Download Imaging cytoskeletal filament organization at the molecular scale

MARIE SKLODOWSKA-CURIE ACTIONS
Co-funding of regional, national and international programmes (COFUND)
DOC2AMU PROJECT 2017 CALL FOR APPLICATIONS
Imaging cytoskeletal filament organization at the molecular scale
1. DESCRIPTION OF THE PHD THESIS PROJECT
1.1 OBJECTIVES OF THE PROJECT BASED ON THE CURRENT STATE OF THE ART
Cell division is a fundamental biological process that is essential for multiple aspects of animal physiology,
including cell proliferation and differentiation, tissue growth and repair. Its dysfunction leads to several human
pathologies, including tumour development and tissue degeneration. Cell division involves strong cell shape
changes and thus heavily relies on the redistribution and cooperation of force-generating cytoskeletal
filamentous proteins, notably actin filaments and microtubules (Fig.1). While the requirement of actin and
microtubules for successful cell division is not questioned, their precise organization and interaction with other
proteins is still poorly understood. In particular, little is known about the functional contribution of septins, a
family of proteins which was recently recognized as a novel component of the animal cytoskeleton. Septins are
essential for the successful completion of cell division: septin depletion leads to unstable and delayed furrow
formation, furrow regression as well as failure to cleave the intercellular bridge. Importantly, septins form
protomers which can polymerize into filaments (Fig.1), and they also associate with actin and with microtubules.
Thus, a complete understanding of how cytoskeletal filaments contribute to biological functions in cell division
and more generally cell shape changes requires a deeper knowledge of how septins, actin filaments and
microtubules organize with respect to each other, which is still lacking.
This project aims at developing optical and biological tools to decipher the organization of septins and their
interaction with actin and microtubules during cell division. The topic addressed is highly interdisciplinary in cosupervision between I. Fresnel (ED 352 Physique et Sciences de la Matière) and Centre de Recherche en
Cancérologie de Marseille (CRCM) (ED 062 Sciences de la Vie et de la Santé). Key open questions will be
addressed: first, there is no direct evidence that septins form filaments in animal cells. Second, septins exist in
different isoforms: how each isoform differs in terms of organization and function is unknown. Third, it remains
a challenge to elucidate how septins organize with respect to actin and microtubules and to identify the
molecular determinants that localize septins to these two cytoskeletal subsystems.
+
septin protomers actin filaments
+
microtubules
Fig.1. Left : schematic representation of cytoskeletal filaments. Right: assembly at the cytokinetic ring during cell division.
Addressing all three questions ultimately boils downs to the challenge of measuring inter-filament organization
(orientation, spacing, alignment); however, quantitative imaging tools that combine molecular specificity, high
spatial resolution and real-time measurements of filament organization are currently limiting.
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To address these questions, dedicated optical imaging methods will be implemented with the project cosupervisor at I. Fresnel, in collaboration with the intersectorial partner HORIBA, with the goal to measure filament
organization at high spatial resolution and potentially in real time. The project is thus inherently fully integrated
in the research axis "imagerie". These measurements will be implemented using polarized fluorescence imaging,
in which I. Fresnel has developed a recognized expertise [1,2], on two systems: in vitro reconstituted cytoskeletal
filaments from purified components, in collaboration with the international partner (AMOLF, The Netherlands),
and dividing mammalian cells, with the co-supervisor at CRCM. In vitro studies will decipher the behaviour and
local structure of single filaments and of filament assemblies, providing reference signatures for studies in cells.
Functional studies in cells will then be performed at different stages of cell division. The molecular mechanisms
at the origin of inter-filament organization will be investigated using septin mutants impaired for polymerization
or with modified selectivity for actin or microtubules.
Impact. This project is of broad scientific interest and will have impact in multiple fields, including cell and
developmental biology, biophysics and mechanobiology. It further promises to benefit society by advancing our
understanding of the role of septins in human disease. There is compelling evidence on links between septin
dysfunction and infertility, neurological diseases and tumorigenesis. Given that the biological function of septins
is inherently linked to their ability to form filaments, this project should provide new means to assess septin
function in health and disease, thus impacting biomedical research. At last, this project will provide a new imaging
tool of interest for cell biologists, with possible extensions in tissues. The polarized fluorescence module
developed in this project, supported by the company HORIBA which is recognized worldwide in instrumentation,
can ultimately be used as a universal add-on, implementable on a large variety of optical microscopes where
structural imaging is required.
1.2 METHODOLOGY
The methodology of this project will involve a combination of optical instrumentation and biological studies, and
will lead to different tools that will be developed in parallel:
Optical tools. Provided that fluorophores are attached to the studied filaments in a sufficiently rigid manner (see
“Biological tools” section below), a polarized excitation/emission provides knowledge on their orientational
behaviour [1,2]. A new polarized fluorescence imaging tool will be implemented in this project based on
preliminary developments at I. Fresnel [2], which showed that polarization splitting provides fluorophore’s
average orientation and extent of angular fluctuations (Fig. 2). Illuminating the sample under wide field or Total
Internal Reflection (TIRF) and splitting the image into several polarized sub-images will allow fast polarized
detection as well as super-resolution imaging based on the single molecule localization techniques STORM/PALM
(Stochastic Optical Reconstruction /Photoactivation Light Microscopy). Such instrument currently does not exist
on the market of optical microscopy.
c)
a)
d
180°
d
50 nm
40°
Fig.2. a) Single molecule orientation detection: the excitation is circularly polarized and the emission is split into sub-images
of localized single molecules, projected on different polarization states. c) Each single molecule is represented by a stick (its
mean orientation) and a color (its angular fluctuation extent). Adapted from [2].
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To optimize the preliminary demonstration performed at I. Fresnel (Fig. 2), the project will benefit from a
collaboration with the inter-sectorial partner (HORIBA, France) in polarization optics. The design will be adapted
from a previous system of HORIBA initially dedicated to spectroscopy, optimizing signal to noise conditions and
measurement precision. First, ensemble measurements will be performed in real time with diffraction limited
resolution (~200 nm). Super-resolution will then be used to reconstruct nanoscale structural orientational maps
of various interacting filaments, in vitro or in cells at various stages of cell division.
Biological tools. Fluorescent labelling of septin, actin and microtubules will allow multicolour imaging specific to
each filaments. To enable polarized imaging, fluorophores need also be rigidly linked to the filament of interest.
To this end, different molecular biology strategies developed at I. Fresnel will be used to generate custom made
molecular constructs where septins and actin are rigidly linked to green fluorescent protein (GFP) variants,
extending this approach to photoactivatable fluorescent proteins to enable PALM super-resolution imaging.
These customized GFP fusion proteins will be used in fixed cells to probe filament organization at specific time
points in cell division, but will also enable for the first time, the real-time measurements of inter-filament
reorganization in living dividing cells. Cell lines expressing specific septin isoforms developed by the co-supervisor
at CRCM will be used in these studies. To relate measurements in cells to the behavior of single filaments or
filament assemblies of controlled geometries, the student will also reconstitute septins, actin and microtubules
in vitro from purified molecules (collaboration AMOLF). In these assays, polarized fluorescence imaging will be
done using purified septin-GFP fusions and actin and tubulin conjugated to small organic fluorophores.
1.3 WORK PLAN
The work plan of this project is divided into several tasks that cover molecular biology, in vitro reconstitution,
cell biology and optical developments. Publication writing and outreach events are indicated as (P) and (O):
Optics
Biology
M1Development of the polarized imaging setup.
In vitro reconstitution of human septin-isoform specific
M6
Definition and dimensioning of the detection system
filaments, actin filaments and microtubules in controlled
(polarization splitter) (I. Fresnel, HORIBA).
geometries (single filaments, higher-order filament
assemblies and hybrid filament co-assemblies) (CRCM, I.
Fresnel, AMOLF).
M6Extension of the method for two/three colour
M9
imaging. Calibration using fluorescent nanobeads.
Development of the adequate data processing and
analysis programming tools (I. Fresnel) (O).
M9M12
M12M18
Time-lapse and super-resolution polarized imaging of filament co-assembly. Measurement of single fluorophore
mobility and filament organization in vitro, used as a reference for further studies in cells (I. Fresnel, CRCM).
Evaluation of potential for patenting and
Biochemical characterization of cell lines expressing
commercialization of the polarized imaging set-up (I.
different septin isoforms; septin labelling with isoformFresnel, HORIBA).
specific reagents; optimization of expression levels of
the different fluorescently-tagged cytoskeletal filaments
(CRCM).
M18M24
Super-resolution and real time polarized imaging in dividing cells. Measurements of inter-filament organization
at different time-points in cell division (cytokinetic ring assembly, ring constriction, intercellular bridge formation
and abscission) in cell lines expressing different septin isoforms (I. Fresnel, CRCM) (P) (O).
Study of the role of septins in actin/microtubule organization. STORM/PALM-polarized imaging in dividing cells
expressing septin mutants that cannot polymerize or that cannot associate with actin or microtubules (CRCM, I.
Fresnel) (P) (O).
Writing up of thesis manuscript, final publications and defense (P).
M24M30
M30M36
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Dissemination / publications / employability: The results of this project are expected to lead to high impact
publications in international peer-reviewed journals, in which both co-supervisors have already shown their
ability to produce high level work in their respective fields in physics and biology. The interdisciplinary nature of
this project should be highly beneficial for enhancing scientific dissemination. In addition, the frequent visits to
HORIBA will permit the student to expand his/her knowledge on the industrial world, from the design of a new
tool to its validation, its patenting if applicable, and its possible commercialization, thus maximizing employability
in both the academic and industrial sectors. The student will furthermore be active in presenting his/her work at
national and international scientific meetings and workshops. He/She will furthermore be involved in public
outreach activities with presentations at high schools, universities and public events, using tools already in place
at both CRCM and I. Fresnel such as Fête de la Science in Marseille that takes place in October every year.
Feasibility: The project is based on preliminary studies (optical imaging, fluorophore rigid labelling of filaments,
in vitro and cell lines preparations) and technical know-how that already exists at I. Fresnel and CRCM. The level
of risk is thus low on many of its aspects, ensuring that the starting point allows rapid work dissemination for the
student. Moreover, the concept of the optical instrumentation is already in place at HORIBA and the core
equipment (laser/microscope/camera) is already present at I. Fresnel.
1.4 SUPERVISORS AND RESEARCH GROUPS DESCRIPTION
This project is part of a starting collaboration between I. Fresnel and CRCM for the use of novel optical imaging
tools to investigate the organization of septins in cells, which has not been funded yet. It involves new
developments on customized protein constructs (I. Fresnel, molecular biology, biochemistry), on septin isoformspecific cell reagents (CRCM), and on optical imaging developments (I. Fresnel) in a complementary supervision.
In parallel to this project, I. Fresnel collaborates since several years with AMOLF on cytoskeletal filament
reconstitution, funded by PHC Van Gogh exchange programs and an ERC StG (G.Koenderink).
Supervisor 1: Institut Fresnel, Marseille, France. The MOSAIC team of I. Fresnel is dedicated to the development
of optical microscopy and endoscopy tools for bio-imaging, from the single molecule scale to tissues. It is in
particular known for its important advances in nonlinear vibrational microscopy and its leading position in
polarized microscopy, in particular for polarized confocal and super-resolution microscopies which exists only in
a few laboratories around the world. Sophie Brasselet (DR2 CNRS) is an optical physicist, with more than 15 years
of experience in the field of optical microscopy and polarized imaging. She will be in charge of the supervision
for the development of the polarized wide field / TIRF and super-resolution imaging modality, together with data
analysis and processing tools. Manos Mavrakis (CR1 CNRS), a biochemist working on septin and actin biology
who joined I. Fresnel in 2015, will supervise the molecular biology and biochemistry aspects of the project.
Supervisor 2: Centre de Recherche en Cancérologie de Marseille (CRCM). The team of Ali Badache works on
the molecular mechanisms of tumor cell motility dependent on microtubules. It deciphered an original signalling
pathway from the ErbB2 oncogenic tyrosine kinase receptor to proteins interacting with the +end of
microtubules. Pascal Verdier-Pinard (CR1 Inserm) has extensive experience in the pharmacology, biochemistry
and cell biology of microtubules and human septins over the last 20 years. He will supervise the cell biology
aspects of the project.
International partner: FOM Institute AMOLF, The Netherlands. Gijsje Koenderink (Professor and group leader)
is an experimental biophysicist focusing on the physical principles that underlie the self-organization of cells.
She will contribute expertise on the reconstitution of septins with hybrid actin/microtubule networks.
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2. 3I DIMENSIONS AND OTHER ASPECTS OF THE PROJECT
2.1 INTERDISCIPLINARY DIMENSION
The project is highly interdisciplinary by nature, since it involves molecular biology and biochemistry (design of
protein constructs, in vitro reconstitution), cell biology (genetic manipulations and imaging in cell lines), and
optical instrumentation (development of a new super resolution fluorescence microscope). The student will
actively participate in all aspects of this project, being closely supervised by experts in their respective fields in I.
Fresnel, CRCM, AMOLF and HORIBA.
2.2 INTERSECTORAL DIMENSION:
The project will actively involve the company HORIBA which is currently developing polarized detection devices
that are conceptually very close to the detection path needed in the novel microscope described in this project.
Currently the scheme developed by HORIBA is based on a splitter of a signal into several polarization projections,
including both linear and circular states, for the purpose of spectroscopic measurements. In this project, the
scheme will be modified to fit the requirement of wide field/TIRF and super-resolution imaging, which is of high
interest for HORIBA (see attached letter of support). If successful, the system developed in the project should set
the basis for the commercialization of a detection product applicable to microscopes currently used in biological
imaging laboratories.
HORIBA will bring a unique expertise in the dimensioning of optical devices dedicated to polarized detection. It
will in particular give access to its know-how on the existing system to evolve towards an imaging-compatible
polarimeter. It will in addition support a possible plan for commercialization of the system if appropriate.
The doctoral candidate will visit frequently the involved colleagues at HORIBA (service of scientific innovation
directed by Olivier Acher), with several goals : (1) learn how to properly dimension a polarimetric detection
system, (2) apply it to super-resolution imaging, (3) expand his/her knowledge on the industrial environment.
Ultimately the candidate will learn about the complete process from the system definition to its demonstration
for a defined biological question, leading to the exploration of commercialization possibilities. This process will
thus enhance the knowledge of the candidate on all processes behind innovation.
The project addresses the SRI_S3 objective ‘santé bien-être’ since important outcomes in terms of the
mechanisms regulating cell division are expected. In particular, research led at CRCM is concentrated on the link
between molecular properties of human septins and cancer development.
2.2 INTERNATIONAL DIMENSION:
I. Fresnel collaborates with G. Koenderink at AMOLF (The Netherlands) on the in vitro reconstitution of
cytoskeletal filament assemblies. During his/her project, the candidate will have the opportunity to go regularly
to AMOLF in order to (1) learn about in vitro reconstitution, (2) prepare his/her own samples to be characterized
at AMOLF and later at I. Fresnel, (3) transfer the necessary knowledge for the completion of the project at I.
Fresnel and CRCM.
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3. RECENT PUBLICATIONS
1. Kress A., Wang X., Ranchon H., Savatier J., Rigneault H., Ferrand P. and S. Brasselet. Mapping the local
organization of cell membranes using generalized polarization resolved confocal fluorescence microscopy.
Biophysical Journal 105, 127-136 (2013)
2. Valades Cruz, C.A, H. A. Shaban, A. Kress, N. Bertaux, S. Monneret, M. Mavrakis, J. Savatier, S. Brasselet,
Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution
microscopy. Proceedings of the National Academy of Sciences of the United States of America 113, E820-828
(2016)
3. Mavrakis, M. Y. Azou-Gros, F-C. Tsai, J. Alvarado, A. Bertin, F. Iv, A. Kress, S. Brasselet, G.H. Koenderink and
T. Lecuit. Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles. Nature
cell biology 16, 322-334 (2014)
4. Mavrakis, M., Tsai, F.C. & Koenderink, G.H. Purification of recombinant human and Drosophila septin
hexamers for TIRF assays of actin-septin filament assembly. Methods in cell biology 136, 199-220 (2016)
5. Mavrakis, M. Visualizing septins in early Drosophila embryos. Methods in cell biology 136, 183-198 (2016)
6. Chanez B., Gonçalves A., Badache A. and P. Verdier-Pinard. Eribulin targets a ch-TOG-dependent directed
migration of cancer cells. Oncotarget. 6, 41667-41678 (2015)
7. Connolly D., Yang Z., Castaldi M., Simmons N., Oktay M.H., Coniglio S., Fazzari M.J., Verdier-Pinard P. and C.
Montagna. Septin 9 isoform expression, localization and epigenetic changes during human and mouse breast
cancer progression. Breast Cancer Res. 13 :R76 (2011)
8. Connolly, D., Abdesselam, I., Verdier-Pinard, P. & Montagna, C. Septin roles in tumorigenesis. Biological
chemistry 392, 725-738 (2011)
4. EXPECTED PROFILE OF THE CANDIDATE
The candidate is expected to be of biophysics background with knowledge in biology and a high interest in
experimental optics. The work involving both optical instrumentation developments and biological sample
preparation, the student will be involved in an interdisciplinary work where in vitro / cell sample developments,
experimental imaging and data analysis will be equally important. A previous experience with interdisciplinarity
during the master thesis is thus preferable, in particular involving optical microscopy. He/She should be
motivated by the exploration of novel questions in biology. He/She should also be interested in the development
of an instrument of interest for industry, since the detection device envisioned in this project can possibly lead
to industrial development. The candidate will work for part of his/her time with an international collaborator
and participate in several national and international conferences: he/she should therefore be capable of
integration in international environments.
5. SUPERVISORS’ PROFILES
Supervisor 1: Sophie Brasselet (DR2 CNRS) has published 100 peer reviewed articles (3100 citations, h index =
33) and given 47 invited conferences in international and national events. She was awarded the Silver Medal of
the CNRS in 2016. After a PhD at CNET Bagneux (1997) on molecular nonlinear optics, she worked for 2 years at
USCD and Stanford U. (USA) in the group of W.E. Moerner (Nobel Prize Chemistry 2014) on single molecule
imaging. She was then recruited at Ecole Normale Supérieure de Cachan (France) as Assistant Professor, and
joined I. Fresnel in 2007 as a CNRS researcher. For the last ten years, she has developed novel microscopy and
super-resolution tools based on fluorescence polarization dedicated to biomolecular structural investigations
using polarized ensemble imaging approaches and recently at the single molecule level.
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She has supervised 23 PhD students (duration 3 years) since 2001. Each supervised PhD project has led to 3
publications on average, in international peer reviewed journals in both physics and biophysics such as Phys. Rev.
Lett, Phys. Rev. A, Opt. Express, Biophys. J., Proc. Natl. Acad. Sci. Last year one student (P. Gasecka) was awarded
the Doctorate Prize of Ecole Doctorale ‘Physique et Science de la Matière’. Among these students. 2 have just
defended in December 2016 (C. Rendon, W. He), 4 are postdoctoral researchers (Univ. Laval Canada, Univ. St
Andrews UK, Univ. Toulouse, Institut Curie), 4 in industry (Quantel Laser, Zeiss, L’Oréal, Bertin Technologies), 1
on intersectorial activities (OPTITEC), 1 is building a company (China), 7 have been recruited in academics (6
Maîtres de conférence in France, 1 CNRS). Currently she is supervising 3 PhD students (2 in co-supervision with
a biologists), that started in 2013, 2015 and 2016.
Manos Mavrakis (CR1 CNRS) was initially trained as a chemist, then as a protein biochemist during his PhD
(EMBL) and cell biologist during his postdoc (NIH), when he investigated the role of the cytoskeleton in cellular
architecture. He was recruited by CNRS in 2008 (IBDM), and developed biochemical, molecular and genetic tools
to study cytoskeletal filaments. In this context, he initiated a collaboration with G. Koenderink in 2011, providing
the first evidence for direct interactions between animal septins and actin. In 2015 he joined I. Fresnel to study
cell morphogenesis in an interdisciplinary environment using cutting-edge imaging approaches. He is currently
co-supervising a phD student with S. Brasselet on actin organization imaging in tissues.
Supervisor 2: Pascal Verdier-Pinard (CR1 Inserm) has produced 58 peer reviewed publications (52 original
articles, 4 reviews, 2 book chapters, 2550 citations, h index = 27). Pascal Verdier-Pinard has acquired extensive
experience on the molecular and cellular pharmacology, proteomics and cell biology of microtubules over the
last 20 years. After a PhD in Molecular Pharmacology in 1994 (Paul Sabatier University, Toulouse, France), he
spent 5 years as a Fogarty Posdoctoral Fellow at the NCI (Bethesda, USA) and 5 years as an Instructor and
Assistant Professor at Albert Einstein College of Medicine (Bronx, NY) before returning to France as a senior
researcher at Inserm in 2007 and presently contribute his expertise to the group of Ali Badache on the role of
microtubules in cell migration. Over the last 10 years he has been working also on Sept9 isoforms in the context
of tumorigenesis.
Before 2007, he has co-supervised 2 PhD students in the USA (6 and 3 published articles, respectively, in cancer
biology journals such as PNAS, Cancer Research, Clin. Cancer Research , Oncotarget) who worked in academic
medical laboratories and a pharmaceutical company, respectively (now working as Research Director at SUNY
Downstate Medical Center, NY, and Senior Researcher at Roche, Basel, Switzerland). In France, between 2007
and mid-2011, he co-supervised 1 PhD student in the field of proteomics and mass spectrometry who published
2 reviews and 6 original articles in Analytical Chem., Chem.Biol., and J. of Proteomics. This PhD laureate
performed two postdocs (one at Liège University in Belgium and one at Harvard Medical School in Boston) and
is now working as a mass spectrometry Scientist in Ambergen inc, a biotechnology company in the Boston area.
In December 2016, 1 PhD student has successfully defended his thesis (3 years, an article in preparation, an
article submitted, two articles in revision for Scientific Reports and PNAS). Currently, Pascal Verdier-Pinard is cosupervising 1 PhD student who started in 2014.
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