Interestingly, we never found SIRT1 in the Aurora B or INCENP IPs, both after analysis by mass spectrometry or by Western blot, suggesting that SIRT1 might interact with a pool of Borealin that is not associated with the other CPC subunits. Recently a role has been proposed for SIRT1 in chromosome condensation during mitosis and ensuring faithful chromosome distribution [4,5]. We are currently investigating whether the SIRT1-borealin association is involved in histone (de)acetylation and how this could affect faithful chromosome segregation [1] Lampson, M. A., & Cheeseman, I. M. (2011). Trends in Cell Biology [2] Poser, I., et al. (2008). Nature Chemical Biology [3] Blander, G., & Guarente, L. (2004). Annual Review of Biochemistry [4] Fatoba, S. T., & Okorokov, A. L. (2011). Cell Cycle. [5] Kim, J.-J., et al. (2015), Journal of Cellular Biochemistry 94 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 58 Investigating the factors regulating condensin association with mitotic centromeres. Catrina Miles, Miao Yu, Jon Baxter Genome Damage and Stability Centre. Department of Life Sciences. University of Sussex, UK. Condensin is a multi-subunit protein complex, essential for chromosome organization, resolution and segregation during cell proliferation. Although condensin is enriched at centromeric regions during mitosis, the full scope of regulation of condensin recruitment remains unclear. In the budding yeast system we have analysed the potential intrinsic and extrinsic factors required for mitotic recruitment of the condensin complex to centromeric regions. Using ChIP to ascertain condensin enrichment levels, we find that the localisation of condensin to centromeric regions is regulated by the extrinsic activities of mitotic kinases and phosphatases, and the intrinsic enzymatic activity of the condensin complex itself. The importance of these data for models of condensin recruitment and activity will be discussed. 95 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 59 Spindle checkpoint signalling from yeast and human kinetochores Jonathan Millar, Virginia Silio, Maria Del Mar Mora-Santos, Andrew McAinsh University of Warwick The spindle assembly checkpoint (SAC) is the major surveillance system that ensures sister chromatids do not separate until all chromosomes are correctly bi-oriented. In early mitosis components of the SAC, including Bub1, Bub3, Mad1, Mad2 and Mad3/BubR1 proteins, are recruited to kinetochores. This induces a conformational change in Mad2 that promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphasepromoting complex (APC/C) until the checkpoint is satisfied. Activation of Cdc20-APC/C activity triggers the destruction of securin and cyclin, which allows the dissolution of sister chromatid cohesion and mitotic progression. We recently showed that phosphorylation of multiple conserved MELT motifs in Spc7 (KNL1) by Mph1 (Mps1) kinase recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal. Conversely, association of PP1 to the N-terminus of Spc7 is necessary to silence the SAC. However the role of the KNL1-Bub3-Bub1 (KBB) pathway in spindle checkpoint signalling in human cells remains unclear. We have examined this issue in immortalised diploid retinal pigment epithelial (RPE1) cells. I will present evidence that, contrary to the situation in yeast, there are at least two distinct receptors for the Mad1-Mad2 complex at human kinetochores and, secondly, that these respond to distinct problems in microtubulekinetochore attachment. 96 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 60 Premature sister chromatid separation is poorly detected by the spindle assembly checkpoint due to system-level feedbacks. Mihailo Mirkovic1, Lukas Hutter2, Bela Novak2, Raquel Oliveira1 1 2 Instituto Gulbenkian de Ciência, Oeiras, Portugal Oxford Center for Integrative Systems Biology, Department of Biochemistry, University of Oxford, Oxford Mitotic errors are prevented by the Spindle Assembly Checkpoint (SAC), a surveillance mechanism that inhibits anaphase onset until all chromosomes are properly aligned and bioriented. Biorientation, in turn, depends on sister chromatid cohesion mediated by the cohesin complex. It should therefore be expected that the SAC arrests mitosis when loss of sister chromatid cohesion occurs prematurely. However, evidence suggests that the SAC is not robust enough to detect and halt cell division in the absence of cohesin. The mechanism behind this poor response is not properly understood. To address this issue we made use of a system to acutely induce sister chromatid separation in Drosophila developing brains. We show that full sister chromatid separation does not elicit a robust checkpoint response and cells abnormally exit mitosis with high segregation errors after a short mitotic delay. Quantitative live-cell imaging analysis reveals that mitotic exit in the presence of single sisters is caused by a gradual stabilization kinetochore-microtubules interactions and consequently weak SAC signalling. Surprisingly, most single sisters attach to the spindle in an end-on fashion, raising the question of how such interactions are escaping the tension-sensitive error-correction mechanisms. To elucidate this, we adopted a mathematical modelling approach to investigate the role of multiple feedback loops in the mitotic signalling network. Our results suggest that frail SAC activation upon cohesion loss is not solely caused by an intrinsic weak signalling capacity, but additionally potentiated by several feedback loops that gradually impair errorcorrection efficiency, which accelerate mitotic exit in response to premature cohesin loss. Our results explain how cohesion defects may escape SAC surveillance and therefore give rise to aneuploid cells. 97 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 61 CDK, PP2A And CDK-dependent PP2A-inhibitory pathway Can Make A Switch-like Response of Mitotic Phosphorylation Satoru Mochida1, Kaori Takaki1, Takeharu Nagai2 1 2 Kumamoto University Osaka University During cell cycle, M phase must be temporally separated from other phases to avoid chromosome instability. This separation is achieved by the abrupt changes of phosphorylation level of CDK substrates. We have shown that a subset of CDK substrates was dephosphorylated by a particular form of PP2A complex including B55 regulatory subunit (PP2A-B55). Enzymatic activity of PP2A-B55 is suppressed in M phase (thereby opposite to CDK activity) by the Greatwall kinase (Gwl)ENSA/ARPP-19 (ENSA collectively) pathway, in which CDK-activated Gwl activates ENSA. Activated ENSA then binds and inhibits PP2A-B55. In addition, recent reports by other groups suggested that both ENSA and Gwl are substrates of PP2A-B55, suggesting double-negative feedback loops. In the current study, we aimed to qualitatively and quantitatively analyse these proteins as a “system” to know if they contribute to the abrupt change of phosphorylation of CDK substrates, especially focusing on mitotic entry. For this purpose, we developed a bioluminescent energy-transfer (BRET) probe, which enabled us to analyse kinetics of phosphorylation/dephopshorylation of a model CDK substrate in real time. By using this probe in our reconstituted reaction, we observed a switch-like behavior on the phosphorylation level of CDK substrate. Together with the established roles of Cdc25 and Wee1, we suggest that PP2A and its regulators contribute to the robustness of the cell cycle. The reconstituted system we built would be a very useful platform to test if and how your protein of interest affects the cell cycle. 98 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 62 Defects in C/D snoRNPs assembly cause premature nucleolar hyper-condensation and impair mitotic exit Ana Isabel de los Santos-Velázquez, Fernando Monje-Casas Anabel de los Santos-Velázquez and Fernando Monje-Casas Dept. Genetics. University of Seville / Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER) Avda. Américo Vespucio, s/n. 41092 Seville (SPAIN) The faithful distribution of the chromosomes during mitosis requires the compaction of the DNA. This is especially important for the segregation of the repetitive ribosomal DNA (rDNA) array, which organizes the nucleolus. Chromosome compaction depends on a protein complex known as condensin. In budding yeast, condensin becomes highly enriched in the nucleolus during anaphase to facilitate chromosome segregation, and this process requires the activity of the Cdc14 phosphatase, a key determinant of mitotic exit. The activation of Cdc14 during the early stages of anaphase promotes condensin enrichment at the rDNA locus through the inhibition of RNA polymerase I transcription. Our data now suggest that not only PolI transcription, but also rRNA maturation interferes with condensin accessibility to the rDNA locus. Specifically, we show that defects in the assembly of C/D snoRNPs, which mediate site-specific modifications of the rRNA, lead to premature nucleolar condensation. Interestingly, this precocious hyper-condensation of the rDNA impairs Cdc14 release from the nucleolus, which is necessary to inactivate mitotic cyclin-CDK complexes throughout the cell and therefore to promote mitotic exit. Unlike most other DNA sequences, the rDNA fully condenses only during anaphase. Our results, together with the fact the same regulatory mechanism is used to couple mitotic exit with the completion of chromosome segregation in budding yeast, also allow us to explain why it is necessary to prevent full condensation of the rDNA until anaphase. 99 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 63 Characterization of a new APC/C subunit in Drosophila melanogaster Ágota Nagy1, Margit Pál2, Olga Nagy2, Levente Kovács1, 2, Péter Deák1, 2 1 2 Department of Genetics, University of Szeged, Szeged, Hungary Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary The Anaphase Promoting Complex (APC/C) plays a key role in controlling the protein level of cell cycle regulators during mitosis and G1 phase. As an E3 enzyme, this large complex determines the specificity of the ubiquitin-dependent destruction of target proteins. The APC/C contains at least 1315 subunits, many of which were identified and characterized by biochemical and genetic means from several model organisms. We have shown recently, that one of these subunits, Apc11, interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme in Drosophila. We have identified another two proteins in Drosophila with high degree of N-terminal sequence similarity to yeast and human Cdc26, a small subunit of APC/C. Mutant alleles of the gene coding for one of these proteins result in pupal lethality accompanied with chromosome overcondensation and mitotic arrest in larval neuroblasts. This phenotype is comparable to that from knockdown of known APC/C subunits. Due to its phenotype and higher similarity to known Cdc26 subunits, we denominate this gene as DmCdc26. Interestingly, the other gene proved to be nonessential for development and fertility, didn’t have any mitotic phenotype but nevertheless, it could complement the loss of function phenotype of DmCdc26, therefore it was dubbed as DmCdc26-like. These findings suggest that the APC/C machinery in Drosophila is somewhat different from those in yeast and human cells and may warrant further analysis. We are investigating the genetic and biochemical relationship between these proteins and the other subunits of the APC/C. 100 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 64 Nuclear dynamics and DNA replication in fission yeast Patroula Nathanailidou1, Anna Maria Rapsomaniki1, Nickolaos Nikiforos Giakoumakis1, Stella Maxouri1, Stavros Taraviras2, Zoi Lygerou1 1 2 Laboratory of General Biology, School of Medicine, University of Patras, Greece Laboratory of Physiology, School of Medicine, University of Patras, Greece DNA replication proceeds under a defined temporal and spatial pattern of origin activation, called replication program. Experimental data support that cells are able to adapt their replication program under different cellular conditions. There are various determinants of origin usage during S phase and among them nuclear structure and the spatial organization of chromatin is considered to be of great importance. We are interested to understand how the replication program is affected with respect to origin positioning in the 3-D space of the nucleus. Combining structural data from 3C studies in fission yeast and data concerning the efficiency of each origin of replication along the fission yeast genome we observed a correlation between positioning with respect to centromeres and origin efficiency. To investigate experimentally how the position of centromeres affects replication, we constructed a strain carrying GFP- tagged (Mis6-GFP), unclustered (Csi1Δ) centromeres and is able to incorporate the thymidine analogue BrdU (hsv-TK,hENT1). We assesed the BrdU incorporation pattern both in Csi1Δ and wild type -for this locus- cells, arrested in early S phase. In wild type cells BrdU foci are located mostly around the centromeres. Upon centromere unclustering BrdU incorporates in a significantly more dispersed manner than in the wild type situation. This result indicates that the position of origins relative to centromeres may affect the efficiency and the timing of their firing. In order to make a direct correlation between the distance from centromeres in the 3-D space and the efficiency of origins at both the population and single cell level specific origins of replication are being tagged using the lacO-lacI system. 101 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 65 Essential role of the atypical Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope Angel Nebreda Institute for Research in Biomedicine (IRB Barcelona) and Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain CDKs are protein kinases normally activated by regulatory subunits named cyclins, which are instrumental for cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout mice infertile. Intriguingly, mice deficient for cyclins that can potentially activate Cdk2 block meiosis earlier (cyclin B1), later (cyclins A1 and A2) or show weaker phenotypes (cyclins E1 and E2), suggesting that Cdk2 might be activated independently of cyclins in meiosis. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are infertile and display meiotic defects virtually identical to those observed in Cdk2 knockout mice including non-homologous chromosome pairing, unrepaired double strand breaks and undetectable sex body. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA knockout spermatocytes display severely affected telomere tethering as well as loss of telomeric Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an essential activator of Cdk2 at meiotic telomeres, which regulates telomere tethering to the nuclear envelope and proper synapsis of homologous chromosomes. This is the first genetic evidence of a physiological function for mammalian Cdk2 that depends on a non-cyclin regulatory subunit. 102 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 66 The Contribution of Cell Size to Functional Decline during Aging Gabriel Neurohr, Angelika Amon David H. Koch Institute, Massachusetts Institute of Technology Functional decline with increasing age has been observed in most known organisms. Genetic and environmental modulators of aging have been identified, yet the mechanisms leading to functional decline remain poorly understood. Conditions that reduce cell growth delay aging from yeast to mammals. Interestingly aging in yeast and mammalian cells correlates with a marked increase in cell size. Whether and how this large cell size itself contributes to functional decline is not clear. I used cell division cycle mutants to generate oversized yeast cells. The resulting decreased DNA:cytoplasm impaired cell proliferation in big cells. Increasing cell size was sufficient to reduce lifespan and slowed cell cycle progression. These delays correlated with impaired induction of cell cycle regulated genes and other inducible transcription systems. Importantly old yeast cells showed the same cell cycle progression and transcription defects as big young cells, indicating that age associated cell cycle defects are indeed a consequence of big cell size. The finding that increasing cell size impairs cell proliferation and contributes to age associated functional decline challenges currently accepted models of aging in budding yeast. 103 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 67 Re-visiting the regulation of centrosome duplication Zsofia Novak, Jordan Raff Sir William Dunn School of Pathology, University of Oxford Centrosomes are the main microtubule organizing centres in animal cells and thereby participate in many key cellular processes, such as cell polarity, motility or bipolar spindle assembly. In most cell types centrosome numbers are carefully controlled, which is critical for the accurate regulation of centrosome-dependent cellular events. Accordingly, numerical centrosome aberrations have been linked to a number of human diseases. A key regulation point of centrosome formation is the assembly of centrioles - the structures that act as both the physical core and the precursor of centrosomes. Centrosomes contain a pair of centrioles, and during a cell division cycle these undergo a single round of semiconservative replication in S-phase to ensure that centrosome duplication occurs strictly once per cell cycle. We have recently shown in Drosophila that the conversion of newly assembled daughter centrioles to duplication-competent mother centrioles requires the centriolar incorporation of Asl/Cep152. The centriolar presence of Asl/Cep152 acts as a primary license for centriole replication, and its recruitment is initiated as new centrioles pass through their first mitosis, prior to their first round of duplication in the subsequent S-phase. The initial incorporation of Asl at new centrioles is dependent on the core centriolar component Sas-4, which itself is present in centrioles from the earliest stages of their assembly at S-phase onset, but appears unable to recruit Asl until late mitosis. Here we investigate the cell cycle-dependent regulation of the Sas-4 –Asl interaction at centrioles in vivo as the key regulation mechanism that permanently converts newly assembled centrioles into a replication-competent form. 104 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 68 Coordinating Cdk1 and Plk1 activation for mitotic entry in early C. elegans embryos and human cells Yann THOMAS1, Luca CIRILLO2, Costanza PANBIANCO2, Lisa MARTINO1, Nicolas TAVERNIER1, Lucie VAN HOVE1, SCHWAGER Françoise2, Anna Santamaria4, Monica GOTTA2, 3, Lionel PINTARD1 1 Jacques Monod Institute, UMR7592, Paris-Diderot University - CNRS, Paris, France Department of Cellular Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1 rue Michel Servet, Geneva 4, Switzerland 3 Swiss National Centre for Competence in Research Program Chemical Biology, Geneva 1211, Switzerland 4 Biomedical Research Unit in Gynecology, Collserola building, Vall Hebron Research Institute (VHIR) 08035 Barcelona, Spain 2 Mitosis is orchestrated by several protein kinases including cyclin-dependent kinases (Cdks), Pololike kinases (Plks) and Aurora kinases. Despite considerable progress toward understanding the individual function of these protein kinases, how their activity is coordinated in space and time during mitosis is less well understood. We have recently shown that CDK-1 regulates PLK-1 activity during mitosis in C. elegans embryos through multisite phosphorylation of the PLK-1 activator SPAT1 (Tavernier et al. 2015). Early embryos expressing SPAT-1 variants mutated on CDK-1 phosphorylation sites presented severe delays in mitotic entry, mimicking embryos lacking spat-1 or plk-1 function. We further showed that SPAT-1 phosphorylation by CDK-1 promotes its binding to PLK-1 and stimulates PLK-1 phosphorylation on its activator T-loop by Aurora A kinase. In human cells, Bora, the ortholog of SPAT-1, regulates several aspects of mitosis (Bruinsma et al. 2014, Chan et al 2008, Macůrek et al. 2008, Seki et al. 2008. Although phosphorylation of Bora is not strictly required to activate Plk1 (Seki et al. 2008), we found that Cdk1 phosphorylation of Bora greatly increases Plk1 activation in vitro (Tavernier et al. 2015). We have now mapped the Cdk1 phosphorylation sites of Bora. Based on the results obtained in C. elegans, we have mutated a number of N-terminal sites and found that Bora ability to activate Plk1 is greatly reduced. We are currently testing the function of these sites in human cells. Our data will help to shed light on the activation mechanisms of Plk1 by Bora. Since Plk1 is overexpressed in many cancers, understanding the precise mechanisms of activation of this kinase will help the design of anti-cancer drugs. References: • Tavernier et al. JCB 2015 • Bruinsma W. et al. J Cell Sci. 2014 • Chan EH. et al. Chromosoma. 2008 • Macůrek L. et al. Nature. 2008 • Seki A. et al. Science. 2008 105 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 69 Cycling to change destiny: dream or reality? Fabienne PITUELLO, Frédéric BONNET, Angie MOLINA-DELGADO, Eric AGIUS Centre de Biologie du Développement UMR5547 CNRS/UPS Our aim is to understand how cell cycle features of neural stem/progenitor cells (NS/PC) time the switch from proliferating progenitors towards differentiating neurons. We previously showed that a positive regulator of the G2/M transition, the phosphatase CDC25B, is promoting neuronal differentiation. CDC25B expression correlates remarkably well with areas where neurogenesis occurs and downregulating the CDC25B phosphatase, results in G2-phase lengthening and defective conversion of NSC to differentiating neurons. We thus identified a new function of the CDC25B phosphatase in promoting neuronal differentiation associated with a G2 phase shortening (Agius et al., 2015; Peco et al., 2012). More recently we began to elucidate how the CDC25B phosphatase switches a proliferating neural progenitor into a differentiating neuron (unpublished data). To help us accomplish this goal we set up a novel high resolution time-lapse imaging technique that allows measuring the duration of each phase of the cell cycle in single neural progenitors in the chicken developing neural tube and to track the fate of daughter cells after mitosis. Neural progenitors can perform three mode of division: self-renewing (one progenitor gives two progenitors, P to PP), asymmetric neurogenic (one progenitor gives a progenitor and a neuron, P to PN), symmetric terminal neurogenic (one progenitor gives two neurons, P to NN). Using specific markers of each division mode, we show that CDC25B promotes neurogenic divisions. Interestingly, asymmetric or symmetric terminal neurogenic divisions will be favored depending on the context. Moreover part of this CDC25B function is independent of its role in the cell cycle suggestive of new targets for the phosphatase linked to the molecular network orchestrating neurogenesis. Our recent unpublished data will be presented. Agius, E., Bel-Vialar, S., Bonnet, F. and Pituello, F. (2015). Cell cycle and cell fate in the developing nervous system: the role of CDC25B phosphatase. Cell Tissue Res 359, 201-13. Peco, E., Escude, T., Agius, E., Sabado, V., Medevielle, F., Ducommun, B. and Pituello, F. (2012). The CDC25B phosphatase shortens the G2 phase of neural progenitors and promotes efficient neuron production. Development 139, 1095-104. This work is supported by the "Fédération pour la Recherche sur le cerveau (FRC)" and the "Fondation ARC pour la Rcecherche sur le Cancer". 106 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 70 Competition between microtubules and MPS1 kinase for kinetochore binding directly regulates spindle checkpoint signaling Yoshitaka Hiruma1, 2, 3, Carlos Sacristan2, 3, Spyridon Pachis2, 3, Athanassios Adamopoulos1, Timo Kuijt2, 3, Marcellus Ubbink4, Eleonore van Castelmur1, Anastassis Perrakis1, Geert Kops2, 3 1 Division of Biochemistry, Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands. 2 Molecular Cancer Research, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands. 3 Cancer Genomics Netherlands, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands. 4 4 Leiden Institute of Chemistry, Leiden University, Post Office Box 9502, 2300 RA Leiden. The Netherlands Cell division progresses to anaphase only after all mitotic chromosomes are connected to spindle microtubules via their kinetochores and the spindle assembly checkpoint (SAC) is satisfied. The mechanism by which the SAC senses kinetochore-microtubule interactions remains unknown. Here we show that microtubules directly compete with the essential SAC kinase MPS1 for binding to the outer kinetochore NDC80 complex. The N-terminal localization module of MPS1 directly interacts with the NDC80 complex in vitro, in a manner dependent on its phosphorylation. That interaction is mediated by residues in the calponin-homology domain of HEC1 and can be disrupted by addition of microtubule polymers. In cells, MPS1 binding to kinetochores or to ectopic NDC80 complexes is prevented by end-on microtubule attachment, independent of phosphatase activity or stripping by dynein. Competition for kinetochore binding between SAC proteins and microtubules may thus represent a direct and evolutionary conserved way to silence the kinetochore-derived SAC signal. 107 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 71 Biochemical Characterization of CENP-N/L complex during Kinetochore assembly Satyakrishna Pentakota, et al. MaxPlanck Institue of Molecular Physiology ABSTRACT: Accurate segregation of chromosomes during mitosis is crucial for the maintenance of genome integrity. A detailed understanding of the mechanisms of chromosomal segregation is very difficult owing to the molecular complexity of this process. Key to proper segregation of the replicated genetic material is the physical attachment of the microtubule-based spindle to each sister chromatid. This attachment is directly mediated by macromolecular structures known as kinetochores. Kinetochores are built on the centromeres, which are the specific centers for Kinetochore assembly. Kinetochores can be conceptualized as having an outer and inner kinetochore. The outer kinetochore consists of the KMN network (Knl1, Mis12 and Ndc80 complex), a 10 subunit complex known to bind to microtubules directly. The inner kinetochore comprises of 16 subunit of the constitutive centromere associated network (CCAN). The CCAN, which is organized in sub complexes, acts as a bridge between the centromeric chromatin and the outer kinetochore complex. Centromeric chromatin contains CENP-A, a histone H3 variant that is heavily enriched at the centromeres that functions as an epigenetic mark for centromere identity. To date, only two subunits of CCAN, namely CENP-C and CENP-N are known to bind to CENP-A. However, the molecular detail of how CENP-C, CENP-N and CENP-A coordinate to bring about faithful chromosome segregation is an ongoing study. In this study, we demonstrate that CENP-N forms a heterodimer with CENP-L, consistent with orthologous proteins in budding yeast. We then confirm that CENP-N/L has a preferential selectivity for the CENP-A nucleosomes when compared with the canonical H3 nucleosomes by employing both GST-pull down and size exclusion chromatography experiments. We produce a fragment of CENP-N lacking the dimerization interface, and show that it binds specifically but weakly to CENP-A nucleosomes when compared with CENP-N/L complex. However, CENP-L does not bind to either CENP-A nucleosomes or canonical H3 nucleosomes indicating that CENP-N could be the prime candidate binding to CENP-A nucleosomes. Our Analytical Ultra Centrifugation experiments (AUC) on CENP-A nucleosomes in complex with CENP-N/L indicate that there are two copies of the CENP-N/L complex that bound to each CENP-A histone within an octameric CENP-A nucleosome. Further biochemical and structural studies with recombinant CENP-C will address the interplay between these two proteins and the CENP-A nucleosome. 108 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 72 CHARACTERISATION OF THE PHOSPHATASE CONTROL SYSTEM THAT PREVENTS PREMATURE MITOTIC ENTRY IN MAMMALIAN CELLS Nisha Peter, Priya Amin, Helfrid Hochegger Genome Damage and Stability Centre, University of Sussex Accurate chromosome segregation during mitosis prevents aneuploidy and cancer. The Wee1Cdc25-CDK1 feedback loop ensures cells enter mitosis in a timely and orderly manner. This signalling system has been proposed to work as a bistable switch that is maintained by the counterbalancing action of kinases and phosphatases. However, the phosphatases critical for this transition in mammalian cells are yet to be identified. Cdc14 is the major phosphatase antagonising CDK in yeast. But its eukaryotic homologue does not appear to play an essential role in mitotic control. Studies in Drosophila and Xenopus have shown PP2A/B55 inhibition is crucial for CDK activation, and that this is brought about by Greatwall kinase and its substrates ENSA/Arpp19. However, it remains unclear, if PP2A/B55 inhibition by phosphorylated Ensa/ARPP is a critical event in regulating mitotic entry in somatic cells. To address this question, we have analysed the effects of ARPP19/ENSA depletion. We found that ENSA depletion causes very similar phenotypes to the depletion of its upstream kinase Greatwall causing mitotic delays and cytokinesis failure. Surprisingly, siRNA depletion of ARPP, which is expressed at 10fold lower levels than Ensa, causes changes in cell shape and spontaneous cell death in interphase cells, but does not affect mitosis. To better understand the dynamics of the G2/M switch we have established a Cdk1 analogue sensitive mutation in HeLa cells that allows a wellcontrolled analysis of mitotic entry in a block/release experiment. Treatment of these cdk1as HeLa cells that are arrested in G2 with the phosphatase inhibitor okadaic acid (OA) causes a rapid trigger of the G2/M switch suggesting that an OA sensitive phosphatase plays a crucial role in preventing mitotic entry. Interestingly depletion of PP2A/B55 and/or inactivation of this phosphatase with constitutively phosphorylated ARPP19 do not overcome this G2 arrest. This suggests that another as yet unknown phosphatase participates in this critical control step. We are currently deploying siRNA screening in our cdk1as HeLa system to identify this additional player in the G2/M switch. Ultimately these experiments will help understand how the balanced activity of the Cdk1 activation switch and its counteracting phosphatases contribute to the regulation of mitotic entry. 109 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 73 Analysis of Drosophila protein interaction networks reveals new centromeric and kinetochore components Marcin Przewloka1, Max Convay2, Pietro Lio2, David Glover1 1 2 Department of Genetics, University of Cambridge Computer Laboratory, University of Cambridge We employed affinity purification – mass spectrometry (AP-MS) method to purify proteins from Drosophila melanogaster cultured cells and syncytial embryos. Baits were chosen from the list of proteins, which are important for the proper cell cycle progression. A very large volume of data was accumulated, so we created a searchable database in order to efficiently analyse the results of our AP-MS experiments. The database was subsequently equipped with custom designed modules for the statistical analysis and visualization of protein-protein interaction networks. Preliminary analyses using these new bioinformatics tools revealed that some novel proteins specifically co-purified with the centromeric or kinetochore baits. We chose few candidates and analysed their localization in cultured cells using GFP fusions or specific antibodies. Interestingly, we found that Drosophila vesicle trafficking protein SNAP29 (or Ubisnap), an adaptor protein Homer and the uncharacterised protein CG15107 indeed localize do centromeres, in some cases in the cell cycle-dependent manner. Centromeric/kinetochore localization of these proteins suggests that previously unknown players and regulatory pathways take part in the kinetochore assembly and function. This finding validates the usefulness of our bioinformatics utility and underscores the importance of the systems biology approach to analyse protein interaction networks. Currently we are selecting new candidates for further studies and in parallel are trying to gain mechanistic insight into the function of newly discovered kinetochore proteins and their role in the cell cycle regulation. 110 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 74 MEN-independent role of Cdc5 polo kinase in the Cdc14 activation during mitosis Jose Antonio Rodriguez-Rodriguez, Ethel Queralt Instituto de Investigaciones Biomédicas de Bellvitge (IDIBELL) During mitosis dividing cells equally segregate their replicated chromosomes to the daughter cells. To complete mitosis, Saccharomyces cerevisiae needs to activate the mitotic phosphatase Cdc14. Before anaphase, Cdc14 is kept inactive in the nucleolus bound to the nucleolar protein Net1. Net1 is kept dephosphorylated by the activity of the PP2ACdc55 phosphatase during metaphase. Separase-dependent downregulation of PP2ACdc55 at anaphase onset allows Cdk-dependent Net1 phosphorylation and Cdc14 release. A number of additional proteins contribute to Cdc14 regulation commonly included in two pathways, FEAR (Cdcfourteen early anaphase release) and MEN (mitotic exit network). Cdc5 polo kinase is a mitotic exit component, which best-characterized role is the inactivation by phosphorylation of the MEN inhibitor Bfa1 in anaphase. However, the role of Cdc5 regulating the Cdc14 release it still not well defined. Here we present new data providing new insight into the mechanism how Cdc5 contributes to the timely Cdc14 release from the nucleolus. We showed that Cdk-dependent Net1 phosphorylation is not required for Cdc5-induced Cdc14 release from the nucleolus and that Cdc5 phosphorylate different residues to Cdk1 within Net1. Apart of regulating MEN activity through Bfa1 phosphorylation, Cdc5 has a parallel role to MEN during anaphase. This MEN-independent Cdc5 function in the Cdc14 activation requires activation by Cdk1-dependent phosphorylation mostly at T242. In addition, Cdc5 requires not only previous Cdk1 activation but also active separase to promote Cdc14 release from the nucleolus. 111 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 75 Self-organization of a Rho GTPase module during polarity establishment Péter Rapali1, 2, Romain Mitteau1, 2, Aurelie Massoni-Laporte1, 2, Julien Meca1, 2, Sylvain Tollis1, 2, Derek McCusker1, 2 1 2 Dynamics of Cell Growth and Division Laboratory, European Institute of Chemistry and Biology, Bordeaux, France Institut de Biochimie et Génétique Cellulaires (IBGC), UMR 5095, Bordeaux, France The most basic function of cells is to grow and divide, thus propagating life via the cell division cycle. Our understanding of the regulatory mechanisms linking cell growth and the cell cycle are surprisingly limited. In budding yeast, Cyclin dependent kinase 1 (Cdk1) triggers polarization of the actin cytoskeleton and growth of a new cell via activation of the Rho GTPase Cdc42 in late G1 of cell cycle. Cdc42 is a key regulator of polarized growth in diverse eukaryotes. The activity of Cdc42 relies on its GDP and GTP nucleotide binding status where the GTP bound state is the active form. The rapid formation of a stable pole of Cdc42 at the cell cortex may be established via feedback mechanisms, wherein the Guanine nucleotide Exchange Factor (GEF) Cdc24 activates Cdc42. It has been proposed that Bem1, a multi-domain scaffold protein is involved in feedback mechanisms underlying Cdc42 regulation, but its exact role has been enigmatic. Here, using a FRET-based spectroscopy assay of Cdc42 activity, we show that Bem1 increases the GEF-catalyzed GDP to GTP exchange rate of Cdc42. In addition, Cla4, a Cdc42 effector kinase, phosphorylates Cdc24 GEF more extensively in the presence of Bem1; moreover, Bem1 accelerates this otherwise slow Cla4mediated phosphorylation of Cdc24 GEF. Since phosphorylation of Cdc24 by Cla4 has been proposed to down-regulate Cdc24 GEF activity, Bem1 may thus also be involved in a delayed negative feedback loop that regulates Cdc42 activity. In conclusion, our results suggest that Bem1 is an active scaffold protein that is directly involved in Cdc42 activation; however, it may also play a role in the late attenuation of Cdc42 activation. This dual function of Bem1 may ensure the rapid establishment and development of an active and stable Cdc42 pole during the cell cycle, while the capacity to down-regulate GEF activity in response to Cdc42 activation suggests that the GTPase module has the propensity to self-organize. 112 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 76 A kinetic model for an irreversible anaphase switch in human cells Scott Rata1, Susanne Hellmuth2, Olaf Stemmann2, Bela Novak1 1 2 Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK Chair of Genetics, University of Bayreuth, Universitatsstraße 30, 95440, Bayreuth, Germany The cleavage of centromeric cohesin by separase, a giant cysteine endopeptidase, is the universal trigger of anaphase onset in eukaryotes. Separase activation is under multiple layers of control, consisting of securin binding, auto-cleavage, and isomerisation. Based on recent findings in human cells [1, 2], a mathematical model is presented demonstrating the irreversible activation of separase at anaphase by the interplay of these mechanisms. During interphase, separase binds to securin and protein-phosphatase 2A (PP2A). Securin inhibits the endopeptidase activity of separase, while PP2A keeps four sites of the bound securin dephosphorylated. At the beginning of metaphase the Anaphase Promoting Complex/Cyclosome (APC/C) becomes activated, which preferentially promotes the degradation of free, phosphorylated securin. Once separase is liberated of securin inhibition it auto-cleaves close to its PP2A binding site, thereby disrupting PP2A binding. With intermolecular cleavage, securin-liberated separase promotes the phosphorylation of other separase-bound securin molecules and makes them a better substrate of the APC/C. Furthermore, securin-liberated separase could be isomerised by the prolyl-directed isomerase Pin1 into a securinresistant configuration, which can be inhibited by cyclin-B binding only. However the mutual inhibition between separase and cyclin-B takes place at late stages of mitosis. This network shows an intriguing similarity to the positive-feedback-promoted separase activation in budding yeast [3]. References 1) Hellmuth, S., Bottger, F., Pan, C., Mann, M., and Stemmann, O. (2014). PP2A delays APC/Cdependent degradation of separase-associated but not free securin. EMBO J. 33, 1134–1147. 2) Hellmuth et al., (2015). Human Chromosome Segregation Involves Multi-Layered Regulation of Separase by the Peptidyl-Prolyl-Isomerase Pin1. Mol. Cell 58: 495 – 506 3) Holt LJ, Krutchinsky AN, Morgan DO. (2008). Positive feedback sharpens the anaphase switch. Nature 454: 353 – 357 113 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 77 ROLE OF THE APC/C COMPLEX-MEDIATED DEGRADATION OF GRK2 IN MITOSIS Clara Reglero1, 2, Verónica Rivas1, 2, Federico Mayor1, Petronila Penela1, 2 1 2 Centro de Biología Molecular Severo Ochoa (CBMSO) - Universidad Autónoma de Madrid (UAM). Madrid. Spain Instituto de Investigación Sanitaria La Princesa. 28006, Madrid, Spain Proteasome-dependent degradation of key kinases represents a major regulatory mechanism in the control of cell cycle. We have recently reported that timely degradation of GRK2, a serine/threonine G protein-coupled receptor kinase, is part of an intrinsic pathway that ensures cell cycle progression. Besides being a key player in the desensitization of manifold GPCR receptors, GRK2 emerges as a signaling node due to its ability to regulate a variety of signaling proteins and cellular functions. We have previously found that GRK2 protein levels progressively decay during G2/M as a result of the sequential cooperation of the E3 ligases Mdm2 and APC/C. Thus, during G2 the functional interaction of Pin1 with the CDK2/cyclinA-dependent phosphorylated GRK2 triggers its ubiquitination by Mdm2. However, at mitosis onset GRK2 decay is promoted by the APC/C-Cdc20 complex that recognizes a D-Box in GRK2, in a spindle checkpoint-independent manner. Impairment of this timely down-regulation of GRK2 by mutation of the D-box motif results in significant alterations in cellular proliferation and mitosis progression. Our data have unveiled the functional interaction of GRK2 with relevant regulatory factors involved in cytokinesis. Thus, we have demonstrated that GRK2 is a key modulator of microtubule dynamics through the phosphorylation of the tubulin deacetylase HDAC6. In addition, we have found that GRK2 influences the extent of PI3K activation by a direct interaction with the regulatory subunit p85, which is involved at the cleavage furrow. Our data reveal the presence of GRK2 in the midbody, a structure wherein HDAC6 and p85 are also found. Dynamic changes in the levels and functionality of GRK2 might help to timely activate HDAC6 or/and PI3K in order to orchestrate microtubule dynamics and to recruit/activate factors involved in organizing the contractile ring and setting the symmetry of the division plane. Our results suggest that defective or sustained degradation of GRK2 during mitosis could impair cytokinesis and contribute to polyploidy and chromosomal instability. 114 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 78 Characterization of the spindle assembly checkpoint in meiosis Julie Rojas, Wolfgang Zachariae Laboratory of Chromosome Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany Faithfull chromosome segregation in both mitosis and meiosis requires coordinating APC/C-Cdc20 activation with correct microtubule-kinetochores attachment. In mitosis, kinetochore-microtubule attachments are monitored by the spindle assembly checkpoint (SAC), which destabilizes incorrect attachments and delays the cell cycle at metaphase until every chromosome is properly bi-oriented (Musacchio and Salmon, 2007). Several studies performed in different model organisms suggest that the SAC is also present in meiosis (Sun and Kim, 2012). However, SAC function and regulation in meiosis are poorly understood. Thus, we study SAC regulation during meiosis in budding yeast. We develop a technique to dynamically assess SAC activity in living cells using time-lapse microscopy. Our results show that the SAC is transiently activated in both metaphase I and metaphase II. We also observed that the SAC remains active longer in several mutants having tension or attachment defects. Current work aims to identify aspects of SAC regulation, which are specific to either the first or the second division of meiosis. Musacchio, A., and Salmon, E.D. (2007). Nat Rev Mol Cell Biol 8, 379-393. Sun, S.C., and Kim, N.H. (2012). Hum Reprod Update 18, 60-72. 115 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 79 Opening the cohesin ring: Asymmetric ATP hydrolysis triggers DNA release Ahmed Elbatsh, Judith Haarhuis, Benjamin Rowland The Netherlands Cancer Institute Sister chromatid cohesion mediated by the cohesin complex is essential for faithful chromosome segregation in mitosis. By resisting the pulling forces of microtubules up to the moment that all chromosomes are correctly attached, cohesin ensures that the sister chromatids separate to the opposite poles of the cell, and that each of the daughter cells receives an equal karyotype. Cohesin stably holds the sister chromatids together by co-entrapping them inside its ring-shaped structure from the moment of cohesion establishment in S phase until mitosis. Cohesin can only stably associate with DNA when it is protected against its cellular antagonist Wapl. This factor is thought to remove cohesin from DNA through the opening up of a DNA exit gate in cohesin rings. How Wapl achieves this ring opening is unknown. In S phase, Eco1 acetylates cohesin’s Smc3 subunit and hereby renders cohesin resistant to Wapl. We used yeast genetics to dissect how Wapl drives cohesin from chromatin. Hereby we identified mutants of cohesin that can entrap, but can’t release DNA. We pinpointed an unexpected role of the highly conserved ABC-like ATPase domain of cohesin’s Smc1 subunit. Using a combination of genetics, microscopy, and in vitro assays, we found that hydrolysis of specifically one of cohesin’s associated ATPs is the trigger for DNA release from cohesin rings. We propose that Eco1 locks cohesin rings around the sister chromatids by counteracting cohesin-mediated ATPase activity. 116 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 80 Bub3-associated BubR1 sequesters Fizzy/Cdc20 at DNA breaks and promotes correct segregation of broken chromosomes Anne Royou CNRS, Université de Bordeaux, IECB/IBGC, UMR 5095, 2 rue Robert Escarpit, 33607 Pessac, France The presence of DNA double-strand breaks during mitosis is particularly challenging for the cell as it produces broken chromosomes lacking a centromere. This situation can cause genomic instability due to improper segregation of the broken fragments into daughter cells. We have recently uncovered a process by which broken chromosomes are faithfully transmitted, via the BubR1dependent tethering of the two broken chromosome ends. However, the mechanisms underlying BubR1 recruitment and function on broken chromosomes were not determined. Here, we show that BubR1 requires interaction with Bub3 to localize on the broken chromosome fragments and to mediate their proper segregation. We also find that Cdc20, a co-factor of the E3 ubiquitin ligase Anaphase-Promoting-Complex/Cyclosome (APC/C), accumulates on DNA breaks in a BubR1 KEN boxdependent manner. A biosensor for APC/C activity demonstrates a BubR1-dependent local inhibition of APC/C around the segregating broken chromosome. Therefore, we propose that the Bub3/BubR1 complex on DNA breaks functions to inhibit the APC/C locally via the sequestration of Cdc20, thus promoting proper transmission of broken chromosomes. 117 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 81 Hi-C analysis of condensin action in budding yeast Stephanie Schalbetter1, Jon-Matthew Belton2, Job Dekker2, Jon Baxter1 1 2 GDSC, University of Sussex, UK University of Massachusetts Medical School, United States Mitotic chromosome condensation is thought to occur through hierarchical folding of the chromatin polymer in mitosis. The activity of condensin and topoisomerase II are required for full compaction of mitotic chromosomes. Here we assess the function of condensin and topoisomerase II in mitotic chromosome compaction by Hi-C analysis in the yeast Saccharomyces cerevisiae. We find that both condensin and topoisomerase II act throughout the yeast genome to resolve yeast mitotic chromosomes from one another and prevent long-range intra-chromosomal interactions during mitosis. However, condensin and topoisomerase II achieve this in distinct manners. Condensin promotes short-range interactions within chromosomes that prevent long-range interactions, whilst topoisomerase II is required to resolve long-range interactions. The impact of these findings on the current models of condensin and topoisomerase II action in mitotic chromosomes will be discussed. 118 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 82 A MEIOTIC CHECKPOINT REGULATES TRANSLATION OF THE SIGNALING FACTOR GURKEN IN DROSOPHILA OOGENESIS. Trudi Schupbach1, Wei Li1, Attilio Pane2 1 2 Princeton University University of Rio de Janeiro, Brazil The spatial patterning of the egg and embryo of Drosophila depends on signaling events that occur during oogenesis. In particular the gene gurken (grk) which encodes a secreted molecule with homologies to TGF-alpha like growth factors, plays an important role in transmitting pattern information from the oocyte to the overlying follicle cells. The levels of Grk are particularly crucial in dorso-ventral patterning, and the production of Grk protein is tightly controlled both at the level of RNA localization, as well as at the level of protein accumulation. We have shown that mutations in genes required for DSB repair in meiosis affect dorso-ventral patterning through activation of a checkpoint that couples progression through meiosis to the translation of Gurken. We have also shown that mutations in genes necessary for the production of piRNAs activate the same checkpoint and, among other effects, lead to a decrease in Gurken protein accumulation. The molecular level at which this meiotic checkpoint affects the translational machinery in oogenesis is under investigation. 119 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 83 The dynamics of Cyclin A2 in the cell cycle: A time and a place for Cyclin A2 Helena Silva Cascales, Erik Müllers, Arne Lindqvist Karolinska Institutet Progression through the cell cycle is a highly regulated process driven by the coordinated action of many enzymes, in particular Cyclin-Cdk complexes. Using a novel method based on accurate quantification of immunofluorescence, we have recently shown that Cdk1 activity starts to accumulate at completion of S-phase. Interestingly, in parallel with Cdk1 activation we observe a change in the localisation of Cyclin A2 from being only nuclear to being both nuclear and cytoplasmic, suggesting a role for Cyclin A2 in the cytoplasm during G2 phase. We find that all cells express cytoplasmic Cyclin A2 in the last hours preceding mitosis. Preliminary results show that perturbations of Cdk activity affect the dynamics of Cyclin A2 localisation to the cytoplasm; however how Cdk activity modulates this process is still an open question. Interestingly, inflicting DNA damage induces a relocalisation of cytoplasmic Cyclin A2 to the nucleus suggesting that the activities needed for a DNA damage response could prevent cytoplasmic localisation of Cyclin A2. We are currently assessing the functional importance of cytoplasmic Cyclin A2 for progression through G2 and mitosis. However our current hypothesis is that the G2 biochemical state brought about by the increase in Cdk activity at the S/G2 transition triggers an unknown mechanism that induces cytoplasmic localisation of Cyclin A2 to allow the phosphorylation of Cdk targets in the cytoplasm needed for correct mitotic entry. 120 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 84 Regulatory mechanism and physiological meaning of anaphase delay during mouse pronuclear formation Shou Soeda1, 2, Miho Ohsugi1, 3 1 Department of Biological Sciences, Graduate School of Sciences, The University of Tokyo Japan Society for the Promotion of Science, Research Fellowship for young Scientists 3 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo 2 Mammalian matured oocytes are arrested at metaphaseⅡ. After the fusion with sperm, oocytes resume meiosis, leading to female chromosome segregation, polar body emission and pronuclear formation. During this process, it takes more than one hour for female chromosomes to be enveloped after chromosome segregation ends. This is in contrast to somatic cells in which nuclear envelope reassembly begins just after chromosome segregation has completed. Here, we investigate the regulatory mechanisms and the physiological importance of this anaphase delay during mouse pronuclear formation. We previously reported that p90RSK, which is a downstream kinase for MOS-MAPK pathway in oocytes, has an activity to delay anaphase during pronuclear formation. However our previous results also demonstrated that pronuclear formation preceded the inactivation of MOS-MAPKp90RSK pathway, indicating that the other factors than p90RSK determine the timing of pronuclear formation. We speculated that the factors that counteract MOS-MAPK-p90RSK pathway are the potent candidates for pronuclear formation accelerators and thus focused on mitotic phosphatases. PP1 and PP2A are reported to facilitate mammalian mitotic exit by counteracting the activities of CDK or other mitotic kinases. We tested the involvement of these phosphatases in temporal regulation of pronuclear formation. Pronuclear formation timing was delayed by okadaic acid in a dose dependent manner, suggesting that pronuclear formation timing is determined by the degree of these phosphatase activities. In agreement with this hypothesis, PP1 over-expression accelerated the timing of pronuclear formation. To address the question of how phosphatase activation delay is achieved, we investigated the CDK-Mastl-Ensa/Arpp19 pathway that inhibits PP2A activity during metaphase in somatic cells. By immunorfluorescent studies we found that phopsho-Ensa was maintained for more than one hour after the onset of anaphase II, but drastically decreased before pronuclear formation. Next, we tested the cross talk between p90RSK pathway and Ensa/Arpp19 pathway. The p90RSK inhibitor BI-D1870 treatment accelerated the decrease of phospho-Ensa as well as pronuclear formation. Importantly, even in this condition, a decrease of phospho-Ensa preceded pronuclear formation. These results suggest that phospho-Ensa decrease is a prerequisite for pronuclear formation and that p90RSK is involved in upstream of Ensa/Arpp19 pathway to delay PP2A activation. We next investigated the physiological importance of delayed pronuclear formation by accelerating the pronuclear formation timing by two ways; PP1 over expression and intra-cytoplasmic sperm injection one hour after parthenogenetic activation (PA-ICSI). We found that PP1 over expressed zygotes formed aberrant small male pronucleus and caused severe chromosome bridges in first cleavage division stage while PP1 over expression didn’t affect pronuclear size in parthenogenetic embryos with two pronuclei and had much less effect on first cleavage division. The similar phenotype was observed in the zygotes obtained by PA-ICSI. Thus we propose that anaphase delay in pronuclear formation is required for sperm chromatin to be reorganized into proper zygotic chromatin. Sperm chromatin remodeling and female chromosome segregation are fulfilled in the same cytoplasm. Our findings revealed that oocytes accomplish these complicated processes by adjusting the cell cycle. 121 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 85 Nek7-interactor RGS2 is required for mitotic spindle organization Edmarcia Elisa de Souza1, Heidi Hehnly3, Arina Marina Perez1, Gabriela Vaz Meirelles1, Juliana Smetana1, Stephen Doxsey3, Jörg Kobarg2 1 Brazilian Laboratory of Biosciences/National Center For Research in Energy and Materials, Campinas, SP, Brazil Institute of Biology, State University of Campinas, Campinas, SP, Brazil. 3 University of Massachusetts Medical School, Program in Molecular Medicine, MA, USA. 2 The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from two centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization. 122 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 86 Dual regulation of Drosophila APC/C activity by Rca1 Frank Sprenger, Matthias Kies University of Regensburg APC/C activity has to be tightly controlled to ensure accumulation of mitotic cyclins in G2. Negative regulation of interphase APC/C activity is achieved by multiple mechanisms: 1) by phosphorylation (and thereby inhibition) of its activator Fzr/Cdh1 by cyclin-dependent kinase activity, and 2) by pseudosubstrate inhibition by Rca1/Emi1. We have analyzed the functional domains in Rca1 in Drosophila Schneider cells and could confirm that the mechanisms proposed for APC/C inhibition by the vertebrate homologue Emi1 are also valid for Rca1 in Drosophila. Further analyses revealed that the F-box domain of Rca1 is also required for efficient APC/C inhibition. Several lines of evidence indicate that Rca1 is part of an SCF complex that can target other proteins for ubiquitination and subsequent degradation and that this function is indespensible for full APC/C inhibition. Dap, a specific CKI inhibitor of CycE/Cdk2, could be identified as a potential target of SCF-Rca1. This suggests that Rca1 contributes to APC/C inhibition in a dual way: as a pseudosubstrate inhibitor, and as part of an SCF-complex, degrading a CKI that would otherwise restrain kinase activity necessary for APC/C inhibition. 123 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 87 Ubiquitin receptor protein UBASH3B mediates a switch-like mechanism of Aurora B localization to microtubules. Ksenia Krupina1, Charlotte Kleiss1, Stephane Schmucker1, Kay Hofmann2, Olivier Poch3, Laurent Brino1, Izabela Sumara1 1 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France. Institute for Genetics, University of Cologne, Cologne, Germany. 3 Computer Science Department, ICube, UMR 7357, University of Strasbourg, CNRS, Fédération de médecine translationnelle, Strasbourg, France. 2 Mitosis ensures equal segregation of the genome and is controlled by variety of ubiquitylation signals on substrate proteins. However, it remains unexplored how the versatile ubiquitin code can be read during mitotic progression. Here, we identify the ubiquitin receptor protein UBASH3B as an important regulator for mitosis. UBASH3B interacts with ubiquitylated Aurora B, one of the main kinases regulating chromosome segregation, and controls its subcellular localization but not protein levels. Importantly, UBASH3B is a limiting factor in this pathway, and is sufficient to target Aurora B to microtubules prior to anaphase, as revealed by super resolution imaging. Moreover, targeting Aurora B to microtubules by UBASH3B is necessary for the timing and fidelity of chromosome segregation in human cells. Our findings uncover an important mechanism how ubiquitin attachment to a substrate protein is decoded during mitosis. 124 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 88 The inner centromere-shugoshin network prevents chromosomal instability Yuji Tanno, Hiroaki Susumu, Miyuki Kawamura, Yoshinori Watanabe Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo Chromosomal instability (CIN) is a major trait of cancer cells and a potent driver of tumor progression. However, the molecular mechanisms underlying CIN still remain elusive. Here, we show that a number of CIN+ cell lines exhibits impaired centromeric localization of SGO1 and Aurora B, which are the core component of the inner centromere-shugoshin (ICS) network. These defects are linked with the hyperstabilization of kinetochore-microtubule attachment, which is a major cause of chromosome segregation error. The defects in ICS network are caused mostly by the loss of histone H3 lysine-9 tri-methylation at centromeres, and sometimes by a reduction in chromatin-associated cohesin; both pathways separately sustain centromeric SGO1 stability. Remarkably, artificial restoration of the ICS network suppresses chromosome segregation errors in a wide range of CIN+ cells. Thus, dysfunction of the ICS network might be a key mechanism underlying CIN in human tumorigenesis. 125 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 89 Polo-like kinase activation through SPAT-1/Bora phosphorylation in human cells and in C. elegans embryos Yann Thomas1, Luca Cirillo2, Costanza Panbianco2, Lisa Martino1, Nicolas Tavernier1, Lucie Van Hove1, Francoise Schwager2, Thibault Leger3, Anna Santamaria5, Monica Gotta2, 4, Lionel Pintard1 1 Jacques Monod Institute, UMR7592, Paris-Diderot University - CNRS, Paris, France Department of Cellular Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1 rue Michel Servet, Geneva 4, Switzerland. 3 Mass Spectrometry Facility, Jacques Monod Institute, UMR7592, Paris-Diderot University - CNRS, Paris, France 4 Swiss National Centre for Competence in Research Program Chemical Biology, Geneva 1211, Switzerland 5 Biomedical Research Unit in Gynecology, Collserola building, Vall Hebron Research Institute (VHIR) 08035 Barcelona, Spain 2 Mitosis is orchestrated by several protein kinases including cyclin-dependent kinases (Cdks), Pololike kinases (Plks) and Aurora kinases. Despite considerable progress toward understanding the individual function of these protein kinases, how their activity is coordinated in space and time during mitosis is less well understood. We have recently shown that CDK-1 regulates PLK-1 activity during mitosis in C. elegans embryos through multisite phosphorylation of the PLK-1 activator SPAT1 (Tavernier et al. 2015). Early embryos expressing SPAT-1 variants mutated on CDK-1 phosphorylation sites presented severe delays in mitotic entry, mimicking embryos lacking spat-1 or plk-1 function. We further showed that SPAT-1 phosphorylation by CDK-1 promotes its binding to PLK-1 and stimulates PLK-1 phosphorylation on its activator T-loop by Aurora A kinase. In human cells, Bora, the ortholog of SPAT-1, regulates several aspects of mitosis (Bruinsma et al. 2014, Chan et al 2008, Macůrek et al. 2008, Seki et al. 2008. Although phosphorylation of Bora is not strictly required to activate Plk1 (Seki et al. 2008, Tavernier et al. 2015), we found that Cdk1 phosphorylation of Bora greatly increases Plk1 activation in vitro. We have now mapped the Cdk1 phosphorylation sites of Bora. Based on the results obtained in C. elegans, we have mutated a number of N-terminal sites and found that Bora ability to activate Plk1 is greatly reduced. We are currently testing the function of these sites in human cells. Our data will help to shed light on the activation mechanisms of Plk1 by Bora. Since Plk1 is overexpressed in many cancers, understanding the precise mechanisms of activation of this kinase will help the design of anti-cancer drugs. References: • Tavernier et al. JCB 2015 • Bruinsma W. et al. J Cell Sci. 2014 • Chan EH. et al. Chromosoma. 2008 • Macůrek L. et al. Nature. 2008 • Seki A. et al. Science. 2008 126 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 90 The budding yeast cell cycle without Cdc14 phosphatase Sandra Touati, Frank Uhlmann The Francis Crick Institute Cell cycle progression is driven by a succession of phosphorylation and dephosphorylation events, mediated by the interplay of kinases and phosphatases. Correct balance of kinase/phosphatase activity is essential to order the cell cycle events. In G1/S-phase, low CDK (cyclin dependent kinases) activity permits accurate DNA replication, whereas high Cdk activity in G2/M is crucial for mitotic entry. The metaphase-to-anaphase transition is a key event of the cell cycle where kinase/phosphatase ratio is gradually reversed. Indeed, mitotic cyclins are degraded, CDK activity is inhibited and phosphatases dephosphorylate CDK substrates. In budding yeast, the main mitotic phosphatase is Cdc14. In anaphase, Cdc14 is released from the nucleolus into the nucleus and cytoplasm and counteracts CDK activity. Substrate dephosphorylation allows anaphase regulation, cytokinesis and mitotic exit. Accordingly, cdc14-1 cells are arrested in anaphase. Glc7 (PP1) and PP2A are also indispensable for cell cycle progression. Indeed, glc7-ts mutants lead to lethality and the cell cycle is highly perturbed upon deletion of either catalytic subunit of PP2A or deletion of the regulatory subunits Cdc55 (B55) and Rts1 (B56). In mammalian cells, Cdc14 is dispensable for mitotic exit and PP2A-B55 seems to be the main phosphatase driving CDK substrate dephosphorylation. In budding yeast, the predominance of Cdc14 could hide fundamental roles for Glc7 and PP2A. Accordingly, we propose to study their roles and substrates after metaphase in a context where Cdc14 is absent. We also intend to investigate if the mitotic arrest observed in Cdc14 depleted cells may be overcome by the modulation of the Glc7, PP2A and expression of its regulatory subunits. 127 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 91 Cytokinetic abscission is an acute G1 event Amit Tzur1, Ofir Gershony2, Tal Pe'er1, Meirav Noach-Hirsh1, Natalie Elia2 1 The Mina and Everard Goodman Faculty of Life Sciences and the Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 5290002, Israel 2 Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University, Beer-Sheva, 8410501, Israel Animal cell division ends with the cutting of the microtubule and membrane intercellular bridge connecting the two daughter cells. This process, known as cytokinetic abscission (abscission), is widely regarded as the last step of cytokinesis, i.e., the last step of the cell cycle. Major breakthroughs have been recently achieved, illuminating mechanistic aspects of abscission; however, the timing of abscission with respect to the mammalian cell cycle remains unclear. In this study, we carefully measured the onset and progression of abscission in dividing cells expressing a G1 reporter. We conclude that abscission commences long after cells enter the G1 phase. Affiliating abscission with G1 is beyond semantics since it essentially postulates that the last step of the cell cycle is regulated in, and probably by, the following cycle. 128 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 92 CDK-1 inactivation and PLK-1 gradient cooperate for the sequential assembly of the Nuclear Envelope during mitotic exit. Paola Vagnarelli, Maria Laura Di Giacinto, Ines Castro, Lorena Ligammari Brunel University London Nuclear Envelope (NE) reformation around the segregating chromatin after mitosis is a very controlled process regulated in both space and time. As with many mitotic exit events it is triggered by inactivation of CDK1/cyclinB and by the activation or re-localisation of several protein phosphatases. Thus far PP1 and PP2A have been both implicated in the de-phosphorylation events necessary for NE reorganisation. Moreover, spatial clues contribute to the coordination of the reassembly process. This spatial information is provided by both the chromatin and a gradient of kinases. Recent work has identified Aurora B kinase as a negative regulator of NE reassembly but little is known about the role of other mitotic kinases that demonstrate a gradient distribution within the anaphase cells. Lagging chromosomes incapable of re-joining the main nucleus after mitosis lead to the formation of micronuclei (MN). These chromatin structures present defects in Nuclear Envelope assembly and provide a great tool to study the role of mitotic kinases enriched at the spindle midzone in the formation of NE. Furthermore, MN undergo rupture during the cell cycle and represent a major source of genomic instability. Using human cell lines and following the destiny and the Nuclear Lamina composition of missegregating chromosomes, we have identified PLK-1 as the kinase that negatively regulates the assembly of Nuclear Pores (but not the Lamina). Moreover we show that LaminA reassembly can be triggered solely by the inactivation of CDK-1 independently of any other spatial clue. We therefore propose that both a clock (CyclinB degradation) and a gradient (PLK, AuroraB) contribute to the timely assembly of the Nuclear Envelope after mitosis. 129 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 93 The phosphatase Cdc14 is essential for anaphase spindle elongation Rosella Visintin, Michela Roccuzzo, Clara Visintin European Institute of Oncology Sister chromatid separation requires the dissolution of the cohesin linkages that hold them together, whilst their segregation relies on the activity of the mitotic spindle and spindle-associated proteins. Cleavage of cohesins and CDK inhibition are thought to be sufficient for triggering chromosome segregation. Here we identified a novel essential requirement for anaphase chromosome movement following cohesin cleavage. We showed that, at anaphase onset, the phosphatase Cdc14 and the polo-like kinase Cdc5 are redundantly required to drive spindle elongation. This essential role of Cdc14 is mediated by the FEAR network, a group of proteins that activates Cdc14 at anaphase onset and we suggest that Cdc5 facilitates both Cdc14 activation and CDK inhibition. We further identify the kinesin-5 motor protein Cin8 as a key target of Cdc14. Indeed, of all the known spindleassociated Cdc14 substrates tested, only a non-phosphorylatable allele of Cin8 is capable of bypassing the cdc14cdc5 double mutant arrest. This result suggests that although stabilization of microtubule dynamics and building a stable midzone are an important requirement for anaphase spindle elongation the limiting step for chromosome segregation is the activation of motor proteins of which Cin8 is an example. 130 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 94 Regulation of origin selection is critical for genome integrity Blanca Gomez-Escoda, Pei-Yun Wu Institute of Genetics and Development of Rennes - CNRS The accurate duplication and transmission of genetic information is critical for cell growth and proliferation as well as for development and differentiation. This is ensured in part by multiple layers of regulation of DNA synthesis and checkpoint pathways. One of the key aspects of this process is the selection and activation of the sites of replication initiation, or origins, across the genome. Interestingly, changes in origin usage have been observed during development and in different pathologies, but the physiological consequences of undergoing S phase with specific replication programs remain largely unexplored. To address this question, we have investigated the effect of alterations in replication initiation induced when checkpoint-defective fission yeast cells encounter replication stress, a situation analogous to that found in cancer. In these conditions, we observe major increases in replication initiation clustered in discrete genomic domains. Strikingly, this origin deregulation leads to the generation of single-stranded DNA and ultimately DNA damage specifically at these loci, constituting a major source of genome instability in these cells. Our findings provide evidence that the organization of DNA replication along the chromosomes plays a critical role in genome maintenance and integrity. 131 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 95 Analysis of prematurely condensed chromosomes in chicken DT40 cells Tao Zhang1, Paul Kalitsis1, 2, Damien Hudson1, 2 1 2 Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Victoria 3052, Australia. Department of Paediatrics, University of Melbourne, Parkville, Melbourne, Victoria 3052, Australia. There are two types of condensins in high eukaryotic cells, which play important roles in chromosome condensation. Condensin I is excluded from the nucleus until prometaphase when nuclear membrane is broken down (NEBD); while condensin II remains in the nucleus throughout the whole cell cycle. There is little information on what role, if any, condensins play at the end of anaphase, during which chromosomes need to be decondensed and condensins to be redistributed into their cellular compartments. In our study in chicken DT40 cells, we focused on the transition between anaphase and interphase and in particular what factors are involved in premature chromosome condensation (PCC). Using quantitative live cell imaging in conjunction with 3D reconstruction, we spatially and temporally dissected the role of condensins in G1 synchronized and PCC cells. We found that shortly upon the completion of anaphase, chromatin bound CAP-H (condensin I) signal dramatically decreased from the nucleus. By contrast, CAP-D3 (condensin II) signal remained and gradually increases in interphase. Surprisingly, PCC led to recruitment of both condensin I and condensin II into G1 premature condensed chromatins without NEBD. However, the characteristic axial staining of the scaffold proteins is lost in G1 PCC chromosomes, and structurally they exhibit heightened fragility. PCC replicated chromosomes show mislocalization of the chromosome scaffold protein KIF4A but not TOPOII and SMC2. Our results show that G1 premature condensed chromosomes require both condensin I and condensin II, but not KIF4A. 132 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Poster presentations Poster 96 Fly-FUCCI - a versatile tool for studying cell proliferation in complex tissues Norman Zielke, et al. Deutsches Krebsforschungszentrum (DKFZ) - Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH) Allianz, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany The development of organs and tissues often involves strictly orchestrated lineages, in which only certain cell-types proliferate at a time. The decision to proliferate is highly dependent on signals from surrounding cells and hence one of the future challenges is to study cell proliferation within its microenvironment. The recently introduced FUCCI system (Fluorescent Ubiquitination-based Cell Cycle Indicator) allows the monitoring of cell cycle phasing in living cells. To enable the specific labeling of small subpopulations of cells we have generated a fly-specific FUCCI system (Fly-FUCCI), whose expression can be spatially and temporally controlled. The Fly-FUCCI system is based on E2F1 and Cyclin B, which are sequentially degraded by the E3-ligases CRL4-Cdt2 and APC/C. Simultaneous expression of both Fly-FUCCI probes allows a distinction of all categories of interphase cells: G1 cells are marked by GFP/CFP; cells in S phase are labeled by RFP/YFP and cells in G2 express both markers. To support a broad range of experimental settings we have generated a toolkit of fly lines expressing the Fly-FUCCI probes under control of UASt, UASp and QUAS promoters. We demonstrate that the Fly-FUCCI system is capable of recapitulating the developmentally programmed cell cycle patterns in eye and wing discs. Furthermore, we have applied the Fly-FUCCI method to the stem cell lineage of the adult midgut, which revealed that the terminally differentiated enterocytes re-enter the cycle during regeneration. Altogether, our work demonstrates that the Fly-FUCCI system is a valuable tool for visualizing cell cycle activity during development and tissue homeostasis. 133 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) List of contributors ALPHABETICAL LIST OF CONTRIBUTORS Adamopoulos, Athanassios, 107 Adriaans, Ingrid, 38 AGIUS, Eric, 106 Akopyan, Karen, 86 Aldea, Marti, 4 Alvarez-Fernández, Mónica, 31 Alves-Rodrigues, Isabel, 41 Amin, Priya, 109 Amon, Angelika, 29, 103 Anger, Martin, 12 Araujo, Ana Rita, 39 Argüello-Miranda, Orlando, 40 Ayte, Jose, 41 Bade, Debora, 42 Bakal, Chris, 43 Bánhegyi, Gábor, 75 Barr, Alexis, 43 Barr, Francis, 34 Barral, Yves, 35, 36, 55, 83 Baxter, Jon, 95, 118 Bayer, Mathias, 55 Bayrak, Sibel, 94 Beaven, Robin, 44 Belton, Jon-Matthew, 118 Benaud, Christelle, 45 Biedermann, Sascha, 19 Binarová, Pavla, 79 Blaukopf, Claudia, 28 Blayney, Martyn, 10 Blythe, Shelby, 1 Boens, Shannah, 56 Bogre, Laszlo, 5, 69 Bohlen, Jonathan, 20 Bollen, Mathieu, 46, 56 Bolognesi, Alessio, 36 Bonaiuti, Paolo, 49 Bong, Seoung Min, 78 Bonner, Amanda, 47 BONNET, Frédéric, 106 Borsos, Mate, 13 Brandeis, Michael, 48 Brino, Laurent, 124 Brown, Nicholas, 81 Bru, Samuel, 66 Buellens, Monique, 46 Buffin, Eulalie, 11 Burgoyne, Robert D, 65 Burkhardt, Sabrina, 13 Cadart, Clotilde, 2 Castro, Anna, 33, 89 Castro, Ines, 129 Chiroli, Elena, 49 Ciliberto, Andrea, 49 CIRILLO, Luca, 105 Cladière, Damien, 11 Clotet, Josep, 66 Cohen, Paula, 13 Collart, Clara, 17 Convay, Max, 110 Coudreuse, Damien, 50 Csikasz-Nagy, Attila, 4 Cundell, Michael, 34 Cuylen, Sara, 28 Davey, Norman, 67 de Boer, Rudolf, 84 de los Santos-Velázquez, Ana Isabel, 99 De Munter, Sofie, 46 de Souza, Edmarcia Elisa, 122 de Vries, Elisabeth, 84 Deák, Péter, 82, 100 de-Carvalho, Jorge, 25 Dekker, Job, 118 Delgado-Barea, María, 51 Deli, Márta, 90 Deneke, Victoria, 22 Denoth-Lippuner, Annina, 83 Deshpande, Ojas, 25 Desvoyes, Bénédicte, 51 Di Giacinto, Maria Laura, 129 Di Talia, Stefano, 1, 22 Dick, Amalie, 52 Ditte, Peter, 53 Dörner, Peter, 19 Doxsey, Stephen, 122 Dozier, Christine, 92 Ducommun, Bernard, 88 Echard, Arnaud, 54 Edgar, Bruce, 20 Eichele, Gregor, 53 El Yakoubi, Warif, 11 Elbatsh, Ahmed, 116 Elder, Kay, 10 Elia, Natalie, 128 Ellenberg, Jan, 28 Endicott, Jane, 81 Farcas, Ana-Maria, 55 Fardilha, Margarida, 56 Ferreira, Monica, 56 Foltman, Magdalena, 57 Françoise, SCHWAGER, 105 Froment, Carine, 92 Frongia, Céline, 88 134 Gallego, Carme, 4 Galli, Matilde, 59 Gérard, Claude, 50 Gerlich, Daniel W., 28, 52 Gershony, Ofir, 128 Giakoumakis, Nickolaos Nikiforos, 101 GIET, Régis, 58 Girard, Juliet, 32 Glover, David, 110 Godfrey, Molly, 60 Godwin, Jonathan, 13 Gomes, Aurélie, 88 Gomez-Escoda, Blanca, 131 Gotta, Monica, 126 Groenewold, Vincent, 42 Gross, Fridolin, 49 Großhans, Jörg, 61 Gruneberg, Ulrike, 30, 63, 91 Guerra-Moreno, Angel, 41 Gueydon, Elisabeth, 14 Gutierrez, Crisanto, 18, 51 Haarhuis, Judith, 116 Haering, Christian, 62 Halada, Petr, 79 Harashima, Hirofumi, 19 Hawley, R. Scott, 47 Haynes, Lee, 65 Hayward, Daniel, 63 Hehnly, Heidi, 122 Heim, Andreas, 64 Helassa, Nordine, 65 Hellmuth, Susanne, 113 Hengeveld, Rutger, 84 Henriques, Rossana, 5 Hernández-Ortega, Sara, 66 Hertz, Emil Peter Thrane, 67 Hidalgo, Elena, 41 Hirai, Kazuyuki, 68 Hiraoka, Daisaku, 8 Hirota, Takayuki, 13 Hiruma, Yoshitaka, 107 Hochegger, Helfrid, 109 Hofmann, Kay, 124 Holubcova, Zuzana, 10 Horváth, Anna, 70 Horváth, Beatrix, 69, 90 Hotz, Manuel, 35 Huang, Jin, 46 Hudson, Damien, 132 Hutter, Lukas, 52, 71, 97 Hyman, Anthony A., 28 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Jang, Young-Joo, 78 JÁTIVA, SORAYA, 74 Jiménez, Javier, 66 Jonak, Katarzyna, 72 Joseph, Stephy, 73 Juanes, Maria Angeles, 36 Kalitsis, Paul, 132 Kapuy, Orsolya, 75 Karasu, Mehmet Erman, 76 Kataria, Meghna, 77 Kawamura, Miyuki, 125 Keeney, Scott, 76 Kermi, Chames, 16 Kies, Matthias, 123 Kim, Jae Hyeong, 78 Kim, Kyungtae, 78 Kirschner, Marc, 85 Kishimoto, Takeo, 8 Klein, Carlo, 62 Kleiss, Charlotte, 124 Klinkert, Kerstin, 54 Kobarg, Jörg, 122 Koff, Andrew C, 76 Kohoutová, Lucie, 79 Kõivomägi, Mardo, 3 Komaki, Shinichiro, 80 Konietzny, Anja, 64 Kops, Geert, 42, 107 Korcsmáros, Tamás, 75 Korolchuk, Svitlana, 81 Kourová, Hana, 69, 79 Kovács, Anita, 90 Kovács, Levente, 82, 100 Kruitwagen, Tom, 83 Krupina, Ksenia, 124 Kruse, Thomas, 67 Kuijt, Timo, 107 Kurucz, Anita, 75 Le Dez, Gaelle, 45 Lee, Byung Il, 78 Lee, Chang-Woo, 78 Leger, Thibault, 126 Lengefeld, Jette, 35 Lens, Susanne, 38, 84, 94 Leontiou, Ioanna, 11 Lesage, Bart, 46 Leviczky, Tünde, 90 Li, Victor, 85 Li, Wei, 119 Ligammari, Lorena, 129 Lindqvist, Arne, 86, 120 Lio, Pietro, 110 Liu, Boyang, 61 Liu, Jian, 87 Lobjois, Valérie, 88 Lopez, María Isabel, 51 Lorca, Thierry, 89 Losada, Ana, 27 Lu, Dan, 32 Lygerou, Zoi, 101 Ma, Sheng, 89 Magiera, Magda, 14 Magyar, Zoltán, 5, 90 Maiorano, Domenico, 16 Makarova, Maria, 6 Malumbres, Marcos, 31 Manenti, Stéphane, 92 Mangat, Davinderpreet, 91 Marcellin, Marlene, 92 Marquina, Maribel, 41 Martin, Mathew, 81 Martino, Lisa, 126 Massoni-Laporte, Aurelie, 112 Matsuda, Muneo, 68 Maxouri, Stella, 101 Mayer, Thomas U., 64 Mayor, Federico, 114 Mazzolini, Laurent, 92 McAinsh, Andrew, 96 McCusker, Derek, 112 Meca, Julien, 112 Melbinger, Anna, 22 Mendoza, Manuel, 93 Mengoli, Valentina, 7, 40 Meppelink, Amanda, 94 Merlini, Laura, 36 Meskiene, Irute, 79 Mészáros, Tamás, 69, 79 Miles, Catrina, 95 Millar, Jonathan, 96 Minakuchi, Yohei, 68 Mirkovic, Mihailo, 71, 97 Mironov, Svetlana, 45 Mitteau, Romain, 112 Miyawaki, Atsushi, 15 Mizrak, Arda, 32 Mochida, Satoru, 98 Mohammed, Binish, 90 Mohammed, Shabaz, 34 MOLINA-DELGADO, Angie, 106 Molist, Iago, 57 Momen Roknabadi, Amir, 1 Mondésert, Odile, 88 Monje-Casas, Fernando, 99 Monnier, Sylvain, 2 Mora-Santos, Maria Del Mar, 96 Moreno, David, 4 Morgan, David, 32, 59 Müller-Reichert, Thomas, 28 Müllers, Erik, 120 Nagai, Takeharu, 98 135 List of contributors Nagy, Ágota, 100 Nagy, Olga, 82, 100 Nagy, Szilvia, 69 Nathanailidou, Patroula, 101 Nebreda, Angel, 102 Nemeth, Edit, 69 Neumann, Beate, 28 Neumann, Heinz, 83 Neurohr, Gabriel, 103 Nilsson, Jakob, 67 Nitzan, Mor, 48 Noach-Hirsh, Meirav, 128 Noble, Martin, 81 Novák, Béla, 43, 50, 52, 71, 97, 113 Novak, Zsofia, 104 Nunes Bastos, Ricardo, 34 O'Farrell, Patrick H, 21 Ohkura, Hiro, 44 Ohsugi, Miho, 121 Oliferenko, Snezhana, 6 Oliveira, Raquel, 71, 97 Olsen, Jesper Velgaard, 67 Otero, Sofía, 51 Pachis, Spyridon, 107 Pál, Margit, 82, 100 Panbianco, Costanza, 126 Pane, Attilio, 119 Papdi, Csaba, 5, 69, 90 Parisi, Eva, 4 Pe'er, Tal, 128 Penela, Petronila, 114 Pentakota, Satyakrishna, 108 Perez, Arina Marina, 122 Perrakis, Anastassis, 107 Peter, Nisha, 109 Peters, Jan-Michael, 26 Petit, Julie, 14 Pettkó-Szandtner, Aladár, 5 Piatti, Simonetta, 36 Piel, Matthieu, 2 Pintard, Lionel, 126 PITUELLO, Fabienne, 106 Poch, Olivier, 124 Ponsioen, Bas, 38 Popescu, Octavian, 82 Poser, Elena, 34 Poser, Ina, 28 Prieto, Susana, 16 Prigent, Claude, 45 Przewloka, Marcin, 110 Qian, Junbin, 46 Queralt, Ethel, 111 Raff, Jordan, 104 Rapali, Péter, 112 Rapsomaniki, Anna Maria, 101 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) Rata, Scott, 113 Reboutier, David, 45 Recolin, Benedicte, 16 Reglero, Clara, 114 Ricco, Natalia, 66 Rivas, Verónica, 114 Robert, Perle, 89 Roccuzzo, Michela, 130 Rodriguez-Rodriguez, Jose Antonio, 111 Rojas, Julie, 115 Rowland, Benjamin, 116 Royou, Anne, 117 Ruiz-Torres, Miguel, 27 Sacristan, Carlos, 107 Saitou, Mitinori, 13 Sajman, Julia, 48 Sanchez-Diaz, Alberto, 57 Sanchez-Perez, Gabino, 69 Santamaria, Anna, 105, 126 Santos, Silvia, 23, 39 Sanz-Castillo, Belén, 31 Sanz-Flores, María, 31 Schalbetter, Stephanie, 118 Scheres, Ben, 69 Schiklenk, Christoph, 62 Schiltz, Odile, 92 Schmoller, Kurt, 3 Schmucker, Stephane, 124 Schnittger, Arp, 19, 80 Schoonen, Pepijn, 84 Schuh, Melina, 10 Schupbach, Trudi, 119 Schwager, Francoise, 126 Schwob, Etienne, 14 Sheriff, Rahuman, 39 Shermoen, Antony W, 21 Shim, Jaegal, 78 Sigurdsson, Jón Otti, 67 Silio, Virginia, 96 Silva Cascales, Helena, 120 Skotheim, Jan, 3 Smetana, Juliana, 122 Smith, Jim, 17 Soeda, Shou, 121 Sprenger, Frank, 123 Stanley, Will, 81 Stemmann, Olaf, 113 Sumara, Izabela, 124 Sung, Hung-wei, 61 Susumu, Hiroaki, 125 Suzuki, Haruka, 68 Sveiczer, Ákos, 70 Szeker, Kathelijne, 56 Tachibana-Konwalski, Kikue, 13 Takahashi, Naoki, 19 Takaki, Kaori, 98 Talarek, Nicolas, 14 Tanno, Yuji, 125 Tapon, Nicolas, 24 Taraviras, Stavros, 101 Tavernier, Nicolas, 126 Teixeira, Antoinette, 42 Telley, Ivo, 25 Thomas, Yann, 126 Tollis, Sylvain, 112 Torok, Katalin, 65 Touati, Sandra, 127 Toyoda, Atsushi, 68 Tsanov, Nikolay, 16 Turner, Jonathan, 3 Tyson, John, 50 Tzur, Amit, 128 Ubbink, Marcellus, 107 136 List of contributors Udvardy, Andor, 82 Uhlmann, Frank, 60, 77, 127 Umeda, Masaaki, 19 Vagnarelli, Paola, 129 Vallot, Antoine, 11 van Castelmur, Eleonore, 107 van der Laan, Siem, 16 Van Eynde, Aleyde, 56 Van Hove, Lucie, 126 van Vugt, Marcel, 84 Vaz Meirelles, Gabriela, 122 Vergassola, Massimo, 22 Vietri, Marina, 37 Vigneron, Suzanne, 89 Visintin, Clara, 130 Visintin, Rosella, 130 Volc, Jindřich, 79 Wang, Jin, 62 Wassmann, Katja, 11 Watanabe, Yoshinori, 9, 125 Weimer, Annika, 19 Wieschaus, Eric, 1 Wilkins, Brian, 83 Winkler, Franziska, 61 Wu, Pei-Yun, 131 Xiang, Jinyi, 20 Yahya, Galal, 4 Yu, Miao, 95 Yu, Xueyang, 20 Yuan, Kai, 21 Zachariae, Wolfgang, 7, 40, 72, 115 Zagoriy, Ievgeniia, 7, 40 Zegerman, Philip, 17 Zenvirth, Drora, 48 Zhang, Tao, 132 Zhang, Tongli, 43 Zielke, Norman, 20, 133 EMBO Cell Cycle Workshop, Budapest, Hungary (2015) List of participants LIST OF PARTICIPANTS ADRIAANS, Ingrid UMC Utrecht Universiteitsweg 100 3584 CG, Utrecht, Netherlands [email protected] BARRAL, Yves ETH Zuerich Institute of Biochemistry IBC HPM D12.2 8093, Zuerich, Switzerland [email protected] ALDEA, Marti Molecular Biology Institute of Barcelona, CSIC Baldiri Reixac 15 3-A16 08028, Barcelona, Spain [email protected] BEAVEN, Robin University of Edinburgh Michael Swann Building EH9 3JR, Edinburgh, United Kingdom [email protected] ALVAREZ-FERNÁNDEZ, Mónica Spanish National Cancer Research Centre (CNIO) C/ Melchor Fernandez Almagro, 3 28029, Madrid, Spain [email protected] AMON, Angelika Koch Institute/HHMI/MIT 77 Mass Ave 02139, Cambridge, United States [email protected] ANGER, Martin Masaryk University Department of histology and embryology Kamenice 3 Brno, Czech Republic [email protected] ARAUJO, Ana Rita MRC-Clinical Sciences Centre, Imperial College London B409, 80 Wood Lane W12 0BZ, London, United Kingdom [email protected] ARGUELLO-MIRANDA, Orlando Max Planck Institute Roentgen Str 1a 82152, Planegg, Germany [email protected] AYTE, Jose Universitat Pompeu Fabra Doctor Aiguader 88 08003, Barcelona, Spain [email protected] BADE, Debora UMC Utrecht Universiteitsweg 100 3584 CG, Utrecht, Netherlands [email protected] BARR, Alexis Institute of Cancer Research Chester Beatty Laboratories SW3 6JB, London, United Kingdom [email protected] BENAUD, Christelle IGDR-UMR6290 2 av Leon Bernard 35043, Rennes, France [email protected] BOGRE, Laszlo Royal Holloway University of London Egham High Hill TW20 0EX, Egham, United Kingdom [email protected] BOLLEN, Mathieu KU Leuven Campus Gasthuisberg 0&N1 3000, Leuven, Belgium [email protected] BONNER, Amanda Stowers Institute for Medical Research 1000 East 50th Street 64111, Kansas City, United States [email protected] BRANDEIS, Michael The Hebrew University of Jerusalem Safra Campus 9190401, Jerusalem, Israel [email protected] CADART, Clotilde Institut Curie 12 rue Lhomond 75007, Paris, France [email protected] CASTRO, Anna CRBM-CNRS 1919 route de Mende 34293, Montpellier, France [email protected] CILIBERTO, Andrea IFOM, Milan Via Adamello 16 Milan, Italy [email protected] COUDREUSE, Damien Institute of Genetics and Development of Rennes 2, avenue du Pr. Léon Bernard 35043, Rennes, France [email protected] 137 CUNDELL, Michael University of Oxford, Department of Biochemistry South Parks Road OX13QU, Oxford, United Kingdom [email protected] CUYLEN, Sara Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) Dr.-Bohr Gasse 3 1030, Vienna, Austria [email protected] DE SOUZA, Edmarcia Elisa National Center For Research in Energy and Materials Giuseppe Máximo Scolfaro 10000 13083-970, Campinas, Brazil [email protected] DESHPANDE, Ojas Instituto Gulbenkian de Ciencia Rua Quinta Grande 6 Oeiras, Portugal [email protected] DESVOYES, Bénédicte Centro de Biología Moleculal Severo Ochoa, CSIC calle Nicolas Cabrera,1 28049, Madrid, Spain [email protected] DI TALIA, Stefano Duke University Medical Center Nanaline Duke Building 27710, Durham, United States [email protected] DICK, Amalie IMBA - Institute of Molecular Biotechnology GmbH Dr. Bohr-Gasse 3 1020, Wien, Austria [email protected] DITTE, Peter Max Planck Institute for Biophysical Chemistry Am Faßberg 11 Göttingen, Germany [email protected] ECHARD, ARNAUD INSTITUT PASTEUR Membrane Traffic and Cell Division Lab 28 RUE DU DOCTEUR ROUX 75724, PARIS CEDEX 15, France [email protected] EDGAR, Bruce University of Heidelberg Im Neuenheimer Feld 282 Heidelberg, Germany [email protected] EMBO Cell Cycle Workshop, Budapest, Hungary (2015) List of participants FARCAS, Ana-Maria Institute of Biochemistry, ETH Zurich Otto-Stern Weg 3 8093, Zurich, Switzerland [email protected] HELASSA, Nordine University of Liverpool Crown Street L69 3BX, Liverpool, United Kingdom [email protected] JOSEPH, Stephy University of Sussex Genome damage and stability centre BN1 9RH, Brighton, United Kingdom [email protected] FERREIRA, Monica KU Leuven Campus Gasthuisberg 3000, Leuven, Belgium [email protected] HERNÁNDEZ-ORTEGA, Sara Universitat Internacional de Catalunya Josep Trueta s/n 08195, Sant Cugat del Vallès, Spain [email protected] KAPUY, Orsolya Semmelweis University Tűzoltó utca 37-47 1094, Budapest, Hungary [email protected] GALLI, Matilde UCSF 600 16th Street 94158, San Francisco, United States [email protected] HERTZ, Emil Peter Thrane Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen Blegdamsvej 3b 2200, Copenhagen, Denmark [email protected] GIET, Régis Université of Rennes 1-CNRS 2, Avenue du Pr Leon bernard 35043, RENNES, France [email protected] GODFREY, Molly The Francis Crick Institute 44 Lincoln's Inn Fields WC2A 3LY, London, United Kingdom [email protected] GROHANS, Jörg University of Göttingen Justus-von-Liebig Weg 11 37077, Göttingen, Germany [email protected] GRUNEBERG, Ulrike University of Oxford South Parks Road OX1 3RE, Oxford, United Kingdom [email protected] GUTIERREZ, Crisanto Centro de Biologia Molecular Severo Ochoa, CSIC-UAM Nicolas Cabrera 1 Madrid 28049, Spain [email protected] HAERING, Christian EMBL Meyerhofstr. 1 69117, Heidelberg, Germany [email protected] HIRAI, Kazuyuki Kyorin University School of Medicine 6-20-2 181-8611, Mitaka, Japan [email protected] HORVATH, Beatrix Royal Holloway, University of London Egham TW20 OEX, Egham, United Kingdom [email protected] HORVÁTH, Anna Budapest University of Technology and Economics, Department of Applied Biotechnology and Food Science Szent Gellért tér 4. H-1111, Budapest, Hungary [email protected] HUNT, Tim Cancer Research UK Rose Cottage EN6 3LH, Potters Bar, United Kingdom [email protected] HUTTER, Lukas Department of Biochemistry/ University of Oxford Lincoln College OX1 3DX, Oxford, United Kingdom [email protected] HAYWARD, Daniel University of Oxford Sir William Dunn School of Pathology OX1 3RF, Oxford, United Kingdom [email protected] JÁTIVA, SORAYA BELLVITGE INSTITUTE OF BIOMEDICAL RESEARCH (IDIBELL) Av. Gran Via de L'Hospitalet 199-203 08908, L'Hospitalet de Llobregat, Spain [email protected] HEIM, Andreas University of Konstanz Universitätsstraße 10 78457, Konstanz, Germany [email protected] JONAK, Katarzyna Max Planck Institute for Biochemistry Am Klopferspitz 18 82152, Martinsried/Munich, Germany [email protected] 138 KARASU, Mehmet Erman MSKCC- Gerstner Sloan Kettering Graduate School 1233 York Ave Apt 16 B 10065, New York, United States [email protected] KATARIA, Meghna The Francis Crick Institute London WC2A 3LY, United Kingdom [email protected] KIM, Kyungtae National Cancer Center, Research Institute 323 Ilsan-ro, Ilsandong-gu, 410-769, Goyang-si, Korea, Republic of (South) [email protected] KISHIMOTO, Takeo Ochanomizu University Ootsuka 2-1-1 112-8610, Bunkyo-ku, Japan [email protected] KOHOUTOVÁ, Lucie Institute of Microbiology AS CR, v. v. i. Videnska 1083 142 20, Prague, Czech Republic [email protected] KOMAKI, Shinichiro University of Hamburg Ohnhorststr. 18 22609, Hamburg, Germany [email protected] KOROLCHUK, Svitlana Newcastle University NICR NE24HH, Newcastle upon Tyne, United Kingdom [email protected] KOVÁCS, Levente Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences Temesvári krt 6726, Szeged, Hungary [email protected] EMBO Cell Cycle Workshop, Budapest, Hungary (2015) List of participants KRUITWAGEN, Tom Institute of Biochemistry, ETH Zurich Otto-Stern-Weg 3 8093, Zurich, Switzerland [email protected] MANGAT, Davinderpreet University of Oxford 16 vicarage farm road TW3 4NW, London, United Kingdom [email protected] NAGY, Ágota University of Szeged Közép Fasor 6726, Szeged, Hungary [email protected] LEGATE, Kyle Nature Communications 4 Crinan Street London, United Kingdom [email protected] MAZZOLINI, Laurent CNRS 2, avenue Hubert Curien 31037, Toulouse, France [email protected] NATHANAILIDOU, Patroula University of Patras Telou Agra 6A 26442, Patra, Greece [email protected] LENS, Susanne UMC Utrecht Universiteitsweg 100 3584 CG, Utrecht, Netherlands [email protected] MENDOZA, Manuel Center for Genomic Regulation (CRG) C/ Dr Aiguader 88 08003, Barcelona, Spain [email protected] NEBREDA, Angel IRB Barcelona C/ Baldiri Reixac 10 08028, Barcelona, Spain [email protected] LI, Victor Harvard Medical School 200 longwood Avenue 02115, Boston, United States [email protected] MEPPELINK, Amanda University Medical Center Utrecht Universiteitsweg 100 3584 CG, Utrecht, Netherlands [email protected] NEUROHR, Gabriel Massachusetts Institute of Technology 68 Wells Rd Lincoln, United States [email protected] LINDQVIST, Arne Karolinska Institutet von eulers v 3 171 77, Stockholm, Sweden [email protected] MILES, Catrina Genome damage and Stability center (Sussex) Science Park road BN1 9QG, Falmer, United Kingdom [email protected] NOVAK, Bela Department of Biochemistry/University of Oxford South Parks Road OX1 3QU, Oxford, United Kingdom [email protected] MILLAR, Jonathan University of Warick Gibbet Hill Road Coventry, United Kingdom [email protected] NOVAK, Zsofia Dunn School of Pathology, University of Oxford South Parks Road OX1 3RE, Oxford, United Kingdom [email protected] LIU, Jian NIH 50 South Drive, Room 3306 Bethesda, United States [email protected] LOBJOIS, Valerie CNRS-ITAV USR3505 1 place Pierre POTIER 31106, TOULOUSE, France [email protected] LORCA, Thierry CNRS 1919 route de mende 34293, Montpellier, France [email protected] LOSADA, Ana Spanish National Cancer Research Centre Melchor Fernandez Almagro 3 E28029, Madrid, Spain [email protected] MAGYAR, ZOLTÁN Institute of Plant Biology, Biological Research Centre, Szeged TEMESVÁRI KRT 62 6726, SZEGED, Hungary [email protected] MAIORANO, Domenico Institute of Human Genetics. CNRSUPR1142. rue de la cardonille 34396 Cedex 5, Montpellier, France [email protected] MIRKOVIC, Mihailo Instituto Gulbenkian de Ciencia Rua Quinta das Palmeiras 45,5f 2780-156, Oeiras, Portugal [email protected] MIYAWAKI, Atsushi RIKEN 2-1 Hirosawa, Wako Saitama, Japan [email protected] MOCHIDA, Satoru Kumamoto university Kyoyotou-Honjo 1 860-0811, Kumamoto, Japan [email protected] MONJE-CASAS, Fernando University of Seville CABIMER. Avda. Americo Vespucio, s/n Seville, Spain [email protected] MORGAN, David University of California, San Francisco UCSF Genentech Hall, Room N312B 94158, San Francisco, United States [email protected] 139 O'FARRELL, Patrick UCSF 600-16th Street 94153, San Francisco, United States [email protected] OLIFERENKO, Snezhana King's College London New Hunt's House SE1 1UL, London, United Kingdom [email protected] PACHIS, Spyridon UMC Utrecht Vredenburg 28C 3511 BC, Utrecht, Netherlands [email protected] PENTAKOTA, satya krishna Max planck institute of Molecular physiology AMRODE 44149, DORTMUND, Germany [email protected] PETER, Nisha Genome Damage and Stability Centre, University of Sussex Science Park Road BN2 4HY, Brighton, United Kingdom [email protected] EMBO Cell Cycle Workshop, Budapest, Hungary (2015) PETERS, Jan-Michael Research Institute of Molecular Pathology Dr. Bohr-Gasse 7 1030, Vienna, Austria [email protected] PIATTI, Simonetta CNRS 1919 Route de Mende 34293, Montpellier, France [email protected] PINTARD, Lionel CNRS 15 rue hélène Brion 75013, PARIS, France [email protected] PITUELLO-BERNIERE, Fabienne Centre de Biologie du Développement UMR5547 CNRS/UPS 118 rte de Narbonne Bât 4R3 31062 cedex 09, Toulouse, France [email protected] PRZEWLOKA, Marcin University of Cambridge Department of Genetics CB2 3EH, Cambridge, United Kingdom [email protected] QUERALT, Ethel Instituto de Investigaciones Biomédicas de Bellvitge (IDIBELL) Avenida Gran via de L'Hospitalet 199203 08908, Barcelona, Spain [email protected] RAPALI, Peter Institut de Biochimie et Génétique Cellulaires, France 14 Rue du Haut Carre 33400, Talence, France [email protected] RATA, Scott Department of Biochemistry, University of Oxford Keble College OX1 3PG, Oxford, United Kingdom [email protected] REGLERO, Clara Centro de Biología Molecular Severo Ochoa (CBMSO) - Universidad Autónoma de Madrid (UAM) C/ Nicolás Cabrera, 1 28049, Madrid, Spain [email protected] ROJAS, Julie Max Planck Institute of Biochemistry Am Klopferspitz 18 82152, Martinsried, Germany [email protected] List of participants ROWLAND, Benjamin The Netherlands Cancer Institute Plesmanlaan 121 1066 CX, Amsterdam, Netherlands [email protected] SOEDA, Shou The University of Tokyo 3-8-1 Komaba 153 8902, Meguro, Japan [email protected] ROYOU, Anne IECB/CNRS 2 rue Robert Escarpit 33607, PESSAC, France [email protected] SPRENGER, Frank University of Regensburg Universitaetsstrasse 31 93053, Regensburg, Germany [email protected] SANCHEZ-DIAZ, ALBERTO UNIVERSITY OF CANTABRIA INSTITUTO DE BIOMEDICINA Y BIOTECNOLOGIA DE CANTABRIA 39012, SANTANDER, Spain [email protected] SUMARA, Izabela IGBMC 1, rue Laurent Fries 67400, Illkirch, France [email protected] SANTOS, Silvia MRC-Imperial College London Clinical Sciences Centre W12 0NN, London, United Kingdom [email protected] SCHALBETTER, Stephanie GDSC, University of Sussex Science Park Road Brighton, United Kingdom [email protected] SCHMOLLER, Kurt Stanford University 337 Campus Drive Stanford, United States [email protected] SCHNITTGER, Arp University of Hamburg Ohnhorststr. 18 22609, Hamburg, Germany [email protected] SCHUH, Melina MRC Laboratory of Molecular Biology Francis Crick Avenue CB2 0QH, Cambridge, United Kingdom [email protected] SCHUPBACH, Trudi Princeton University Washington Road 08544, Princeton, United States [email protected] SCHWOB, Etienne CNRS Institute of Molecular Genetics IGMM CNRS 34293, Montpellier, France [email protected] SILVA CASCALES, Helena Karolinska Institutet Ölandsgatan 52 116 63, Stoclholm, Sweden [email protected] 140 TACHIBANA-KONWALSKI, Kikue IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences Dr. Bohr Gasse 3 1030, Vienna, Austria [email protected] TANNO, Yuji IMCB, University of Tokyo Yayoi 113-0032, Bunkyo-ku, Tokyo, Japan [email protected] TAPON, Nicolas Cancer Research UK London Research Institute 5 Hartswood Road W12 9NQ, London, United Kingdom [email protected] THOMAS, Yann Institut Jacques Monod/ Universite Paris Diderot 15 rue Helene Brion 75205, Paris, France [email protected] TOUATI, Sandra CRICK 44 Lincoln's Inn Fields London, United Kingdom [email protected] TZUR, Amit Bar-Ilan Keren Hayesod 15/a Givat Shmuel, Israel [email protected] UHLMANN, Frank The Francis Crick Institute 44 Lincoln's Inn Fields WC2A 3LY, London, United Kingdom [email protected] VAGNARELLI, Paola Brunel University London Heinz Wolff Building UB8 3PH, Uxbridge, London, United Kingdom [email protected] EMBO Cell Cycle Workshop, Budapest, Hungary (2015) VIETRI, Marina 2Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital Montebello 0379, Oslo, Norway [email protected] VISINTIN, Rosella European Institute of Oncology Via Adamello, 16 Milan, Italy [email protected] WASSMANN, Katja IBPS- Institut de Biologie Paris Seine 9 quai St. Bernard 75005, Paris, France [email protected] WATANABE, Yoshinori University of Tokyo Yayoi 1-1-1, Bunkyo-ku 113-0032, Tokyo, Japan [email protected] WIESCHAUS, Eric Princeton University 435 Moffett Laboratory 08544, Princeton, United States [email protected] WU, Pei-Yun Jenny Institute of Genetics and Development of Rennes - CNRS 2 av. du Pr. Léon Bernard 35043, Rennes, France [email protected] ZACHARIAE, Wolfgang Max Planck Institute of Biochemistry Am Klopferspitz 18 D-82152, Martinsried, Germany [email protected] ZEGERMAN, Philip Gurdon Institute, University of Cambridge Tennis Court Road Cambridge, United Kingdom [email protected] ZHANG, Tao Murdoch Childrens Research Institute Royal Childen Hospital 3052, Melbourne, Australia [email protected] ZIELKE, Norman DKFZ Im Neuenheimer Feld 282 69120, Heidelberg, Germany [email protected] 141 List of participants PREVIOUS EUROPEAN CELL CYCLE CONFERENCES Year Location Organizer 1st 1969 Copenhagen (Denmark) Erik Zeuthen 2nd 1972 Innsbruck (Austria) Wilhelm Sachensenmaier 3rd 1974 Zürich (Switzerland) Gerhard Braun 4th 1976 Edinburgh (Scotland) Murdoch Mitchison 5th 1978 Madrid (Spain) Jorge Lopez-Saez 6th 1981 Prague (Czechoslovakia) Eva Streiblova 7th 1984 Heidelberg (Germany) Christian Petzelt 8th 1987 Bath (England) Allan Wheals 9th 1990 Stockholm (Sweden) Anders Zetterberg 10th 1993 La Rochelle (France) Christian Petzelt 11th 1997 Gardone (Italy) Lilia Alberghina 12th 2001 Mayerhofen (Austria) Wilhelm Sachsenmaier 13th 2004 Salamanca (Spain) Sergio Moreno 14th 2007 Spetses (Greece) Stathis Gonos 15th 2011 Montpellier (France) Etienne Schwob 16th 2015 Budapest (Hungary) Béla Novák & Frank Uhlmann registration desk MAP OF THE HOTEL GROUND FLOOR Stairs to Auditorium Jázmin corridor Platán