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UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1220 ISSN: 0346-6612 ISBN: 978-91-7264-670-4 Editor: The Dean of the Faculty of Medicine Regulation of tubulin heterodimer partitioning during interphase and mitosis Per Holmfeldt Department of Molecular Biology Umeå University, Sweden 2008 Copyright © Per Holmfeldt Printed by Print & Media Umeå 2008 TABLE OF CONTENTS 1. 2. 3. LIST OF PUBLICATIONS LIST OF ABBREVIATIONS ABSTRACT INTRODUCTION 4. INTRODUCTION TO THE CYTOSKELETON 4.1 The cytoskeleton 4.2 Evolution of cytoskeletal systems that organize the cell 4.3 The cytoskeleton reorganizes during the cell cycle 4.4 Lateral bonds between adjacent protofilaments are required for the stability of cytoskeletal filaments 4.5 Nucleation is the rate limiting step for a polymerization reaction 5. BASIC STRUCTURE AND PROPERTIES OF MICROTUBULES Microtubules are polar polymers of α/β-tubulin heterodimers Microtubules are nucleated from microtubule-organizing centers The spatial organization of the microtubule system may differ between specialized cell types 5.4 The energy of GTP hydrolysis drives the intrinsic dynamic instability of microtubules PROTEINS RELEVANT TO THE STRUCTURE AND FUNCTION OF THE MICROTUBULE SYSTEM 6.1 Regulation of microtubule dynamics in intact cells 6.2 Microtubule regulatory proteins 6.3 Unidirectional movement of molecular motors along microtubules MICROTUBULE FUNCTION DURING THE INTERPHASE AND MITOSIS OF THE CELL CYCLE 7.1 A brief introduction to the mammalian cell cycle and its check points 7.2 Microtubule dependent molecular motors organize the intracellular space 7.3 Microtubules segregate the duplicated chromosomes during mitosis 7.4 The role of kinetochore microtubule treadmilling to ensure bipolar kinetochore attachment THE IMPORTANCE OF CHROMOSOMAL INSTABILITY FOR TUMOR PROGRESSION 8.1 Tumor progression requires on genomic instability 8.2 Genomic instabilities of tumors depends on two distinct mechanisms MAJOR REGULATORS OF MICROTUBULE POLYMERIZATION 9.1 MCAK is a conserved catastrophe-promoting protein 9.2 The function of MCAK during the cell cycle 9.3 The evolutionary conserved XMAP215/TOGp protein family 9.4 Function of XMAP215/TOGp family proteins during mitosis 9.5 Counteractive activities between MCAK and XMAP215 family members 9.6 Op18 - a developmentally regulated microtubule-destabilizing protein 9.7 Phosphorylation-inactivation of Op18 during interphase and mitosis 9.8 Dysregulation of Op18 in human malignancies 5.1 5.2 5.3 6. 7. 8. 9. 1 1 2 2 2 3 3 4 4 5 6 6 7 7 8 8 9 9 11 11 12 12 12 13 14 14 15 15 16 9.9 9.10 9.11 Relations between Op18 and the other Op18/Stathmin family members Microtubule stabilization by the ubiquitous expressed MAP4 Regulation of MAP4 during interphase and mitosis RESULTS AND DISCUSSION 10. AIMS 11. PROLOGUE 12. THE MCAK AND XMAP215/TOGp PROTEINS ARE NOT IMPORTANT FOR THE CONTROL OF TUBULIN SUBUNIT PARTITIONING BUT HAVE ESSENTIAL INTERCONNECTED FUNCTIONS DURING SPINDLE ASSEMBLY 12.1 Background 12.2 XMAP215/TOGp is not essential to enhance the polymerization rate in human somatic cells 12.3 Differential phenotypes of XMAP215/TOGp and MCAK depletion in human cells and Xenopus egg extracts 12.4 The overexpression phenotypes of MCAK and TOGp reveal an unexpected interplay 12.5 Confirmation that MCAK function is important for spindle fidelity 12.6 TOGp provides protection from centrosomal MCAK activity, which is essential for bipolar spindle formation 18 18 18 19 19 20 20 20 12.7 13. 14. 15. 16. 17. 18. The functions of both CaMKIIγ and TOGp are essential for protection of spindle microtubules from centrosome-directed MCAK activity PHOSPHORYLATION-REGULATION OF Op18 AND MAP4 CONTROLS THE PARTITIONING OF TUBULIN SUBUNITS IN THE INTERPHASE MICROTUBULE SYSTEM 13.1 Background 13.2 Evidence for the importance of MAP4 and Op18 as interphase-specific counteractive regulators of tubulin subunit partitioning 13.3 The significance of MAP4 and Op18 as phosphorylation-responsive regulators of tubulin subunit partitioning IDENTIFICATION OF Op18 AS A MAJOR SIGNAL TRANSDUCTION RESPONSIVE REGULATOR OF TUBULIN SUBUNIT PARTITIONING 14.1 Background 14.2 Identification of an Op18 dependent signal transduction chain for microtubule polymerization in response to T cell antigen receptor triggering EXCESSIVE Op18 ACTIVITY DURING MITOSIS RESULTS IN SPINDLEDESTABILIZATION AND CHROMOSOMAL INSTABILITY 15.1 Background 15.2 The relationship between the oncogenic Q18ÆE substitution of Op18 and leukemia-associated up-regulation of the wild-type protein 15.3 The significance of life-span of animals for the demand for increased chromosomal instability during tumor-progression FINAL CONCLUSIONS ACKNOWLEDGMENTS REFERENCES 16 16 17 21 21 22 22 23 23 24 24 25 25 25 25 1. LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to in the text by their roman numerals: I. Holmfeldt, P., Stenmark S., and Gullberg M., (2004) Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis. EMBO Journal 23:627-637. II. Holmfeldt P., Zhang X., Stenmark S., Walczak C., and Gullberg M., (2005) CaMKIIgamma-mediated inactivation of the Kin I kinesin MCAK is essential for bipolar spindle formation. EMBO Journal 24:1256–1266. III. Holmfeldt P., Brännström K., Stenmark S., and Gullberg M., (2006) Aneugenic activity of Op18/stathmin is potentiated by the somatic Q18->E mutation leukemic cells. Molecular Biology of the Cell 17:2921-30 IV. Holmfeldt, P., Stenmark S., and Gullberg M., (2007) Interphase-specific phosphorylation-mediated regulation of tubulin dimer partitioning in human cells. Molecular Biology of the Cell 18:1909-17 2. LIST OF ABBREVIATIONS MARK, Microtubule-Affinity Regulating Kinase MCAK, Mitotic Centromere-Assoociated Kinesin Mor1, Microtubule ORganisation gene-product 1 Msps, Mini spindles MT, MicroTubule NuMA, Nuclear Mitotic Apparatus protein Op18, Oncoprotein of 18 kDa PAR1, PARtitioning-defective mutant 1 PI-3 kinase, PhosphatidylIonositol-3 kinase PKA, Protein Kinase A (also known as cAMP dependent protein kinase) SCG10, Superior Cervical Ganglion 10 SCLIP, SCG10-LIke Protein shRNA, short hair-pin RiboNucleic Acid Stu2, Suppressor of a TUbulin mutation 2 Ras, Rat sarcoma virus oncogene Ran, Ras-related nuclear protein Rb, Retinoblastoma susceptibility protein RB3, Rat homolog of the Xenopus gene XB3 Rho, Ras homology RNA, RiboNucleic Acid TACC, Transforming Acidic Coiled-Coil TBCE, TuBulin-folding Cofactor E +TIP, plus-TIP(/end) binding protein TOGp, Tumour Overexpressing Gene protein TPX2, Targeting Protein for Xklp2 XMAP215, Xenopus Microtubule Associated Protein of 215 kDa ZAP-70, Zeta-chain-Associated Protein kinase of 70 kDa Zyg-9, ZYGote defective 9 APC, Adenomatous Polyposis Coli APC/C, Anaphase Promoting Complex/Cyclosome ADP, Adenosine DiPhosphate Alp14, ALtered Polarity gene-product 14 ATP, Adenosine TriPhosphate CaMKII, Ca2+/CalModulin-dependent protein Kinase II CaMKIV, Ca2+/CalModulin-dependent protein Kinase IV cAMP, cyclic Adenosine MonoPhosphate CDC6, Cell Division Control mutant 6 CDC42, Cell Division Control mutant 42 CDK, Cyclin-Dependent Kinase CLASP, CLIP-ASsociated Protein CLIP, Cytoplasmic LInker Protein Dis1, Defect In Sister chromatid disjoining 1 DdCP224, Dictyostelium discoideum Centrosomal Protein of 224 kDa DLG1, Drosophila discs LarGe 1 DNA, DeoxyriboNucleic Acid EB1, End-Binding protein 1 EBV, Epstein-Barr Virus ELKS, protein rich in E (glutamic acid), L (leucine), K (lysine) and S (serine) FtsZ, Filamenting temperature-sensitive mutant Z GDP, Guanosine DiPhosphate GTP, Guanosine TriPhosphate KIF17, KInesin Family member 17 KIN I, KINesin Internal motor domain MAP2, Microtubule-Associated Protein 2 MAP4, Microtubule-Associated Protein 4 MAPK, Mitogen-Activated Protein Kinase 1 evolutionary context, the cooperation of these filaments was first required for segregation of duplicated chromosomes, and has subsequently evolved to facilitate complex cell shapes and evolution of multi-cellular organisms. The cytoskeleton encompasses microtubules, intermediate filaments and actin filaments (also known as microfilaments), which are polymers of evolutionary conserved protein subunits. Given that polymerization of these subunits in general is rapid and reversible, the cytoskeleton can rapidly reorganize during the cell cycle and in response to changes of the environment. A large set of accessory proteins has evolved to facilitate specific nucleation, assembly and the dynamic behavior of the cytoskeleton. In addition, proteins that serve as molecular motors are central for the function of the microtubule and actin cytoskeletons. These motor proteins generate forces by directional movement along polymers and by this mean moves cargo to selected cellular locations. Thus, the cytoskeletal systems involve many more proteins than their structural subunits. While the cytoskeleton systems have been described in eukaryotic cells, recent reports on structural similarities with prokaryotic proteins important for cell division have lead to the insight that the microtubule and actin cytoskeletons have an ancient origin (reviewed in Erickson, 2007). 3. ABSTRACT The microtubule cytoskeleton, which consists of dynamic polymers of α/β tubulin heterodimers, organizes the cytoplasm and is essential for chromosome segregation during mitosis. My thesis addresses the significance and potential interplay between four distinct microtubuleregulatory proteins. The experimental approach included the development of a replicating vector system directing either constitutive expression of short hairpin RNAs or inducible ectopic expression, which allows stable depletion and/or conditional exchange of gene-products. Based on the originally observed activities in frog egg extracts, MCAK and TOGp have been viewed as major antagonistic proteins that regulate microtubule-dynamics throughout the cell cycle. Surprisingly, while my thesis work confirmed an essential role of these proteins to ensure mitotic fidelity, tubulin subunits partitioning is not controlled by the endogenous levels of MCAK and TOGp in human somatic cells. Our major discovery in these studies is that the activities of both CaMKIIγ and TOGp are essential for spindle bipolarity through a mechanism involving protection of spindle microtubules against MCAK activity at the centrosome. In our search for the major antagonistic activities that regulates microtubule-dynamics in interphase cells, we found that the microtubule-destabilizing activity of Op18 is counteracted by MAP4. These studies also established Op18 and MAP4 as the predominant regulators of tubulin subunit partitioning in all three human cell model systems studied. Moreover, consistent with phosphorylation-inactivation of these two proteins during mitosis, we found that the microtubule-regulatory activities of both MAP4 and Op18 were only evident in interphase cells. Importantly, by employing a system for inducible gene product replacement, we found that site-specific phosphorylation-inactivation of Op18 is the direct cause of the demonstrated hyperpolymerization in response to T-cell antigen receptor triggering. This provides the first formally proven example of a signal transduction pathway for regulation of interphase microtubules. Op18 is frequently upregulated in various types of human malignancies. In addition, a somatic mutation of Op18 has recently been identified in an adenocarcinoma. This thesis work revealed that the mutant Op18 protein exerts increased microtubule-destabilizing activity. The mutant Op18 protein was also shown to be partially resistant to phosphorylation-inactivation during mitosis, which was associated with increased chromosome segregation aberrancies. Interestingly, we also observed the same phenotype by overexpressing the wild type Op18 protein. Thus, either excessive levels of wild type Op18 or normal levels of mutated hyper-active Op18 seems likely to contribute to tumor progression by exacerbating chromosomal instability. 4.2 Evolution of cytoskeletal systems that organize the cell During the early evolution of the eukaryotic cell the genome increased both in size and complexity. This was associated with evolution of strategies to ensure correct transmission of the genetic material to the two daughter cells during cell division, such as splitting up the genome into chromosomes that are segregated by microtubules of the mitotic spindle during cell division (Fig. 1A (I)). This process has been coined mitosis based on the thread-like appearance of the condensed chromatin (from Greek mitos, thread)(reviewed in Paweletz, 2001). In addition to division of the genetic material, the contractile ring built from actin filaments has evolved to execute the cellular division, a process known as cytokinesis (Fig. 1A (II)). A) B) I II II III I IV V INTRODUCTION Fig. 1. Cellular functions that depend on the cytoskeleton 4. INTRODUCTION TO THE CYTOSKELETON 4.1 The cytoskeleton All eukaryotic cells contain three distinct types of filament forming proteins, collectively termed the cytoskeleton. In an During interphase, the period in between two division events, cytoskeletal filaments serve a primordial role to protect against mechanical stress, which made the cell wall redundant in the ancient eukaryotic cell. This was probably a 2 facilitate the contraction of the whole cell. During cell division, actin filaments together with myosin form the contractile ring that is positioned just beneath the plasma membrane at the cellular equator (Fig. 2). prerequisite for the development of actin-filament based phagocytosis (Fig. 1B (I)) and for subsequent acquisition of endosymbiotic organelles, such as mitochondria. Thus, the loss of the cell wall paved the way for the current organization of the eukaryotic cells into distinct compartments. To allow a cytoskeleton dependent spatial organization of the cytoplasm, the molecular motor proteins evolved. Through the utilization of energy released during ATP hydrolysis, a molecular motor can move directionally along either microtubules or actin filaments. By attachment to motor proteins, various cargos such as organelles, vesicles and macromolecules is moved to specific locations in the cell (Fig. 1B (II)). Alterations of cell shape depends on the protruding action of polymerizing actin-filaments exerted on the plasma membrane (Fig. 1B (III)). The resulting protrusions are thereafter in general stabilized by intermediate filaments. If the protrusive activity of actin-filaments at one side of a cell is coupled to simultaneous actin-filament dependent contractions at the opposite side, this will result in directional movement of the cell (Fig. 1B (IV)). A central aspect during evolution of multicellular organisms is cell surface molecules that facilitate specific adhesion between neighboring cells. These connections depends on anchorage of the adhesion molecules to the intermediate filaments, which provides resistance towards mechanical stress (Fig. 1B (V)). Microtubules Actin filaments Intermediate filaments Interphase Mitosis Fig. 2. Reorganization of the three distinct cytoskeleton systems during the cell cycle 4.4 Lateral bonds between adjacent protofilaments are required for the stability of cytoskeletal filaments Many of the functions of the cytoskeleton require filaments to assemble over long distances and still have the potential to disassemble rapidly to allow recycling of subunits at different cellular locations, for example during reorganization of cells in response to signaling events. The small size of the protein subunits, in the range of a few nanometers, enables rapid diffusion over long distances. Soluble subunits are thereby available for polymerization at any given location within the cell. To generate polymers with the potential of rapid disassembly, the subunits are held together by weak noncovalent bonds (Fig. 3A). However, a polymer containing just a single row of subunits, held together by weak interactions, has a high probability to break at any given point of the polymer, which would prevent formation of polymers of the required length (Fig. 3B). Thus, to combine the requirements of rapid disassembly with stability, all cytoskeletal filaments are stabilized by lateral interactions between adjacent rows of subunits, termed protofilaments. This arrangement provides a stable core with dynamic ends (Fig. 3C). 4.3 The cytoskeleton reorganizes during the cell cycle As illustrated in Fig. 2, all parts of the cytoskeleton undergo major reorganizations between interphase and mitosis. The microtubule system, which during interphase is organized into a single array that originates from the centre of the cell, transforms into the bipolar mitotic spindle. The interphase microtubule system constitutes the track system on which molecular motors move to localize cellular components over long distances. In certain cell types specialized microtubules are essential for the motile behavior of cellular appendages, called cilia and flagella. During mitosis, the microtubules of the mitotic spindle organize the duplicated chromosomes onto the cellular equator and thereafter segregate them such that each daughter cell receives a complete set of chromosomes (Fig. 2). Nuclear intermediate filaments termed lamins are localized just beneath the inner nuclear membrane and are important for the integrity of the nuclear envelope during interphase in all eukaryotic cells. Cytosolic intermediate filaments have only been identified with certainty in metazoan cells, in which they form a web-like network throughout the cell. These cytosolic intermediate filaments provide mechanical resistance and stabilize connections with neighboring cells or the extracellular matrix (Fig. 2). Actin filaments are generally found directly beneath the plasma membrane. These filaments provide structural integrity of the plasma membrane and are important for formation of cellular protrusions, thus serving a central role in establishment of cell shape. Actin filaments also have the ability to form contractile structures together with bipolar filaments of the motor protein myosin-II. These contractile structures can either be positioned just beneath the plasma membrane and mediate contractions of specific parts of the cell, or, as in muscle cells, fill the interior of the cell and A) Monomers + B) C) + Polymer + + Breakage of longitudinal monomer binding due to ambient thermal energy Stabilizing lateral bindings between adjacent protofilaments Fig. 3. Cytoskeletal filaments stabilized by laterally interacting protofilaments In the case of microtubules, lateral interactions are provided by the arrangements of 13 protofilaments that interact to form a hollow tube. This tubular structure is stiff 3 1967), α- and β-tubulin were subsequently identified as the subunits of microtubules (Stephens, 1970). The α- and βtubulin subunits are ~40% identical in sequence and are highly conserved among eukaryotes (Little & Seehaus, 1988). The atomic structure of tubulin heterodimers, solved by electron crystallography of zinc induced tubulin sheets, revealed a very similar globular structure of the α- and βtubulin subunits (Fig. 6B; Nogales et al., 1998). but brittle, which provides the ability to generate pushing forces. Intermediate filaments are composed of eight protofilaments, which wrap around each other to form a ropelike bundle. This arrangement is strong and flexible and obviously well suited to withstand stress without rupturing. In actin filament two protofilaments twist around each other to form a stable but yet dynamic structure (Fig. 4). Microtubule Intermediate filament Actin filament A) Protofilament B) β-tubulin α-tubulin Subunits: 25 nm 10 nm 7 nm Tubulin heterodimer Variable Actin Fig. 6. Microtubule and tubulin structure Fig. 4. Structure of the three cytoskeletal filaments of eukaryotic cells The α- and β-tubulin subunits do not exist as monomers and their folding and subsequent assembly into the tubulin heterodimer is dependent on several specific chaperonin cofactors (reviewed in Lopez-Fanarraga et al., 2001). The significance of this folding pathway is illustrated by the finding that a mutation in one of its components, TBCE, is the genetic defect in the human inherited disease KennyCaffey syndrome (Parvari et al., 2002). Both α- and β-tubulin subunits are encoded by multiple genes, with a pronounced cell-type specific expression in animals. The tubulin heterodimer isotypes appear largely functionally redundant and co-polymerize into mixed microtubules in vitro. It is still unclear how the different isotypes affect the microtubule system in intact cells; however, it seems clear that the neural specific isotypes are important for the properties of neural cells in which the microtubule system has highly specialized roles during e.g. axon outgrowth (reviewed in Katsetos et al., 2003). Moreover, the presence of isotypes is also of clinical significance, since changes in isotype composition can mediate resistance to tubulin directed drugs in tumor cells (reviewed in Verrills & Kavallaris, 2005). Tubulin heterodimers form polar protofilaments through head-to-tail assembly (Fig. 6A). Parallel protofilaments form hollow tubes by lateral interactions. Microtubules assembled under in vitro conditions may contain between 8 and 19 protofilaments (Chretien & Wade, 1991), suggesting flexibility in the interactions between adjacent protofilaments. Cellular microtubules, however, always contain 13 protofilaments (Evans et al., 1985), a number probably explained by the mechanism of nucleation, which is based on a template (see Fig. 7). Microtubules are polar polymers with α-tubulin exposed at one end and β-tubulin at the opposite and the polymerization rate differs ~5-fold between opposite ends. The faster growing end is termed the plus end while the slower growing end the minus end. By docking the crystal structure of the tubulin heterodimer into a microtubule model it has been established that α-tubulin is exposed at the minus end and β-tubulin consequently is exposed at the plus end of the microtubule (Nogales et al., 1999). 4.5 Nucleation is the rate limiting step for a polymerization reaction During in vitro assembly of filaments (Fig. 5A), formation of an initial aggregate of subunits, the nucleus, is the first step of the polymerization reaction. This process is called nucleation and depends on the formation of short oligomers. Since the subunits will only be able to interact with few other subunits, the short oligomers are unstable and are likely to dissociate. This creates a kinetic barrier for spontaneous polymerization, which provides a mean for a cell to control the localization of cytoskeletal assembly. To facilitate nucleation, cells are equipped with dedicated proteins, termed nucleation factors, which serve as templates on to which subunits can efficiently form polymers (Fig. 5B). The subcellular localization and activation of nucleation factors will therefore be a major determinant for the location of cytoskeletal assembly. A) Polymerization Spontaneous nucleation B) Nucleation factor Template facilitated nucleation Polymerization Fig. 5. Initiation of polymerization is regulated by nucleation factors that decrease the kinetic barrier of nucleation 5. BASIC STRUCTURE AND PROPERTIES OF MICROTUBULES 5.1 Microtubules are polar polymers of α/β-tubulin heterodimers The α/β-tubulin heterodimer is the subunit of microtubules. It consists of two homologous proteins, α- and β- tubulin, each of a molecular weight of 55 kDa (Fig. 6A). Tubulin heterodimers were initially identified because of their affinity to the spindle poisoning drug colchicine (Borisy & Taylor, 4 Tubulin heterodimers are subject to complex posttranslational modifications (reviewed in Westermann & Weber, 2003), but the physiological significance is unclear and the extent of modifications seems largely to reflect the age of individual microtubules. Most of these modifications affect the carboxy-terminal region, which in both α- and βtubulin is located on the outside of the microtubule and therefore positioned to influence interactions between the microtubule and other proteins. The prokaryotic protein FtsZ has a limited sequence homology with α- and β-tubulin (reviewed in Erickson, 1995) and is virtually identical in structure (Lowe & Amos, 1998). FtsZ can assemble into protofilaments (Mukherjee & Lutkenhaus, 1994), which form a contractile ring at the division site of the bacterial cell (Ma et al., 1996). Inhibitors of FtsZ function is currently developed as novel therapeutic agents against pathogenic bacteria (reviewed in Vollmer, 2006). In the bacteria Prosthecobacter dejongeii two genes with sequence homology to α- and β-tubulin have been identified (Jenkins et al., 2002), which have probably been acquired through horizontal gene transfer (Schlieper et al., 2005). These bacterial versions of tubulin assemble into protofilaments, which form bundles rather than tubular microtubule structures (Sontag et al., 2005). The function of the Prosthecobacter tubulin-like proteins is still unclear. these centrioles, termed mother, is characterized by two distinct sets of nine appendages at distal and subdistal locations (Paintrand et al., 1992). In the currently favored model for γ-tubulin mediated microtubule nucleation, the γ-tubulin ring complex forms a lock-washer shaped template, on which tubulin heterodimers can readily polymerize (Fig. 7) (reviewed in Raynaud-Messina & Merdes, 2007). Following their nucleation by γ-tubulin ring complexes, the minus end of microtubules are believed to be anchored to the appendages of the mother centriole inside the centrosome (Fig. 8A; Piel et al., 2000), through a ninein dependent mechanism (Mogensen et al., 2000), leaving the minus ends inert. Thus, the γ-tubulin ring complex has potential to be recycled for many round of nucleation. 5.2 Microtubules are nucleated from microtubuleorganizing centers Provided that nucleation is facilitated by e.g., volumeexcluding glycerol or the microtubule-stabilizing drug taxol, tubulin heterodimers have potential to self-assemble in vitro. In intact cells, however, a template structure is required to initiate polymerization. A key structural element for microtubule nucleation in all eukaryotes is provided by γtubulin, a paralogue of α- and β-tubulin (reviewed in Oakley, 2000). Multiple γ-tubulin molecules associates laterally to form a ring –shaped structure, first detected by electron microscopy studies of purified complexes from Xenopus eggs (Fig. 7) (Zheng et al., 1995). Many proteins are important for assembly, localization and the structure of the γ-tubulin ring complex (reviewed in Raynaud-Messina & Merdes, 2007). Cytosolic γ-tubulin, which constitutes the major part of cellular γ-tubulin, does not exert significant microtubule nucleation activity (Moudjou et al., 1996). Active γ-tubulin is primarily located at structures termed microtubule organizing centers, exemplified by the animal centrosome and the spindle pole body of Saccharomyces cerevisiae. Based on morphological studies, the centrosome has been characterized as a large, ~1 μm in diameter, non-membranous organelle, which is attached to the cell nucleus in what appears to be a microtubule dependent mechanism (reviewed in Gonczy, 2004). Centered on a pair of perpendicular arranged centrioles and surrounded by a cloud of pericentriolar material, the centrosome is believed to contain at least 200 different proteins. Centrioles, which are required for centrosomal integrity (Bobinnec et al., 1998), are barrel shaped structures with walls made of nine parallel triplet microtubules, with each triplet tilted inward like the blades of a turbine. The centrioles are duplicated once during the cell cycle (reviewed in Azimzadeh & Bornens, 2007) and the individual centrioles consequently differ in age. The older of During mitosis, centrosomal microtubules are excised by the microtubule severing protein katanin (Fig. 8B; McNally & Thomas, 1998) and released from the centrosome. The minus ends of the microtubules are thereafter focused by the molecular motor dynein (Gaglio et al., 1997) and re-attached to the centrosome, by a mechanism that leaves the minus end exposed (Fig. 8A). This arrangement allows for a flux of tubulin heterodimers through the polymer, with addition of subunits at the plus end coupled to simultaneous disassociation at the minus end, a process termed treadmilling. γ-tubulin ring complex Fig. 7. Cellular microtubule formation is facilitated by templated nucleation mediated by γ-tubulin ring complexes A) Interphase Mitosis Centrosome B) Microtubule severing by katanin Fig. 8. Microtubule nucleation, release and anchorage at the centrosome Generation of mitotic microtubules is also dependent on extra-centrosomal nucleation (reviewed in Walczak & Heald, 2008), believed to occur primarily in the vicinity of chromosomes through either a Ran regulated TPX2 dependent mechanism (Carazo-Salas et al., 2001; Gruss et al., 2002) or a γ-tubulin mediated mechanism 5 extracellular fluids or the cells themselves (Fig. 9D). These appendages, termed cilia and flagella, respectively, are formed around a central core, the axoneme, consisting of nine doublet microtubules arranged in a circle surrounding two singlet microtubules (reviewed in Downing & Sui, 2007). Through sliding of the axonemal microtubules, mediated by specific molecular motors belonging to the dynein family, the appendage will acquire an oscillating motion. The axonemal microtubules are nucleated from a structure termed the basal body, which is situated right beneath each individual appendage during their assembly. The basal body contains a single centriole, which is analogous to the mother centriole of the centrosome, and may mediate the actual nucleation event (reviewed in Dawe et al., 2007). (Mahoney et al., 2006). The randomly nucleated microtubules are sorted into bipolar arrays by the action of microtubule dependent molecular motors (reviewed in Gadde & Heald, 2004). This mechanism allows spindle formation to occur even in cells with ablated centrosomes (Khodjakov et al., 2000). In vitro studies have revealed that many proteins, such as dynein (Malikov et al., 2004), EB1 (Vitre et al., 2008) and XMAP215 (Popov et al., 2001), posses significant microtubule nucleation activity. Whether this is of physiological significance remains unclear. 5.3 The spatial organization of the microtubule system may differ between specialized cell types Most mammalian cells exhibit a radial array of microtubules with minus ends focused at the centrosome (Fig. 9A). However, in certain specialized cells, a non-radial array of microtubules lacking centrosomal attachment may dominate (reviewed in Bartolini & Gundersen, 2006). + A) B) + + + + C) - - - - -- 5.4 The energy of GTP hydrolysis drives the intrinsic dynamic instability of microtubules Tracking of individual microtubules in vitro, through dark field video microscopy, revealed that microtubules exhibit non-equilibrium dynamics, characterized by periods of polymerization and depolymerization, with stochastic transitions between them (Fig. 10; Horio & Hotani, 1986). The transition between polymerization and depolymerization is termed a catastrophe and the opposite event a rescue. Similar dynamic behavior was recorded in intact cells, but with a general increase in dynamicity (Fig. 10; Cassimeris et al., 1988). The observed dynamic behavior had indeed previously been postulated, based on analysis of the length distribution of fixed microtubules, and termed dynamic instability (Mitchison & Kirschner, 1984a; Mitchison & Kirschner, 1984b). The energy released during GTP hydrolysis provides the driving force for the dynamic instability of microtubules (Hyman et al., 1992). Both α- and β-tubulin have a GTPbinding site termed the N-site (Non-exchangeable) and E-site (Exchangeable), respectively (Fig. 6B). While α-tubulin binds GTP prior to assembly and the GTP molecule is buried at the monomer-monomer interface within the tubulin heterodimer, the β-tubulin GTP is exposed on the longitudinal end of the heterodimer. This leaves the E-site bound GTP of soluble tubulin heterodimers freely exchangeable and in equilibrium with the free GTP pool. During polymerization of tubulin, the E-site GTP will be buried at the newly formed heterodimer interface, making it non-exchangeable and at the same time exposed to a catalytic loop on α-tubulin that promotes its hydrolysis (Nogales et al., 1999). + - - -- +- ++ + + + D) + + + + + + ++ Fig. 9. Cell type specific organization of microtubules Length of MT (μM) Neurons have a combination of centrosomal attached microtubules, that form a radial array in the cell body, and non-centrosomal microtubules that form bundles in both axons and dendrites (Fig. 9B). The non-centrosomal bundles are thought to provide both structural support and the transport tracks needed for the delivery of various cargos by molecular motors. All neuronal microtubules are believed to be nucleated at the centrosome and that the severing activity of katanin (Ahmad et al., 1999) and spastin (Evans et al., 2005) serves to release microtubules from the centrosome. The released microtubules are subsequently arranged and localized by molecular motors and accessory proteins. In polarized epithelial cells, microtubules are nucleated at the centrosome and are thereafter released to organize into an array of parallel microtubules along the apical-basal axis of the cell (Keating et al., 1997), with minus ends at the apical side and the plus ends at the basal side (Fig. 9C). The minus ends are potentially stabilized by ninein (Mogensen et al., 1997) through a mechanism that requires functional adherence junctions (Chausovsky et al., 2000). This microtubule organization is important for the establishment and maintenance of the highly polarized structure of the epithelial cell, through polarized transport of cellular components along the microtubules by molecular motors. Certain specialized epithelial cells and sperms are equipped with motile appendages that are used to either move 8 Catastophe 6 Rescue In In vivo intact cells 4 2 In vitro vitro In 0 0 20 40 60 80 100 120 Time (seconds) Fig. 10. Dynamic behavior of microtubules in vitro and in intact cells GTP hydrolysis at the E-site induces a straight-tocurved conformational change in the tubulin heterodimer (reviewed in Nogales & Wang, 2006) and the classical GTP- 6 cap model postulates that this generates an energetically unstable microtubule lattice, stabilized by a layer of straight GTP loaded heterodimers at the plus-end (Fig. 11). The stochastic losses of the GTP-cap, due to either hydrolysis or heterodimer disassociation, allows the GDP-containing protofilaments to relax into a more energetically favorable curved conformation, which disrupts the stabilizing lateral interactions between adjacent protofilaments. The individual protofilaments will consequently rapidly depolymerize due to that longitudinal interactions alone are not sufficient to maintain the polymer (see Fig. 3). During this depolymerization-phase, the ends of microtubules have a distinct appearance revealed by electron microscopy, i.e. frayed ends in which the curling protofilaments have a ram horn like appearance (Fig. 11; Mandelkow et al., 1991). The difference in microtubule dynamics in intact cells and during assembly of purified tubulin in vitro is primarily due to an array of microtubule regulatory proteins. A recent proteomic search in Drosophila embryos identified 270 microtubule interacting proteins (Hughes et al., 2008), out of which about 100 was not previously known to bind microtubules. The task of determining which ones of these proteins that are important for regulation of microtubule function and dynamics is challenging. 6.2 Microtubule regulatory proteins Several evolutionary conserved regulatory mechanisms important for microtubule dynamics have been identified, primarily through work on Xenopus egg extracts. These mechanisms can generally be divided into microtubule stabilizing and destabilizing activities. Microtubule stabilizing proteins are subdivided in two main categories, namely proteins that interact alongside microtubules or proteins that specifically interact with microtubule ends. Among the proteins interacting along the outside of microtubules the best characterized are the so called classical Microtubule-Associated-Proteins (MAPs), which include the neuronal members MAP2 and tau and the ubiquitously expressed MAP4. These proteins are thought to stabilize microtubules by crosslinking adjacent protofilaments and thereby prevent depolymerization (Gustke et al., 1994), which explains the rescue promoting activity of these proteins (Ookata et al., 1995; Pryer et al., 1992). Proteins that specifically interact with the plus end of microtubules are termed +TIP-proteins (reviewed in Akhmanova & Steinmetz, 2008). Members of the +TIP family include XMAP215/TOGp, APC and CLASP. The latter two proteins have been show to be involved in the selective stabilization of microtubules at specialized cellular locations, such as the kinetochores of the mitotic chromosomes and polarized sites in the plasma membrane (Fig. 13). It appears therefore that APC and CLASP allow dynamic microtubules to probe the cellular space only to be stabilized at special sites (see section 7.2-3; reviewed in Akhmanova & Steinmetz, 2008). Given that MAP4 and XMAP215/TOGp have been in focus during my thesis work, a more detailed account of their structure and function will be given in Chapter 9. GTP GTP-tubulin GDP + Depolymerization Polymerization GDP-tubulin Catastrophe Rescue - Fig. 11. Dynamic instability implies stochastic switches between polymerization and depolymerization that depend on hydrolysis of a stabilizing GTP-cap Rescues, i.e., the transition from depolymerization to polymerization, are also frequent in a system consisting only of pure tubulin heterodimers and GTP. It can not be excluded that rescues are the result of contaminating microtubule stabilizing proteins. Nevertheless, rescues in the presumed absence of microtubule-binding proteins are thought to involve breakage of protofilaments close to the microtubule wall and the simultaneous association of GTP loaded heterodimers to generate a new GTP-cap. 6. PROTEINS RELEVANT TO THE STRUCTURE AND FUNCTION OF THE MICROTUBULE SYSTEM 6.1 Regulation of microtubule dynamics in intact cells The dynamicity of cellular microtubules differs distinctively compared to microtubules assembled from purified tubulin (Fig. 10). Importantly, during entry into mitosis microtubule dynamic is altered such that very short microtubules become predominant during prometaphase (Fig. 12A). This alteration of microtubule length distribution can be fully explained by an ~7-fold increase of catastrophe-promotion in mitotic frog egg extracts (Fig. 12B). A) Interphase Long, less dynamic microtubules Mitosis Short, more dynamic microtubules 1 Xenopus egg extractsa B) Interphase Mitosis Catastrophe frequency (sec-1) 0.018 0.12 Rescue frequency (sec-1) 0.011 0.02 Growth rate (μm/min) 9.3 12.3 Shrinkge rate (μm/min) 12.8 15.3 2 3 Fig. 13. Selective stabilization of microtubules through interaction with +TIP proteins Microtubule destabilizing proteins can be subdivided into three categories, i) severing proteins, which facilitate breakage of the microtubule (reviewed in Quarmby, 2000), exemplified by katanin (see Fig. 8B), ii) catastrophe promoters, which interact with microtubule ends and destabilize the GTP-cap and thereby promoting catastrophes, a (Belmont et al., 1990) Fig. 12. Dynamic parameters of microtubules during interphase and mitosis 7 apparatus (Corthesy-Theulaz et al., 1992) and mitochondria (Varadi et al., 2004), ii) vesicles (Gilbert & Sloboda, 1989) and iii) specific proteins (reviewed in Kamal & Goldstein, 2002) and iv) specific mRNAs (Schnorrer et al., 2000). Motor proteins translocate along microtubules through conformational changes driven by cycles of ATP binding, hydrolysis and product release (Fig. 14). Since at least one of the two motor domains of both kinesins and dyneins is attached to the microtubule at any given time point, the stability of attachment and processivity of these motors is high, i.e. translocation can occur over great lengths without de-attachment (reviewed in Amos, 2008). 6.3 Unidirectional movement of molecular motors along microtubules Microtubule dependent molecular motors are proteins that through cycles of conformational changes have the ability to move directionally along microtubules. The two classes of structurally distinct microtubule dependent motor proteins translocate to either the plus-end, which are prototype members of the large kinesin superfamily, or the minus-end, which are members of the dynein family. Kinesins and dyneins share the same general features, even though they are structurally unrelated. Each motor unit is centered on two heavy chains, each with a globular head motor domain and an elongated tail, which is responsible for the dimerization of the heavy chains. Accessory light and intermediate chains are primarily used to facilitate the association of different cargos to the motor unit. The kinesin superfamily constitutes a diverse array of related proteins, with about 45 members in human, grouped into 14 distinct subfamilies (Lawrence et al., 2004). Most kinesins have the motor domain of the heavy chain localized to the carboxy-terminal part of the protein, which will facilitate plus-end translocation. However, members of the kinesin-14 subfamily have the motor domain localized to the amino terminal of the heavy chain, which alters the direction of translocation to the minus-end. Members of the kinesin-13 subfamily have the motor domain localized to the central part of the heavy chain, which results in loss of the original motor activity and instead facilitate the binding and destabilization of microtubule ends. The best characterized kinesin-13 protein is MCAK, which is described in detail in Chapter 9. Through the direct interaction via their light chains or a connection mediated by various adaptor proteins kinesins can bind to, i) organelles, such as the endoplasmic reticulum (Dabora & Sheetz, 1988), lysosomes (Hollenbeck & Swanson, 1990) and mitochondria (Nangaku et al., 1994), ii) vesicles (Sheetz et al., 1986), iii) specific proteins (reviewed in Wozniak et al., 2004) and iv) specific mRNAs (reviewed in Tekotte & Davis, 2002). The critical question regarding the regulation of motor-cargo binding has only recently started to be unfolded when it was shown that the kinesin KIF17 ability to interact with cargo was inhibited through CaMKII phosphorylation (Guillaud et al., 2008). Dynein comprises a small homogeneous family compared to the kinesins and are either located in the cytoplasm or in cilia/flagellas as a part of the axoneme. Cytoplasmic dyneins are homodimers of heavy-chains and are expressed in all eukaryotes, while the axonemal dyneins are heterodimers or heterotrimers and are only expressed in specialized cells containing cilia or flagella. Cytoplasmic dyneins have been shown to interact directly or through adaptor proteins with, i) organelles, such as the Golgi 1. 2. ATP 3. ADP A A B - 4. ATP ADP ADP ATP A B + - B B + - 5. + - A i.e. the transition from polymerizing to depolymerizing (reviewed in Howard & Hyman, 2007) and iii) tubulin heterodimer binding proteins, which function by sequestering tubulin from the polymerization process (reviewed in Howard & Hyman, 2007). In this thesis I have focused on functional analysis of two prominent microtubule destabilizing proteins, MCAK and Op18, and a detailed description on their structure and function will be given in Chapter 9. ATP B A + - + Fig. 14. ATP-dependent unidirectional translocation of kinesin along a microtubule 7. MICROTUBULE FUNCTION DURING THE INTERPHASE AND MITOSIS OF THE CELL CYCLE 7.1 A brief introduction to the mammalian cell cycle and its check points The term cell cycle is used to describe the series of events that culminates in cell division and the term interphase refers to all stages of the cell cycle except mitosis (Fig. 15). During the G1-phase the cell commits itself to enter the cell cycle and increase in size. DNA replication together with centriole duplication occurs during S-phase, followed by quality control of the replicated DNA during the G2-phase (Fig. 15). Cellular division compromises two main events, the segregation of the duplicated genome, termed mitosis, and the subsequent cytoplasmic division of the cell, termed cytokinesis (Fig. 15). G0 3. M G1 1. 2. G2 Interphase S 1. G1/S checkpoint Sense: Surroundings and DNA status Block: DNA replication 2. G2/M checkpoint Sense: DNA status and cell size Block: Mitotic entry 3. Spindle assembly checkpoint Sense: Chromosome attachment to the mitotic spindle Block: Chromosome separation and cytoplasmic division Mitosis Fig. 15. The cell cycle and its major checkpoints Various surveillance systems, collectively termed checkpoints, ensure the correct sequential order of cell cycle phases. Check points also monitor that each cell cycle phase is accurately completed before progression into the next phase is allowed. The mammalian cell cycle contains three major checkpoints (Fig. 15) that are in principle conserved among all eukaryotes. Progression through the cell cycle is regulated by the family of cyclin dependent kinases (CDKs). Both the activity and substrate specificity of the constitutively expressed CDK-proteins depend on specific cyclin partners, which are proteins that oscillate during the 8 interior for membrane proximal stabilization cues, i.e. +TIP proteins (Fig. 18). cell cycle. The temporal pattern of cyclin expression and degradation allows for the correct sequential order of the cell cycle. It is notable from the outline of key molecular events in Fig. 16 that the CDKs important for G1 and S-phases are primarily involved in transcriptional regulation, while the mitotic CDK1/cyclin B kinase is an executor of mitosis and many structural proteins are direct or indirect targets. 1. External stimuli Golgi 2. Selective stabilization of microtubules that randomly encounter membrane localized tip stabilizing proteins, which have been activated at the signaling site. Expression induced by mitogen signaling S Production of cyclin E 3. Cyclin E CDK 2 Golgi P Rb P Cyclin D Cyclin A CDK 1 Cyclin A Production of S phase components and cyclin A Dynein dependent re-orientation of the centrosome. Firing of replication origins 4. Golgi CDK 2 P Rb P P P Golgi G1 CDK 4/6 G2 P Prevention of re-replication CDC6 P Cyclin CDK 1 B M P Nuclear lamins CDC42 is a member of the Rho-GTPase family and has been shown to be essential for polarization of astrocytes (Etienne-Manneville & Hall, 2001). After local activation at the leading edge, CDC42 activates a signaling pathway that de-repress the activity of the +TIP protein APC in the proximity of the leading edge (Etienne-Manneville & Hall, 2003), which mediates local stabilization of microtubules (general principle outlined in Fig. 18). These microtubules are anchored to the plasma membrane through the interaction between APC and DLG1 (Etienne-Manneville et al., 2005). Stabilized microtubules at the leading edge are used for the re-orientation of the centrosome towards the leading edge by the dynein dependent pulling force (Etienne-Manneville & Hall, 2001; Palazzo et al., 2001). Migration of fibroblasts involves different regulatory proteins but polarization is achieved according to the same search and capture mechanism as in astrocytes. In fibroblasts microtubules are stabilized at the leading edge by members of the CLASP family of +TIP proteins, which are locally activated by a phosphatidylionositol-3 kinase (PI-3 kinase) dependent pathway (Akhmanova et al., 2001). The stabilized microtubules are thereafter anchored to the plasma membrane through the interaction between CLASPs and LL5beta or ELKS (Lansbergen et al., 2006). T-lymphocyte recognition of antigens on virally infected cells or specialized antigen presenting cells is associated with centrosome re-orientation (Geiger et al., 1982). In the case of a cytotoxic T-lymphocyte, the secretory apparatus is thereby localized in proximity to the target cell. Centrosomal reorientation in T-lymphocytes depends on Tcell antigen receptor mediated activation of the downstream effectors CDC42 (Lowin-Kropf et al., 1998; Stowers et al., 1995), ZAP-70 (Blanchard et al., 2002) and dynein (Combs et al., 2006). Fig. 16. The sequential activation of a family of cyclin-dependent kinases drive cell cycle progression 7.2 Microtubule dependent molecular motors organize the intracellular space Interphase microtubules are organized by the centrosome in most cells (see section 5.3 for exceptions), with the minus ends anchored within the centrosomal matrix and the plus ends extending out towards the cell periphery, resulting in a microtubule array focused at the center of the cell. The major function of the interphase microtubule system is to facilitate intracellular organization, which to a large extent involves kinesin and dynein mediated transport of different cargos along microtubules (see section 6.3). By this mean, microtubules organize cellular organelles and facilitate vesicle transport, e.g. in the secretory- and endocytotic pathways (Fig. 17). + 1. Nucleus 2. Endoplamic reticulum 3. Golgi apparatus 4. Mitochondrion 5. Vesicle in the secretory pathway 6. Vesicle in the endocytotic pathway 1. + 2. + 3. 6. Kinesin + 5. + 4. Stable microtubules serve as rail tracks, transporting membrane vesicles and actin regulatory proteins to support directional cell migration. Fig. 18. Selective stabilization of interphase microtubules results in cellular polarization Chromatin condensation and nuclear envelope disassembly, i.e. mitotic entry Condensin Chomosome separation and mitotic exit triggered by activation of APC/C Dynein In a non-polarized cell, microtubules exhibit dynamic instability, i.e. they constantly grow and shrink in a random manor. + Fig. 17. Microtubule mediated organization of organelles during interphase Polarized intracellular transport of various cargos, e.g. membrane vesicles or actin regulatory proteins, is important during cellular migration. Cell polarization during migration involves re-orientation of the centrosome, which is accompanied by the Golgi apparatus, towards the leading edge of the cell. The search-and-capture mechanism of microtubules provides the cell with a system to probe its 7.3 Microtubules segregate the duplicated chromosomes during mitosis During mitosis, chromosomes duplicated during the S-phase are segregated into two daughter cells. This is accomplished by assembly of an elaborate microtubule structure, termed the 9 and attach to all kinetochores, which is the hallmark of the prometaphase (Fig. 19 & 22; reviewed in Walczak & Heald, 2008). mitotic spindle, that is responsible for organizing the sister chromatid pairs onto the cellular equator and subsequently segregate them to the opposite cell poles (Fig. 19; reviewed in Walczak & Heald, 2008). At the entry of mitosis, the interphase microtubule system depolymerizes and the centrosomes duplicated during the S-phase separate to initialize the formation of two separate microtubule arrays. During the initial phase of mitosis, termed prophase (Fig. 19), the two arrays are separated by sliding of anti-parallel microtubules by members of the kinesin-5 family (Fig. 20A&C; Kapitein et al., 2005). The positioning of the division plane is thereafter determined by interactions between specifically located dyneins and the astral microtubules as outlined in Fig. 21 (reviewed in Dhonukshe et al., 2007). Interphase 1. Astral microtubules are stabilized by tip binding proteins at cell poles 2. Membrane anchored dynein translocates along astral microtubules - 1. + 2. 3. Dynein mediated pulling forces relocate spindle poles to generate the correct division plane 3. Prophase Telophase/ cytokinesis Fig. 21. Establishment of correct division plane by astral microtubules and dynein Prometaphase 1 Microtubules restlessly search the cellular space... Anaphase 2 ...and are stabilized at the Metaphase kinetochores of the chromosomes AstralOverlapKinetochore MT Fig. 19. Microtubule dependent processes during different stages of mitosis Prophase A) * * * * 3 Finally both kinetochores capture microtubules from opposite centrosomes and the sister chromatid pair is positioned at the cellular equator by the polar ejection forces generated by microtubules marked with an asterisk Anaphase B B) Fig. 22. Search and capture of chromosomes by microtubules during the assembly of the mitotic spindle C) Plus end During prometaphase, kinetochores of sister chromatid pairs will progressively become attached to microtubules from the opposite spindle poles. Sister chromatid pairs are thereafter positioned near the cellular equator to form the metaphase plate (Fig. 19). This process is primarily thought to be mediated by pushing forces on the chromatids by non–kinetochore attached spindle microtubules; a phenomenon termed polar ejection forces (Fig. 22; Rieder et al., 1986). Since these forces are inversely proportional to the distance to the spindle poles, sister chromatid pairs will line up close to the cell equator, i.e. at an equal distance to each spindle pole. Prometaphase proceeds into metaphase at a stage in which all sister chromatid pairs are aligned at the cellular equator (Fig. 19). This is followed by anaphase, at which sister chromatids segregate to opposite poles (Fig. 19). Segregation requires dissociation of sister chromatids, which is executed by proteases that are activated by the Anaphase Promoting Complex (also known as the cyclosome). Following dissociation, individual sister chromatids are transported to respectively cell pole by two distinct mechanisms, namely i) depolymerizing kinetochoremicrotubules pull sister chromatids toward the pole (Fig 19), Bipolar kinesins Plus end Fig. 20. Bipolar kinesins mediate sliding of anti-parallel microtubules during mitosis A protein complex, termed the kinetochore, assemble during prophase on distinct structures on the chromosomes, termed centromeres. Each sister chromatid contains only one centromere and consequently only have one kinetochore. Several +TIP proteins localize to the kinetochore and are thought to selectively stabilize microtubules that randomly encounter the kinetochore during prometaphase (Fig. 22; reviewed in Maiato et al., 2004). At the entry to mitosis the microtubules become more dynamic (see section 6.1), which facilitates probing of the cytosol for sister chromatid pairs by the search-andcapture mechanism. By this stochastic trial and error mechanism, microtubules have potential to rapidly localize 10 exposed, which is indeed a characteristic of microtubules during mitosis (see Fig. 8A). Defects in the spindle checkpoint increase the probability for aneuploidy, i.e. abnormal chromosome content, which is a characteristic of >90% of all human tumors. It is therefore not surprising that several of the components in the spindle checkpoint have been found to be mutated in cancer (reviewed in Kops et al., 2005). ii) sliding of anti-parallel overlap-microtubules pushes spindle poles further apart (Fig. 20B&C). The genetic cell division is completed in telophase (Fig. 19), when most of the microtubules return to their interphase configuration and the remnants of the overlapmicrotubules determine the position of the actin containing contractile ring, which will mediate the final stage of cell division, cytokinesis (reviewed in Barr & Gruneberg, 2007). Labeled tubulin 7.4 The role of kinetochore microtubule treadmilling to ensure bipolar kinetochore attachment The spindle checkpoint is a control mechanism that ensures that all sister chromatid pairs are aligned on the cellular equator before initiation of anaphase. The high degree of fidelity of this check point is explained by the fact that a single non-attached kinetochore is sufficient to prevent activation of the Anaphase Promoting Complex (reviewed in Kops, 2008). The spindle checkpoint is also responsive to errors in the attachment of microtubules at kinetochores, e.g. if microtubules from the same spindle pole are attached to both kinetochores of the sister chromatid pair, i.e. syntelic attachment (Fig. 23). Kinetochores distinguish between correct and incorrect attachments by a sensing mechanism that register the amount of tension applied over the kinetochores. Thus, given that bipolar attachment is required for tension, syntelic attachment will be sensed and the repair involves induced depolymerization at the kinetochore followed by a new round of search-and-capture (reviewed in Tan et al., 2005). Kinetochores with incorrect microtubule attachment recruit and activate proteins that cause depolymerization of the incorrectly attached microtubules. One of these depolymerizing proteins is the kinesin-13 member MCAK (Kline-Smith et al., 2004), which has been in focus during my thesis work, and detailed account of the structure and function of MCAK will be given in Chapter 9. Kinetochores under tension Metaphase - + Fig. 24. Treadmilling of microtubules visualized by labeled tubulin 8. THE IMPORTANCE OF CHROMOSOMAL INSTABILITY FOR TUMOR PROGRESSION 8.1 Tumor progression requires genomic instability Formation of human tumors is a multi-step process (Fig. 25), termed tumor progression, which requires alteration at several distinct levels of cell function. 1.- Uncontrolled cellular growth and avoidance of cellular death X X X X X X X X X X X X Anaphase 2.- Formation of new blood vessels 3.- Metastasis STOP Aneuploidy Kinetochores without tension Fig. 25. Key events during the development of a metastasizing tumor Genomic instability In solid tumors, alterations at six levels of cell function appear essential for malignant growth (Fig. 26), and these functional alterations have been termed the “Hallmarks of cancer” by Hanahan and Weinberg (Hanahan & Weinberg, 2000). These alterations are the result of accumulation and selection of random mutations in genes operationally termed oncogenes and tumor suppressor genes. Mutations in unrelated proteins may result in the same distinct change of cellular physiology. While certain mutations are frequent among various diagnostic groups of tumors, it has still been established during recent years that each tumor is unique with respect to the whole set of accumulated mutations (Fig. 27; Wood et al., 2007). Fig. 23. Tension dependent detection of misaligned chromosomes during prometaphase The source of tension is the treadmilling behavior of kinetochore microtubules. Treadmilling implies simultaneous association of subunits at the plus end and dissociation of subunits at the minus end, which result in an intrinsic flux of subunits without altering the length of the microtubule (Fig. 24; reviewed in Rogers et al., 2005). A prerequisite for treadmilling is that the minus ends at the centrosome are 11 also microtubule-regulatory proteins that are normally not involved in spindle-formation may acquire alterations in expression levels or function that serve to promote chromosomal instability in tumors (See section 9.8). Mutation 1 Self-sufficiency in proliferative signals 1. - Defective DNA repair 2 Insensitivity to anti-growth signals 2. Normal cell 3. 3 Evading cell death Tumor cell 4. 4 Limitless replicative potential 5 Sustained blood vessel formation 5. - Errors during chromosome segregation 6 Metastasis capability 6. Fig. 26. Essential traits that are acquired during tumor progression of solid tumors Fig. 28. Two distinct mechanisms for genomic instability in tumor cells 2 X X 5 3 6 X 4 5 X 1 4 X 3 X 2 X 1 9. MAJOR REGULATORS OF MICROTUBULE POLYMERIZATION 9.1 MCAK is a conserved catastrophe-promoting protein The conserved MCAK protein belongs to a unique group of kinesins, the kinesin-13 family (previously known as KIN I for their internal motor domain), which depolymerize microtubules rather than translocate along them (reviewed in Kinoshita et al., 2006). Human MCAK is preferentially expressed in proliferative cells (Kim et al., 1997) and has also been shown to be overexpressed in various solid tumors (Nakamura et al., 2007; Shimo et al., 2008). The pioneering study on the microtubule regulatory activity of MCAK was done using the Xenopus ortholog, originally termed XKCM1 (Walczak et al., 1996). The inactivation of MCAK in mitotic Xenopus egg extracts was shown to be accompanied by longer, less dynamic microtubules, and quantification of dynamic parameters revealed a 4-fold decrease in the catastrophe frequency (Walczak et al., 1996). Studies on purified MCAK revealed an interaction with both minus and plus ends of microtubules (Desai et al., 1999) and it has been proposed that ATP-independent diffusion along the sides of microtubules facilitates rapid targeting of MCAK to the microtubule ends (Helenius et al., 2006). The binding of MCAK to the microtubule ends promotes the curling of protofilaments, thereby structurally destabilizing the GTPcap, thus promoting a catastrophe, i.e. the transition from polymerization to depolymerization (Fig. 29; Desai et al., 1999). For recycling of MCAK, the energy from ATP hydrolysis is utilized to release the protein from the curling protofilament (Desai et al., 1999). Cancer #1 6 Cancer #2 Fig. 27. Mutations in many types of proteins are required for tumor progression 8.2 Genomic instabilities of tumors depend on two distinct mechanisms Given random acquisition of oncogenic mutations and that the mutation frequency in a normal cell is very low, cancer development is statistically improbable. However, mutations in genes encoding proteins involved in different types of surveillance and repair systems, which normally protect against genomic instability, are known to cause an increase in mutational frequency in tumors (reviewed in Cahill et al., 1999). Thus, inactivating mutations in genes encoding for such gene-products can be considered as a prerequisite for tumor progression. The most common causes for increased mutational frequency in tumor cells are mutations that either alter the function of proteins involved in DNA repair or proteins involved at some levels of chromosome segregation during mitosis (Fig. 28). The later mechanism behind genomic instability, termed chromosomal instability, is thought to be the major factor that causes genomic instability in human tumors (reviewed in Kops et al., 2005). Chromosomal instability seems primarily to be a consequence of defective microtubule attachments to kinetochores that evade detection by the spindle checkpoint. Several +TIP proteins, e.g. APC, which has been implicated in stabilizing microtubule attachments to kinetochores have been found mutated in tumors, which seems likely to promote chromosomal instability (Fodde et al., 2001; Kaplan et al., 2001). In this thesis, by analyzing the phenotype of a tumor specific point mutation in the Op18 gene, I will argue that 9.2 The function of MCAK during the cell cycle Studies in Xenopus egg extracts suggest that the activity of MCAK is low during interphase, which has been proposed to be an effect of the dominant counteractive activity of the microtubule-associated protein XMAP215. Moreover, the predominant microtubule-destabilizing activity of MCAK during mitosis has been explained by phosphorylation– inactivation of XMAP215 by mitotic kinases (Tournebize et al., 2000). The predominant and counteractive activities of 12 Beside its association to microtubules, TOGp, the human homologue of XMAP215, has been shown to bind tubulin heterodimers in vitro (Spittle et al., 2000) and XMAP215 coimmunoprecipitates with tubulin heterodimers in Xenopus egg extracts (Niethammer et al., 2007). XMAP215 and MCAK in frog egg extracts have formed the current paradigm on how the microtubule-system is regulated during interphase and mitosis. This paradigm is also presented in the most influential of current text books (e.g., Alberts et al., Molecular biology of the cell 5th edition, page 1080 and Lewin et al., Cells, page 341). However, our own studies in human cell-lines depleted for MCAK and the human homologue of XMAP215, TOGp, fail to reproduce the importance of these counteractive activities during both interphase and mitosis (Paper I). As will be extended on in the “Result and Discussion” section, I believe that our results from human cells refutes that MCAK represents a major microtubule-destabilizing activity. TOG-domain TOGp 1972 a.a. XMAP215 2065 Msps 2050 2015 DdCP224 Mor1 1979 Zyg-9 MCAK 1415 Dis1 882 Alp14 809 Stu2p Fig. 29. MCAK destabilizes both microtubule ends During mitosis, MCAK concentrates to kinetochores and spindle poles (Wordeman & Mitchison, 1995). Inactivation of MCAK, which was achieved by either microinjection of a dominant negative mutant (Kline-Smith et al., 2004; Maney et al., 1998) or RNA interference mediated protein knockdown (Paper I; Cassimeris & Morabito, 2004; Ganem et al., 2005) has only small effects on mitotic spindle assembly. Significantly, however, an increase of lagging chromosomes during both prometaphase and anaphase was observed (Kline-Smith et al., 2004; Maney et al., 1998; Andrews et al., 2004; Lan et al., 2004; Walczak et al., 2002). The current well established model implies that MCAK is specifically recruited to kinetochores with incorrect microtubule attachments, and subsequently destabilize these faulty attachments, see model in Paper II (Fig. 7), (reviewed in Walczak & Heald, 2008). The regulation of recruitment and activation of MCAK during this process is complex and is shown to involve the Aurora B kinase at multiple levels (reviewed in Walczak & Heald, 2008). 888 Fig. 30. The XMAP215/TOGP family of microtubule regulators. In vitro studies using purified tubulin heterodimers have revealed that XMAP215 causes a 7-fold increase in the polymerization rate specifically at the plus end (Gard & Kirschner, 1987). Two distinct mechanisms and mutually exclusive models have been proposed to explain the accelerated polymerization rate, namely i) XMAP215 serves as a template for tubulin oligomers and thereby facilitates delivery of preassembled protofilaments to the plus-end of microtubules (Fig. 31A; Kerssemakers et al., 2006; Slep & Vale, 2007) ii) XMAP215 binds processively to the plus end of the microtubule and facilitates repeated rounds of addition of tubulin heterodimers into polymerizing protofilaments (Brouhard et al., 2008), possibly through the recruitment of tubulin heterodimers by high affinity binding (Fig. 31B). 9.3 The evolutionary conserved XMAP215/TOGp protein family Members of the XMAP215/TOGp family are +Tip-proteins (see section 6.2) and are found in all eukaryotes (Fig. 30). In most species XMAP215/TOGp proteins are represented as a single essential gene. The most conserved feature of the XMAP215/TOGp family proteins is amino-terminal TOG domains, which mediate binding to tubulin (Al-Bassam et al., 2007). XMAP215/TOGp has been implicated in many levels of microtubule regulation, potentially by multiple distinct mechanisms (reviewed in Howard & Hyman, 2007). Electron microscopy studies have revealed an elongated shape for both Xenopus XMAP215 (Cassimeris et al., 2001) and the S. cerevisiae family member Stu2p (Al-Bassam et al., 2006). The crystal structure of a TOG-domain of the C. elegans family member Zyg-9 reveals a flat and paddle-shaped structure (Al-Bassam et al., 2007). The number of TOG domains in the protein varies between two and five, depending on species (reviewed in Ohkura et al., 2001). A) B) Pre-assembled protofilament High affinity surface Fig. 31. Possible mechanisms for the XMAP215 dependent increase in polymerization rate at microtubule plus ends Decreased amounts of XMAP215 in Xenopus egg extracts (Wilde & Zheng, 1999; Tournebize et al., 2000) has been shown to result in shorter microtubules, which was also observed when the protein was inhibited in intact Xenopus oocytes (Becker et al., 2003). Given the high degree of conservation among the XMAP215/TOGp family members (reviewed in Kinoshita et al., 2002), it has been assumed that the prominent microtubule stabilizing activity of XMAP215 observed in Xenopus model systems is general among eukaryotes. This concept was supported by the pronounced decrease of microtubule stability caused by mutations of the Arabidopsis homologue Mor1 (Whittington et al., 2001), the C. elegans homoluge Zyg-9 (Srayko et al., 2005), and the Drosophila homologue Msps (Moon & Hazelrigg, 2004). 13 However, depletion of the human homologue of XMAP215, TOGp, does not reveal detectable effects on the levels or stability of microtubules (Gergely et al., 2003; Paper I; Cassimeris & Morabito, 2004). By studies using purified components, it have also been shown that XMAP215 (Shirasu-Hiza et al., 2003) and Stu2p (van Breugel et al., 2003) have the potential to destabilize microtubules. This paradoxical finding might be explained by the documented ability of XMAP215/TOGp family members to bind to soluble tubulin heterodimers and possibly sequester them from microtubule polymerization under certain conditions. In intact cells, the microtubule destabilization activity has also been documented for overexpressed TOGp (Paper I). Interestingly, this activity was shown to be dependent on the microtubule destabilizer MCAK, but the mechanism behind this phenomenon remains unclear. Thus, current literature on the function of XMAP215/TOGp family members in various species is clearly difficult to boil down into a simple coherent picture. This is in part due to that these family members found in evolutionary divergent species are quite different with respect to size and numbers of the microtubule-binding TOG domains, which makes it unclear whether all of these proteins are true orthologs or just paralogs. 9.5 Counteractive activities between MCAK and XMAP215 family members A series of classical experiments in Xenopus egg extracts have had major influence over the current perception on the regulation of microtubule-polymer mass and dynamics through the cell cycle (reviewed in Andersen & Wittmann, 2002; Heald, 2000). In these experiments, XMAP215 and MCAK were shown to be the major counteractive microtubule regulatory activities. Thus, based on antibody mediated depletion of egg extracts, their relative activities appeared to largely determine the dynamic properties and polymer-content of the microtubule system (Figure 32; Tournebize et al., 1997). Interphase extract Mitotic extract Control 1 9 XMAP215 inhibited 7 21 2 3 (only 60% depletion) XMAP215 and MCAK inhibited 9.4 Function of XMAP215/TOGp family proteins during mitosis XMAP215/TOGp family proteins concentrate to spindle poles and spindle microtubules during mitosis (reviewed in Gard et al., 2004). This localization of XMAP215 family members has been shown to be dependent on TACC proteins (reviewed in Gergely, 2002) and possibly regulated by the Aurora A kinase (Barros et al., 2005; Giet et al., 2002). Studies on cells from various species lacking the XMAP215/TOG protein have revealed a prominent role in the organization of the mitotic spindle. Disorganized spindle poles and aberrant spindle morphology have been recorded in human cell lines (Gergely et al., 2003), Drosophila (Cullen et al., 1999) and C. elegans (Matthews et al., 1998). In human cells, depletion of TOGp results in multipolar spindles (Gergely et al., 2003), which is a phenotype that depends on the microtubule destabilizing activity of MCAK (Paper I; Cassimeris & Morabito, 2004). To explain the TOGp depletion phenotype in human cells, we have proposed a model in which TOGp is essential for inhibiting MCAK at the spindle poles, thereby ensuring the bipolarity of the spindle. We have subsequently extended our model to include the CaMKIIγ, which we have shown has an equal essential role in suppressing the activity of MCAK at the spindle poles (Paper II). Studies on XMAP215/TOGp family members in the two diverse yeast species S. pombe (Garcia et al., 2001) and S. cerevisiae (Kosco et al., 2001) have revealed a prominent kinetochore localization, which has not been observed in animal cells. Loss-of-function mutational studies in these two yeast model systems showed impaired microtubule attachment to kinetochores and failure to form bipolar spindles. These studies thereby reveal an essential role at the opposite end of microtubules compared to the human XMAP215/TOGp family member TOGp, which indicates major differences in the function of these proteins in diverse species. Relative catastrophe frequency Fig. 32. Counteractive activities of MCAK and XMAP215 in Xenopus egg extracts (adopted from Heald, 2000) The proposed model implies that the dominant microtubule stabilizing activity of XMAP215 generates the long and modestly dynamic microtubules seen during interphase. At the entry into mitosis, inactivation of XMAP215 is thought to relieve inhibition of MCAK, which would explain how the catastrophe promoting activity of MCAK mediates formation of short and highly dynamic microtubules during mitosis, but not during interphase (Fig. 32 and 33). Decreased activity of XMAP215 during mitosis is thought to be mediated by activation of Cdk1/Cyclin B, since XMAP215 is an in vitro substrate for this kinase (Vasquez et al., 1999). The polymerization rate of purified tubulin is slower than observed in intact cells and the frequency of catastrophes is low as depicted in Fig. 12. However, if purified XMAP215 and MCAK are added to purified tubulin, both the microtubule growth rate and catastrophe frequency will increase and under certain conditions be virtually identical to what is observed in intact cells (see Fig. 12) (Kinoshita et al., 2001). Taken together, these data were conceived as compelling evidence for a model in which microtubule dynamics in cells are primarily determined by the combined actions of XMAP215 and MCAK (Kinoshita et al., 2002). According to this model on collaborating activities of XMAP215 and MCAK, the counteractive activities earlier described in Xenopus frog egg extracts can be perceived as primarily important to determine the average length of microtubules. Subsequent studies in human cells (Paper I; Cassimeris & Morabito, 2004) have provided a very different picture of both the functions and interplay of XMAP215/TOGp and MCAK. The implications of these findings will be discussed in the “Results and Discussion” section (see section 12). 14 (Sellin et al., 2008) to regulate the level of tubulin heterodimers, which in the Drosophila study was shown to be an essential role. CDK1/Cyclin B MCAK TOGp A) B) N II I α β C Fig. 34. Microtubule destabilization by Op18 Fig. 33. Proposed model of counteractive microtubule-regulating activities of MCAK and XMAP215/TOGp during the cell cycle 9.7 Phosphorylation-inactivation of Op18 during interphase and mitosis Op18 is phosphorylated at four Ser residues (Ser-16, -25, -38, and -63) by multiple signal transducing kinases and mitotic kinases, which results in a partial or complete inactivation (Figure 35A; reviewed in Cassimeris, 2002). Expression of kinase-site deficient mutants of Op18, combined with detailed analysis of the phosphorylation state of endogenous Op18, indicate that spindle formation requires phosphorylation-inactivation of Op18 at the onset of mitosis (Figure 35B; Larsson et al., 1997; Larsson et al., 1995; Marklund et al., 1996) 9.6 Op18 - a developmentally regulated microtubuledestabilizing protein The ubiquitously expressed cytosolic protein Op18, also known as Stathmin, was initially isolated due to its phosphorylation in the response to external signals and upregulation in various malignancies (reviewed in Sobel, 1991). Op18 is developmentally regulated with expression levels up to 50 fold higher in neonatal tissue compared with the corresponding adult tissue (Koppel et al., 1990). Op18 is highly conserved among vertebrates and has also been identified in Drosophila (Ozon et al., 2002). Op18 was identified as a microtubule destabilizer in Xenopus egg extracts (Belmont & Mitchison, 1996) and in intact human cells (Marklund et al., 1996). Studies with purified components demonstrated that Op18 can specifically stimulate microtubule catastrophes (Fig. 34A (I); Belmont & Mitchison, 1996) or sequester tubulin heterodimers (Fig. 34A (II); Curmi et al., 1997). These discrepancies were resolved when it was shown that Op18, indeed, excesses both these activities, which in vitro are dependent on buffer conditions and can furthermore be attributed to distinct regions of the protein (Howell et al., 1999b). The Op18–tubulin complex consists of two tubulin heterodimers arranged head-to-tail (Steinmetz et al., 2000) with each of the two tandem helical repeats of Op18 binding alongside one heterodimer (Figure 34B; Gigant et al., 2000). Consistent with its microtubule regulatory activity, Op18 has been implicated in cell migration during Drosophila development (Borghese et al., 2006; Ozon et al., 2002) and invasive behavior and metastasis of solid tumor cells (Baldassarre et al., 2005; Belletti et al., 2008). Surprisingly, however, mice with the Op18 gene inactivated by gene targeting develop normally (Schubart et al., 1996), which contrasts to the essential role of Op18 during Drosophila development (Borghese, et al., 2006). Nevertheless, later studies revealed subtle neuronal phenotypes such as impaired regulation of innate and learned fear (Shumyatsky et al., 2005) and age-dependent axonopathy of both the central and peripheral nervous systems (Liedtke et al., 2002). The physiological relevance of Op18 was initially established by the finding that antibody mediated inactivation of Op18 in newt lung cells reduced catastrophe promotion and increased microtubule polymer content (Howell et al., 1999a). In human cells, depletion of Op18 by RNA interference is associated with a substantial shift of the tubulin pool towards the polymeric state (Paper III and IV). Furthermore, the Op18 protein has been shown both in Drosophila (Fletcher & Rorth, 2007) and human cell lines A) CaMKIV MAPK PKA Ser-16 Ser-25 Ser-38 PKX Signal transduction Ser-63 CDK PKX Op18 (149 a.a.) Cell cycle regulation B) Interphase (active) Op18 P P P P Mitosis Op18 (inactive!?) Fig. 35. Phosphorylation dependent regulation of the microtubule-destabilizing activity of Op18 Despite a consensus that Op18 is phosphorylationinactivated in human cell lines during mitosis, it appears that Op18 is sub-stoichiometrically phosphorylated in mitotic Xenopus egg extracts. Based on this model system, a model on phosphorylation gradients around condensed chromosomes was proposed, which implied that local phosphorylation-inactivation of Op18 occurs in the vicinity of condensed chromosomes (Andersen et al., 1997; Tournebize et al., 1997). However, the Op18 mouse knockouts are viable and fertile and there is no indication of chromosomal instability, as evidenced by unaltered incidence of tumors (Schubart et al., 1996). Finally, my own studies in human cell lines extensively depleted of Op18 by stable expression of interfering RNA do not reveal any alteration in cell growth or distinguishable spindle defects (Paper III). The basal level of Op18 phosphorylation is low in interphase cells and increases in response to stimulation of various signal-transducing receptor systems. Initial experiments in our research group demonstrated that phosphorylation of ectopically expressed Op18 on specific sites results in inactivation (Melander Gradin et al., 1997; Gradin et al., 1998). By co-expression of constitutively active protein kinases involved in signal transduction events, it was shown that site-specific Op18 phosphorylation may provide a 15 responsible for generating local accumulation of tubulin heterodimers as previously proposed by us (Holmfeldt et al., 2003a). direct mechanism to stabilize the microtubule system in response to external signals but the physiological significance was unclear. In the present thesis, this was addressed by a gene-replacement strategy combined with a physiologically relevant signaling event, as represented by T cell antigen receptor stimulation. This study revealed that cell surface receptor stimulation and activation of cognate kinase systems result in a dramatic microtubule stabilization that is strictly dependent on site-specific phosphorylation-inactivation of Op18 (Paper IV). Op18 P P P SCG10 P P P SCLIP P P P RB3 P Ubiquitous Neuronal P Palmitylated membrane localization domain 9.8 Dysregulation of Op18 in human malignancies Op18 has frequently been reported as overexpressed in various malignancies (reviewed in Mistry & Atweh, 2002). In prostatic adenocarcinomas it has been reported that low Op18 expression correlates with higher levels of differentiation of the tumor cells and increased patient survival (Friedrich et al., 1995). Recently, a somatic missense mutation in the Op18 gene was isolated in a human tumor (Misek et al., 2002). The mutation results in substitution of glutamine (Q) to glutamic acid (E) at amino acid position 18 and cells expressing the Q18ÆE mutant form of Op18 were also shown to form tumors when injected in immunodeficient mice (Misek et al., 2002). From previous studies on leukemia and lymphomas it is clear that Op18 expression levels are uncoupled from cellular proliferation (Brattsand et al., 1993; Nylander et al., 1995) and two recent studies have implicated Op18 mediated regulation of microtubules as important for tumor invasion and metastasis (Baldassarre et al., 2005; Belletti et al., 2008). Finally, we have reported that excessive Op18 activity during mitosis may serve to promote chromosomal instability (Paper III), a hallmark of most tumors, and the significance of our findings will be discussed in the “Results and Discussion” (see section 15). Fig. 36. The Op18/stathmin family 9.10 Microtubule stabilization by the ubiquitous expressed MAP4 Many diverse proteins have been found to associate with microtubules and in most cases the significance of the observed association is unclear. However, the classical microtubule-associated proteins (MAPs), MAP2, MAP4 and tau, have common features in their organization in distinct functional domains and are evolutionary related. MAPs bind via a conserved region to the outer wall of microtubules and stabilize them (reviewed in Mandelkow & Mandelkow, 1995). Only MAP4 is ubiquitously expressed, while the others are abundant proteins in neuronal tissues. MAP4 appears conserved among mammals and Xenopus expresses a protein with similar overall structure but with weak sequence homology (~30% identity) (reviewed in Andersen, 2000). The MAP4 primary-transcript can be spliced into three different isoforms, which differ by encoding tree, four or five 18 amino-acids imperfect repeats in the microtubule binding domain of the protein (Chapin et al., 1995). All three isoforms are large proteins with a molecular mass exceeding 200 kDa, which all share the potential to bind to microtubules. MAP4 can promote polymerization of purified tubulin (Bulinski & Borisy, 1980) and overexpressed MAP4 stabilizes microtubules in intact cells (Holmfeldt et al., 2002; Nguyen et al., 1997). Studies on the dynamic behavior of microtubules assembled from purified tubulin documented that MAP4 have the potential to increase the rescue, i.e. the transition from depolymerization to polymerization, frequency without significantly altering the other dynamic parameters (Fig. 37; Ookata et al., 1995). The microtubule stabilizing activity of MAP4 has been shown to counteract the destabilizing activity of katanin in Xenopus egg extracts (McNally et al., 2002), MCAK and the catastrophepromoting activity of Op18 in intact human cells (Holmfeldt et al., 2002). The physiological significance of MAP4 for the stability of microtubules is unclear, however, since antibody depletion of MAP4 in human fibroblasts and monkey epithelial cells failed to generate any observable effect on the microtubule system through the cell cycle (Wang et al., 1996). Nevertheless, depletion studies in human suspension growing cell model systems suggest that MAP4 and Op18 are the major counteractive microtubule regulating activities in human cells (Paper IV), and the significance of these findings will be discussed in the “Results and Discussion” (see section 13). 9.9 Relations between Op18 and the other Op18/ Stathmin family members Op18 is the founding member of a small family of conserved proteins, in which Op18 is ubiquitously expressed, while the other members, RB3, SCG10 and SCLIP, are expressed almost exclusively in the nervous system (Fig. 36; reviewed in Curmi et al., 1999). The neural-specific family members are expressed about 100-fold less compared to Op18 in brain cells (Bieche et al., 2003). In contrast to the cytosolic Op18 protein, all other members of the Op18/Stathmin family have a membrane targeting region, which mediates binding to intracellular membranes and concentrates preferably at the Golgi apparatus and to vesicle-like structures along neurites and at growth cones (Gavet et al., 2002). The tubulin binding affinity is much higher for the neural-specific family members compared to the Op18 protein, shown both in vitro (Charbaut et al., 2001) and in intact cells (Holmfeldt et al., 2003a). Tantalizing as it might seem, it is unlikely that the neuronal-specific family members influence the microtubule system by the same principle mechanism as the abundant and cytosolic Op18 protein. Given their low abundance, the neuronal-specific family members can only be expected to bind such small amounts of tubulin heterodimers that the effect on the tubulin monomer-polymer partitioning of the cell would be negligible. Considering their subcellular localization, it is instead possible to envision them as proteins 16 MAP4 MAP4 MAP4 MAP4 MAP4 MAP4 Fig. 37. Potential mechanism for MAP4 mediated rescue promotion 9.11 Regulation of MAP4 during interphase and mitosis Members of the PAR1/MARK family of kinases (reviewed in Hurov & Piwnica-Worms, 2007) phosphorylate conserved KXGS motifs that are present in all three classical microtubule associated proteins, which results in their detachment from microtubules (Fig. 38; reviewed in Drewes et al., 1998). MARK kinases phosphorylate MAP4 on Thr802, Ser-914 and Ser-1046 (Fig. 38; Illenberger et al., 1996). Studies on the cellular effects of MARK mediated phosphorylation have been centered on neurons, primarily explained by the Alzheimer´s disease specific phosphorylation on tau at Ser-262, and its significance in non-neuronal cells have only recently been addressed. In human cells we have demonstrated that MARK2 phosphorylation-inactivation of MAP4 has a profound effect on the tubulin partitioning status in cells (Paper IV). Negative regulation of MAP4 has also been shown to be mediated by members of the septin family of proteins (Fig. 38; Kremer et al., 2005). Septins are conserved proteins found among all eukaryotes except plants and have been described to have functions during cell division, cytoskeletal organization and membrane-remodeling events (reviewed in Weirich et al., 2008). In mammals, at least 12 distinct septins with multiple splice variants have been identified. A heterotrimer of Septins 2, 6 and 7 was shown to bind MAP4 and thereby inhibit MAP4 association to microtubules (Kremer et al., 2005). Interphase Mitosis PAR1/MARK family kinases CDK1 MAP4 Septins Septins Fig. 38. Proposed post-translational regulation of MAP4 during the cell cycle Phosphorylation of MAP4 by CDK1/cyclin B on Ser-696 and Ser-787, decreases its affinity for microtubules (Fig. 38; Ookata et al., 1997). The significance of this mitotic phosphorylation has been investigated in Xenopus cells by over-expressing a kinase-site deficient mutant of MAP4, which was shown to interfere with chromosome movement during anaphase (Shiina & Tsukita, 1999). Depletion of septins in human cells generates a similar phenotype, which is suppressed by the by co-depletion of MAP4 (Kremer et al., 2005). Together these data suggest the existence of two different pathways that are import for inhibition of MAP4 during mitosis, which based on the study of MAP4-septin interactions appear to be required for proper spindle function. Consistent with these recent reports, it was earlier shown that inhibition of MAP4 by injection of inactivating antibodies is not associated with any detectable effects on the mitotic spindle (Wang et al., 1996). 17 permitted gene-product replacement with finely tuned expression levels, which required alterations of codons to prevent targeting by the co-expressed shRNA. We have dedicated much time to optimize design of shRNAs in a shuttle vector context, and during initial experiments I scored with excitement many interesting phenotypes suggesting a cell cycle block during G1. However, it turned out in all cases that the observed growth arrest did not correlate with gene-product depletion, but rather that all shRNA sequences caused more or less a G1arrest and that some sequences for obscure reasons were much more potent in this respect than others. I therefore dedicated a lot of efforts to screen shRNA sequences and to adjust shRNA expression levels to achieve optimal depletion with minimal “non-sequence” specific G1-arrest. It seems likely that the observed G1-arrest is a consequence of inhibition of protein synthesis by, e.g., activation of double stranded RNA activated protein kinase (Sledz et al., 2003). It is notable that these artificial effects of both small interfering RNA and shRNA appear to be generally ignored in the community. Thus, there are several published examples of a claimed specific G1-arrest in response to RNA interference that I believe are artifacts. During optimization of this dual system for inducible gene-product replacement, I included many microtubule-regulatory proteins with “known” loss-offunction phenotypes. With the ambition to identify the major regulators of the microtubule-system during interphase and mitosis, it also became obvious to include the most intensively studied of all regulators, namely MCAK and TOGp in the initial optimization of the system. RESULTS AND DISCUSSION 10. AIMS: - To elucidate which microtubule-regulatory proteins are the major determinants of tubulin subunit partitioning - To explore the implications of site-specific Op18 phosphorylation-inactivation as a mechanism to stabilize the interphase microtubule-system in response to external signals - To search for mechanisms behind selection of Op18 overexpressing clones during tumor progression 11. PROLOGUE During my early period as a Ph.D. student, I was involved in mechanistic studies concerning Op18 mediated microtubule destabilization. At that time, two different activities of Op18, tubulin sequestering and microtubule end-specific catastrophe promotion, were suggested to either individually or in combination contribute to microtubule destabilization. However, it was notable that the physiological function of Op18 was largely unknown at that time and there were disparate ideas concerning an important role during either interphase or mitosis (see section 9.6), or alternatively, that Op18 may not be important at all since the Op18 deficient mouse strain developed normally and only subtle neurological defects had been reported. Based on these disparate views on the relevance of Op18 and which proteins were the major regulators of the microtubule system during interphase and mitosis, I refocused to establish a system that allowed gene-product depletion and exchange. My aim was a system that allowed counter-selection to provide stable and homogeneous cell populations that could be monitored for phenotypes for extended periods. In addition, it seemed also important to combine gene-product depletion with inducible gene-product exchange, to allow temporal studies of reversal of a loss-offunction phenotype. Inducible gene-product exchange was also essential since kinase-target site deficient mutants of Op18 were known to block cells during mitosis. This was at the early time of RNA-interference in mammalian cells, which implied a revolution within the field of molecular cell biology. Inspired by the initial report on expressed short hair-pin (sh)RNA (Brummelkamp et al., 2002), driven by RNA polymerase III promoters, we established this system in our EBV-based shuttle vectors (Fig. 39A). Due to the stringent replication control of the EBV-based vectors we could simultaneously express up to four different derivatives in cells, thereby generating cells depleted for multiple gene-products. We had previously used EBV-based vectors for inducible expression of cDNAs under the control of the hMTIIa promoter and developed protocols for optimized induced expression and for efficient suppression of this promoter (Fig. 39A). The present system of co-transfected EBV-based shRNA vectors and pMEP expression proved indeed very versatile for semi-stable depletion of multiple gene-products combined with inducible expression as depicted in Fig. 39B. This system also A) B) Inducible promoter cDNA X Resistance EBV Ori Constitutive promoter shRNA Y Resistance EBV Ori Fig. 39. EBV-based shuttle vector system used for manipulation of multiple gene-products in leukemic cells 12. THE MCAK AND XMAP215/TOGp PROTEINS ARE NOT IMPORTANT FOR THE CONTROL OF TUBULIN SUBUNIT PARTITIONING BUT HAVE ESSENTIAL INTERCONNECTED FUNCTIONS DURING SPINDLE ASSEMBLY 12.1 Background Previous studies in Xenopus egg extracts had suggested that MCAK and XMAP215/TOGp exerts predominant counteractive activities on the microtubule system throughout the cell cycle (see section 9.5). It therefore felt important to document the role of these regulators in intact human cells and thereby allow a comparison with the regulatory importance of Op18 and other microtubule regulators. However, although we found clear-cut evidence for MCAK and XMAP215/TOGp interplay in human cells, the functional importance of this interplay appeared completely different as compared with Xenopus egg extracts. Most importantly, these two regulators do not have any significant 18 microtubule-content, it is clear that depletion of XMAP215/TOGp has completely different consequences in human somatic cell lines and Xenopus egg extract model systems. I therefore conclude that TOGp does not have a general role to stabilize microtubule polymers in human cells, but instead performs a localized counteraction of MCAK at microtubule minus ends during mitosis (Fig. 40A-B). role in the regulation of the interphase microtubule systems. Thus, our initial experiments to understand the significance of the claimed counteractive activities of MCAK and XMAP215/TOGp expanded and resulted in the discoveries presented in Papers I and II. In this thesis I will take the opportunity to focus on aspects that are not already discussed in these reports. Mitosis Physiological levels Interphase A) B) MCAK TOGp Overexpressed 12.2 XMAP215/TOGp is not essential to enhance the polymerization rate in human somatic cells As outlined in the introduction section, an elegant series of recent structural and functional characterization has established XMAP215/TOGp as a prototype for a protein that has potential to facilitate increased rates of both polymerization and depolymerization of microtubules. It is notable that a modest 60% depletion in Xenopus egg extracts significantly reduced the polymerization rate at least in interphase cells. It seems reasonable to assume that the high rate of polymerization is required for the complex process of spindle formation. If this assumption is correct, it follows that the TOGp depletion phenotypes in mammalian cells indicate that this function of TOGp is redundant (note that codepletion of TOGp and MCAK allows bipolar spindle formation (see Paper I, Fig. 2), which implies that other as yet unidentified proteins may serve to facilitate the high rates of polymerization/depolymerization in TOGp/MCAK codepleted cells. The alternative interpretation, namely that >95% depletion is not sufficient to abolish this function of TOGp, seems less likely. C) Fig. 40. Model of observed interplay between MCAK and TOGp in human cells The discrepancy concerning the role of TOGp may be explained by differences in species, cell types or experimental system (i.e. Xenopus egg extracts versus human somatic cell lines). There are several lines of evidence that microtubules are regulated differently in embryonic and somatic cells, but a direct comparison within a single species is lacking. For example, the alteration of microtubule dynamics at mitotic entry appears very different since there is an ~7 fold increase in catastrophe-frequency in the embryonic egg extracts compared to only an ~2 fold increase in somatic mammalian cells (Belmont et al., 1990; Rusan et al., 2001). Independent evidence that TOGp is of major importance for the general microtubule stability in embryonic cell systems but not in somatic cells has been provided by studies in the nematode C. elegans and the fruit fly Drosophila. Thus, the XMAP215/TOGp family member Zyg-9 appears essential for the stability of meiotic spindle microtubules (Matthews et al., 1998), while the phenotype of partial depletion of the Drosophila XMAP215/TOGp family member Msps in larval brain cells is reminiscent of phenotypes of somatic mammalian cells (Cullen et al., 1999), i.e. spindle disorganization without a clear-cut decrease of microtubule stability. It may be speculated that an embryonic cell, i.e. an egg cell with a very large intracellular space, requires spindle microtubules with higher dynamicity to probe the cellular space within a reasonable time frame. In Xenopus egg extracts the large increase in catastrophes at mitotic entry has been shown to be dependent on MCAK (see Fig 32). In human cell lines, however, we find no evidence for a general role of (cytosolic) MCAK for microtubule dynamicity during mitosis (see below). It seems therefore likely that MCAK is less active in somatic cells, which would explain why TOGp mediated counteraction is not important for the general microtubule stability. 12.3 Differential phenotypes of XMAP215/TOGp and MCAK depletion in human cells and Xenopus egg extracts Analysis of the microtubules in Xenopus egg extracts has revealed that even a partial depletion (60%) of XMAP215/TOGp cause a striking increase of catastrophepromotion (Tournebize et al., 2000). By codepletion/inactivation of MCAK, it was evident from this report that a prime importance of XMAP215/TOGp was to counteract MCAK dependent catastrophe-promotion. Moreover, with an efficiency of depletion approaching 90%, very short or no microtubules were observed, which precluded analysis of microtubule dynamics. Thus, XMAP215/TOGp is certainly a dominant microtubule stabilizer in this embryonic model system. At a first glance at data, it also appears reasonable to assume that this dominance reflects the requirement to counteract MCAK activity, which seems to provide a major destabilizing activity during both interphase and mitosis. Given the dominance by which XMAP215/TOGp suppresses catastrophe-promotion in Xenopus egg extracts (see Fig. 32) it was surprising that an essentially complete depletion of the human homologue TOGp does not produce detectable alterations in tubulin partitioning during interphase or mitosis (Paper I, Fig. 6A and our unpublished data). Furthermore, even though depletion of TOGp resulted in a dramatic phenotype manifested as dissorganized spindles (Paper I, Fig. 2), it is notable that co-depletion of MCAK reversed this phenotype since the spindle poles in double depleted cells were well organized and at least 75% of all cells formed bipolar spindles (Paper I, Fig. 2). Thus, since TOGp-depletion does not alter tubulin partitioning, i.e. 19 abnormality was monoastral spindles, which was found in ~25% of all mitotic cells (Paper I, Fig. 2). The monoastral spindle phenotype in MCAK depleted cells has been reported by others (Kline-Smith & Walczak, 2002). The mechanism behind these monoastral spindles is unclear but may involve i) failure to destabilize the interphase microtubule array at mitotic entry, ii) nonsuccessful centrosome separation during prophase, or iii) failure to repair syntelic microtubule attachment to chromosomes. While we were unable to detect a significant overexpression or depletion phenotype in interphase cells, the first alternative appears less likely. However, since MCAK to some extent seems to accumulate to the centrosome in mitotic cells, it seems plausible that a local MCAK mediated increase of microtubule dynamicity may be of importance for centrosome separation. It seems also plausible that excessive syntelic microtubule attachment may cause regression of a bipolar spindle to an apparent monopolar state. Taken together, our studies seem very clear in that MCAK does not exert significant global destabilizing/catastrophe-promoting activities during either interphase or mitosis. It follows that the important function of MCAK in mammalian somatic cells is as a locally acting depolymerase that is involved in a regulatory net-work, required for high fidelity spindle formation. However, future studies are required to establish whether the apparent insignificance of globally acting MCAK also applies to mammalian egg cells with a large cytosolic space and lack of cell cycle check-point controls. 12.4 The overexpression phenotypes of MCAK and TOGp reveal an unexpected interplay The differences in phenotypes of XMAP215/TOGp depleted Xenopus egg extracts and human cell lines might, as argued above, be the result of different levels of MCAK activity in embryonic and somatic cells. It was therefore surprising to us that the MCAK overexpression phenotype was unaltered by TOGp-depletion (Paper I, Fig 6C). It was even more surprising that TOGp overexpression resulted in a significant decrease of polymeric tubulin and that this phenomenon depends on endogenous MCAK (Fig. 40C; Paper I, Fig. 7) While these data are consistent with interplay between TOGp and MCAK, the dependency on MCAK for the observed unexpected TOGp mediated microtubule destabilization reveals that the simple model on counteractive activities (see Fig. 33) is not correct. It is notable that ~25-fold overexpression of MCAK in human cells results in a quite modest destabilization of both interphase and mitotic microtubules, which appears surprising since MCAK clearly exerts a potent microtubule destabilizing activity as analyzed in Xenopus egg extracts and in vitro (Walczak et al., 1996). It is also notable that the presence of endogenous TOGp does not influence the MCAK overexpression phenotype (Paper I, Fig. 6C). The only evidence for counteractive activities between MCAK and TOGp that we observed was that overexpressed TOGp to some extent rescued spindle formation in MCAK overexpressing cells, which appears consistent with the model derived from Xenopus egg systems. However, while this rescue effect was evident on the level of the functionality of spindles (Paper I, Fig. 5), it was not manifested on the microtubule polymer content (data not shown). The result discussed above indicates the presence of microtubule-stabilizing protein(s) in my cell model system that efficiently counteracts the documented potent catastrophe-promoting activity of MCAK. While the identity of this counteractive protein(s) is unclear, I believe that my reports on MAP4 mediated counteractions of catastrophe promoting proteins show that MAP4 at least in certain human cell types will be of significance (Holmfeldt et al., 2002). 12.6 TOGp provides protection from centrosomal MCAK activity, which is essential for bipolar spindle formation Previous reports have revealed an essential role for XMAP215/TOGp family members for spindle organization in Drosophila (Cullen et al., 1999) and human cells (Gergely et al., 2003), which is manifested by disorganized multipolar spindles in cells lacking XMAP215/TOGp. Our depletion studies of TOGp confirmed the multipolar spindle phenotype, and moreover showed that formation of the multipolar spindles was dependent on the activity of endogenous MCAK (Paper I, and Fig. 2). Co-depletion of MCAK not only suppressed the formation of multipolar spindles but also restored cell proliferation to a level observed after depletion of MCAK alone (Paper I, Fig. 1). The demonstrated MCAKdependence for the multipolar spindle phenotype in TOGpdepleted cells was concurrently shown in a study using HeLa cells (Cassimeris & Morabito, 2004). A possible mechanism for the formation of multipolar spindles in TOGp depleted cells may be nucleation of microtubules from disintegrated centrosomes. Indeed, Gergely et al. found γ-tubulin, a protein primarily concentrated at the centrosome, at all spindle poles in multipolar spindles formed in TOGp depleted cells (Gergely et al., 2003). However, time-lapse analysis of mitosis in TOGp depleted cells revealed that chromosomes aligned near the cellular equator before reverting to a more disorganized state, which indicate that the multipolar spindles are formed from disintegrating bipolar spindles (Gergely et al., 2003). Our analysis of the localization of the centrosomal protein pericentrin revealed that multipolar spindles, formed in 12.5 Confirmation that MCAK function is important for spindle fidelity Previous reports have characterized MCAK as a globally acting and major catastrophe promoting activity in mitotic Xenopus egg extracts (Tournebize et al., 2000; Walczak et al., 1996). More recent studies in mammalian cells have also implicated MCAK as a locally acting microtubule depolymerase, which facilitates repair of incorrectly attached microtubules at kinetochores (see Fig. 23; Kline-Smith et al., 2004; Maney et al., 1998). Since either of these functions depends on the depolymerizing/catastrophe-promoting activity of MCAK, these functional roles of MCAK are not mutually exclusive. Our studies on tubulin partitioning during interphase or mitosis did not reveal any significant effect of >90% depletion of MCAK (Paper I, Fig. 6A, data not shown). However, determination of cell growth still revealed about 50% inhibition (Paper I, Fig. 1), which was associated with a modest increase of the mitotic index and spindle abnormalities (Paper I, Fig 2). The most prominent spindle 20 from the spindles observed in TOGp-depleted cells (Paper II, Fig. 1). In addition, using the CaMKII specific drug KN93, we observed similar types of multipolar spindles. Finally, we also demonstrated that the phenotype of CaMKIIγ-depletion was dependent on endogenous MCAK, which firmly established the significance of this phosphorylation-pathway (Paper II, Fig. 2). TOGp depleted cells, only contain two centrosomally associated microtubule asters and additional acentrosomal microtubule asters (Paper I, Fig. 3A-B). Furthermore, through the microtubule minus end marker NuMa we established that all microtubule asters of the multipolar spindles were gathered at the microtubule minus ends (Paper I, Fig. 3C). Taken together, these data indicate that the dissorganized multipolar spindles in TOGp-depleted cells evolve by the gradual release of spindle microtubules from centrosomal microtubule asters, with the subsequent focusing of non-centrosomal microtubules by dynein molecular motors. Given that TOGp appears to be enriched at the centrosome (Charrasse et al., 1998), we have proposed that TOGp is essential for the protection of exposed microtubule minus ends against MCAK mediated destabilization at the centrosome during mitosis. (Paper I). Thus, according to our model, removal of TOGp allows access to MCAK at the centrosome, which causes destabilization of minus-ends and subsequent release of microtubules as depicted in Fig. 41, II. This model readily explains why co-depletion of MCAK suppresses the formation of multipolar spindles and that the phenotype of MCAK/TOGp co-depleted cells appears indistinguishable from the phenotype of cells depleted of MCAK alone, i.e. bipolar spindles with potential missattachments (Fig. 41 (IIIa)) or monoastral spindles (Fig. 41 (IIIb)). In addition, this model is also consistent with my proposal in section 12.5, namely that MCAK in mammalian somatic cells primarily acts as a locally acting de-polymerase involved in regulatory net-works that is required for high fidelity spindle formation. Fig. 41. Model depicting the role of kinetochore localized MCAK during spindle assembly and the regulatory mechanisms that ensure suppression of MCAK at the centrosome. Besides the significance of discovering the importance of negative regulation of MCAK activity, which may be exerted locally at centrosomes, our findings also provided a tool that allowed an independent confirmation of our model of TOGp and MCAK interplay at the centrosome. Thus, using a system of inducible TOGp expression in cells depleted of CaMKIIγ, or alternatively, inducible CaMKIIγ expression in cells depleted of TOGp, I believe that we could provide formal evidence that our model on the protective function of TOGp at the centrosome presented in Paper I is in principle correct (Paper II, Fig. 6). A simplified version on this model is given in Fig. 41. 12.7 The functions of both CaMKIIγ and TOGp are essential for protection of spindle microtubules from centrosome-directed MCAK activity An objection to the model outlined in section 12.6, as presented in our first report on MCAK/TOGp interplay (Paper I), was that the model predicts that excessive MCAK activity would result in the same phenotype as TOGp depletion, which according to the literature is not the case. However, in Paper II we prepared an MCAK derivative that was expressed at 5-fold higher levels than the previously used derivatives (Paper II, Fig. 3). Under these conditions our data clearly demonstrated that excessive MCAK activity has potential to generate the same phenotype as TOGp depletion, namely, dissorganized multipolar spindles (Fig. 41 (IV)). Our studies on CaMKIIγ mediated regulation of MCAK derived from the observation by our colleague Claire Walczak that MCAK is phosphorylated by this kinase in vitro. Ironically, we have in Paper I used a CaMKIIγ-specific shRNA derivative as a negative control, which seemed appropriate since this derivative allowed ~60% depletion. My initial experiment indeed documented that a constitutively active CaMKIIγ derivative suppresses the microtubuledestabilizing activity of ectopic MCAK (Paper II, Fig. 4). This evidence prompted us to reassess the phenotype of CaMKIIγ-depletion using new highly efficient shRNA derivatives that resulted in >95% specific depletion of CaMKIIγ. Under these conditions of extensive CaMKIIγ depletion we observed, to our delight, abundant dissorganized multipolar spindles that were indistinguishable 13. PHOSPHORYLATION-REGULATION OF Op18 AND MAP4 CONTROLS THE PARTITIONING OF TUBULIN SUBUNITS IN THE INTERPHASE MICROTUBULE SYSTEM 13.1 Background As shown in Papers I and II, the MCAK and TOGp proteins are not of any significance for tubulin monomer-polymer partitioning in mammalian cells. Given the original aim to identify regulators of tubulin partitioning, we carried on by evaluating the physiological significance of two other potentially important microtubule regulatory proteins, Op18 and MAP4, which had previously been shown to exert counteractive activities (Holmfeldt et al., 2002). Both of these proteins have previously been inferred as significant regulators of microtubule content (Drewes et al., 1997; Marklund et al., 1996; Melander Gradin et al., 1997). While there has been a consensus that both Op18 and MAP4 have the potential to regulate the interphase array, the literature concerning a potential role of these microtubule 21 kinetochores in the large intracellular space of egg cells (Andersen et al., 1997; Tournebize et al., 1997). An obvious argument against this model is that Op18 mouse knockouts are viable and breeds normally (Schubart et al., 1996). Moreover, the reports on spindle-defects in K562 cells partially depleted of Op18 using antisense RNA inhibition (Iancu et al., 2001; Luo et al., 1994), I am convinced represent artifacts that derive from the use of anti-sense RNA to decrease Op18 content. regulators during spindle assembly has been, to say the least, confusing (Chang et al., 2001; Iancu et al., 2001). However, results presented in Paper II (Fig. 2) and Paper III (Fig. 1) show that depletion of either MAP4 or Op18 has no detectable phenotypes during spindle assembly. In the case of Op18, this would be consistent with the previously proposed phosphorylation-inactivation of Op18 during mitosis (see Fig. 42; Marklund et al., 1996). Moreover, analysis of cells partially (Paper I, Fig. 6A) or extensively (Paper III, Fig. 1) depleted of Op18 clearly established Op18 as the predominant regulator of microtubule-content in the K562 leukemia cell line. These previous publications from us and others provide the background to the experimental approaches and analysis of tubulin partitioning presented in Paper IV. I will use this thesis as an opportunity to focus on aspects that are not discussed in this publication. 13.3 The significance of MAP4 and Op18 as phosphorylation-responsive regulators of tubulin subunit partitioning To evaluate the physiological importance of counteractive MAP4 and Op18 activities, we used the principle genetic approach described in Papers I and II. We have spent a lot of efforts in screening targeting sequences for shRNA shuttlevector derivatives. Most of these shRNAs mediated only partial downregulation of Op18 and MAP4 in the range 50 to 90%. In the case of Op18, 70% depletion was sufficient for a dramatic increase of microtubule-content (Paper I, 6A). However, we found that a partial depletion of MAP4 had no detectable effect and it required extensive screening and modifications of MAP4 shRNA derivatives to establish a condition in which depletion exceeded 85% and that was not associated with non-specific growth inhibitory effects by shRNA. Under these conditions of optimized extensive MAP4 depletion, we indeed observed a substantial decrease of microtubule content without significant effects on cell growth. Thus, while a partial depletion of Op18 is sufficient for a clear phenotype, the significance of MAP4 for microtubule stability is only evident under conditions of essentially complete depletion. These findings were not commented on in Paper IV but are still worth considerations in a discussion of mechanisms by which Op18 and MAP4 exert their counteractive activities. By a combination of extensive shRNA mediated depletion and ectopic expression of Op18, we have established the dose-response of Op18 mediated microtubuledestabilization in K562 cells (Paper III, Fig. 5 and Paper IV, Fig. 4) and Jurkat T cells (Sellin et al., 2008). These studies have shown that the microtubule-system rapidly responds to changes of Op18 levels within the range of expression levels found in normal and malignant cells. However, by comparing the microtubule-phenotypes at various levels of MAP4 depletion, we noted that >80% depletion was required to even detect a decrease in microtubule content. This unpublished peculiarity of the dose-response of MAP4 depletion, indicates that the endogenous MAP4 level is in great excess over the level required for its microtubule stabilizing function. Op18 has been shown to have potential to rapidly regulate the microtubule-system by three distinct mechanisms, namely catastrophe-promotion, tubulinsequestering and by regulating the synthesis of tubulin heterodimers (see section 9.6). However, the relative importance of the mechanisms in various cell types remains unclear. Nevertheless, the demonstrated responsiveness of cells to variations within the physiological relevant range of Op18 levels is fully consistent with the conclusion in Paper IV that phosphorylation-inactivation is a relevant mechanism for alterations of the microtubule-system in response to signal transduction events. 13.2 Evidence for the importance of MAP4 and Op18 as interphase-specific counteractive regulators of tubulin subunit partitioning It is shown in Paper IV that MAP4 and Op18 are interphasespecific principal determinants of tubulin subunit partitioning in three human suspension growing model system (Paper IV, Fig. 1, and suppl. Fig. 1). It is also shown that MAP4 and Op18 exert phosphorylation-responsive counteractive regulation of the microtubule-system as depicted in Fig. 42 (Paper IV, Fig. 2-3 and suppl. Fig 3). Since we have developed a method to quantitate microtubule polymercontent in distinct cell cycle phases (Holmfeldt et al., 2003b), we were also able to address the importance of MAP4 and Op18 during the assembly of the mitotic spindle. Our results provided a clear-cut demonstration that these microtubuleregulatory proteins are only of significance during the interphase of the cell cycle (Paper III, Fig 1; Paper IV, Fig. 1). Thus, in all cell types in which Op18 and MAP4 are expressed at sufficient abundance, it seems reasonable to assume that these proteins are the principal determinants of tubulin subunit partitioning of the interphase microtubule system. CaMKIV PKA Op18 MARK2 MAP4 Fig 42. Model showing the phosphorylation-regulated balance between MAP4 and Op18 that determines the partitioning of tubulin during interphase in human cells Our evidence that MAP4 and Op18 have no functional role during spindle assembly are consistent with several previous reports and with that both MAP4 and Op18 appear to become phosphorylation-inactivated during mitosis (see section 9.7 and 9.11). However, published evidence suggest that Op18 is only partially phosphorylated in mitotic Xenopus egg extracts. In this embryonic model system, a model has been proposed in which local phosphorylationinactivation of Op18 occurs in the vicinity of condensed chromosomes, which would facilitate microtubule capture of 22 for gene product exchange. Generally this involves the exchange of the wild type protein with a protein in which the specific phosphorylated residue is exchange to a similar but non-phosphorylated amino-acid. In my thesis work, shRNA based gene product depletion was combined with inducible expression of mutant derivatives (see section 11). Since phosphorylation-site deficient Op18 derivatives are incompatible with cell growth (phosphorylation-inactivation is essential during mitosis, see Fig. 43), the rapid induction of the mutated gene product combined with well defined expression levels is an essential feature of the experimental system. The versatility of this system for inducible geneproduct exchange is illustrated by the data in Fig. 4 in Paper IV. Given the homology of MAP4 to neural classical MAPs (see section 9.10), it seems reasonable to assume that MAP4 increases microtubule content by direct stabilizing interactions with polymers. It seems therefore surprising that this stabilizing effect only requires a small fraction of the endogenous MAP4, as evidenced by our unpublished studies of human suspension growing cell model systems. Since the MAP4 levels of our hematopoetic cell model systems appear to be very similar to an epithelial cell line such as HeLa (Holmfeldt et al., 2002), which implies ~0.1% of total cellular proteins (Bulinski & Borisy, 1979), our findings are likely to have general implications. While I can not explain the apparent “excess” of MAP4, our findings have clear implications for the potential to regulate the microtubulesystem by posttranslational regulation of MAP4 activity. Thus, it is difficult to envisage that sub-stoichiometric phosphorylation-inactivation of MAP4 is of significance for alterations of the microtubule system. Moreover, the recent identification of septins as negative regulators of MAP4 (see section 9.11), makes it difficult to speculate on the importance of post-translational regulation of MAP4 activity. Interphase P P Op18 Op18 Op18 Regulatory phosphorylations in response to signal transduction events 14. IDENTIFICATION OF Op18 AS A MAJOR SIGNAL TRANSDUCTION RESPONSIVE REGULATOR OF TUBULIN SUBUNIT PARTITIONING 14.1 Background Previous works from our group have shown that expression of constitutively active derivatives of the signal transducing kinases CaMKIV and PKA results in increase of tubulin polymer levels (Melander Gradin et al., 1997; Gradin et al., 1998; Paper IV, Fig 2A-B). Since both CaMKIV and PKA have been shown to mediate phosphorylation-inactivation of overexpressed Op18 (Melander Gradin et al., 1997) (Gradin et al., 1998), it appeared likely that the increase in tubulin polymer levels seen in cells expressing constitutively active CaMKIV or PKA is a consequence of site-specific phosphorylation-inactivation of endogenous Op18. Reports from other laboratories have inferred many protein kinases as negative regulators of Op18 activity (le Gouvello et al., 1998; Parker et al., 1998; Daub et al., 2001). It is important to distinguish the role of signal transduction mediated Op18 phosphorylation and the apparently complete inactivation of Op18 by multi-site phosphorylation during mitotic entry. Thus, while Op18 phosphorylation during interphase may be of importance for cell communication, I view the complete phosphorylation-inactivation by mitotic kinase systems as a necessary action to protect the mitotic spindle from the otherwise deleterious destabilizing activity of Op18 as depicted in Fig. 43. As argued in section 13.3, the interphase microtubule system is clearly highly responsive to alterations within the range of Op18 expression levels observed in normal and malignant cells. Thus, Op18 has an obvious potential to function as a phosphorylation responsive regulator of the microtubule system in cell types in which it is expressed at sufficient abundance. Mitosis P P P Op18 Active P P P P Op18 Stoichiometric Multi-site phosphorylation Fig. 43. Model depicting the role of phosphorylationinactivation of Op18 during interphase and mitosis Using this system, we first established a causal relationship between site-specific Op18 phosphorylation and the previously reported increase in microtubule content in cells induced to express constitutively active CaMKIV or PKA (Paper IV, Fig. 5). It should be noted that these experiments reflect the maximal impact of Op18 phosphorylation since the induced constitutively active kinases result in stoichiometric Op18 phosphorylation (Melander Gradin et al., 1997; Gradin et al., 1998). In all cases investigated, however, Op18 is sub-stoichiometrically phosphorylated by cell surface receptor mediated activation of cognate kinase systems (Brattsand et al., 1994; Marklund et al., 1993). The T cell antigen receptor activates an array of kinase cascades (reviewed in Matthews & Cantrell, 2006) but previous studies indicate that only about half of all Op18 molecules become phosphorylated (Marklund et al., 1993). Moreover, it has never been shown that T-cell activation is associated with alterations in microtubule content. In addition, phosphorylation occurs on two Ser-residues, namely Ser-16 and Ser-25, and the functional significance of Ser-25 has not been established. It was therefor important to evaluate the consequences of T cell antigen receptor stimulation for both tubulin subunit partitioning and the potential role of Op18 phosphorylation in such a response. In paper IV it is demonstrated that site-specific phosphorylation of Op18 is both necessary and sufficient for T-cell activation mediated microtubule-stabilization (Paper IV, Fig. 6). To my knowledge, this provides the first formally proven example of a signal-transduction chain that mediates changes of microtubule-density. 14.2 Identification of an Op18 dependent signal transduction chain for microtubule polymerization in response to T cell antigen receptor triggering To establish the causal relation between a specific phosphorylation event and a cell response requires a system 23 T-cell antigen receptor stimulation by an antigenpresenting cell results in the formation of an immunological synapse between the T-lymphocyte and the antigenpresenting cell. The minus end directed molecular motor dynein is recruited to the immunological synapse and is responsible for relocalization of the centrosome towards the antigen-presenting cell (Combs et al., 2006). Centrosomal relocalization will position the Golgi apparatus in the proximity of the antigen-presenting cell and the subsequent polarized secretion of effector molecules from the Golgi apparatus is essential for the correct immunological response (see section 7.2). I believe that the phosphorylationinactivation of Op18 after T-cell antigen receptor stimulation and the consequent increase in tubulin polymers may be important for microtubule targeting to the immunological synapse (Fig. 44). However, the significance of phosphorylation-inactivation of Op18 for centrosomal relocalization after T-cell antigen receptor stimulation still remains unclear and is currently under investigation in our laboratory. Selective stabilization by + Tip proteins P Op18 Tubulin heterodimer 15.2 The relationship between the oncogenic Q18ÆE substitution of Op18 and leukemia-associated upregulation of the wild-type protein Based on the pattern of an apparent synergistic action of the Q18ÆE mutation and the microtubule-stabilizing drug paclitaxel, it was originally proposed that the mutant form of Op18 may enhance the stability of microtubules (Misek et al., 2002). This proposal is clearly refuted in Paper III in which we show that the Q18ÆE mutation greatly enhances the microtubule-destabilizing activity of Op18. This increase in activity is manifested during both interphase and mitosis (Paper III, Fig. 2, 3 and 5). Interestingly, the consequence of this general increase of microtubule-destabilizing activity was found to be exacerbated during mitosis, because the mutation also provides a partial resistance to phosphorylation-inactivation (Fig. 45B; Paper III, Fig. 2G). Failure to inactivate Q18ÆE mutated Op18 during mitosis was found to be the cause of the observed aneugenic activities, manifested as spindle abnormalities (Paper III, Fig. 2D-E), polyploidization (Paper III, Fig. 5D) and chromosome loss (Paper III, Fig. 6). The aneugenic effects of the Q18ÆE mutant can be predicted to contribute to chromosomal instability in tumors, thus promoting tumor progression (see section 8). Interestingly, the potential destabilization of kinetochoremicrotubules during anaphase by uncontrolled Op18 activity can also be predicted to generate chromosomal instability without the requirement of a compromised spindle assembly checkpoint (Fig. 46). This implies a conceptional difference to the previously established role of oncogenic mutations of proteins involved in the spindle-assembly checkpoint (reviewed in King, 2008). T-cell receptor P P P P P P P P P Antigen presenting cell P P Fig. 44. Model of the potential physiological significance of phosphorylation-inactivation of Op18 during T-lymphocyte activation 15. EXCESSIVE Op18 ACTIVITY DURING MITOSIS RESULTS IN SPINDLE-DESTABILIZATION AND CHROMOSOMAL INSTABILITY 15.1 Background The name “Oncoprotein 18” was coined based on upregulated protein levels in human lymphoma/leukemias relative to non-transformed but actively proliferating lymphocytes (Hailat et al., 1990). As outlined in section 9.8, it has been proposed that increased Op18 levels may facilitate tumor invasion and metastasis. The general relevance of this proposal is questionable since suspension growing human leukemias are often found to express the most extreme levels of Op18 (~1% of total proteins; Brattsand et al., 1993). For a protein to formally qualify to be termed an “oncoprotein”, it should according to current standards contain tumor-specific mutations. These oncogenic mutations may either alter the expression level or the function of the gene-product. Luckily enough, a recent example of a tumorspecific mutation of Op18 has indeed been identified in a single clinical isolate of a human esophageal adenocarcinoma (Misek et al., 2002). This mutation, Q18ÆE, causes a shift in the mobility on 2D-gels due to the negative charge of glutamic acid. A demonstrated tumor-specific mutation provides a very strong link to tumor-progression (see section 8.1). However, it was not discussed in the original report how this single case of a tumor-specific Op18 mutation may relate to the fact that the wild type Op18 protein is frequently upregulated in an array of human malignancies. A) Normal cells Interphase Mitosis P P P P wt wt Active Inactive B) Tumor cells Interphase Mitosis Interphase P P P P Q18E Q18E Hyperactive Partially active Mitosis P P wt wt P P P P wt wt I II Fig. 45. Two distinct mechanisms that may cause chromosomal instability as a result of incomplete phosphorylation-inactivation of Op18 during mitosis Analysis of cells expressing Op18 at the excessive levels occasionally observed in leukemias revealed that also the wild-type Op18 protein under these conditions exert aneugenic activities (Paper III, Fig. 5-6). The mechanism behind this phenomenon may reflect that excessive Op18 levels saturate mitotically active kinase systems (Fig. 45B (I)) and/or that phosphorylation does not imply a complete inactivation (Fig. 45B (II)). Thus, both the hyperactive 24 significance of Op18 overexpression in human tumors is to increase chromosomal instability. This because mouse cells probably have close to “optimal” levels of chromosomal instability already prior to transformation, which would imply that any oncogenic alteration that functions to increase chromosomal instability in human tumors will not be found in mice. Q18ÆE mutant and overexpressed wild-type Op18 generates chromosomal instability by exerting uncontrolled microtubule-destabilization during both the early and late phases of mitosis. 16. FINAL CONCLUSIONS Chromosomal loss (encapsulated into a ”micro-nucleus”) - The essential roles of TOGp and CaMKIIγ involve protection of spindle microtubules against MCAK activity at the centrosome (Fig. 47A) - Abundantly expressed MAP4 and Op18 are the principle regulators of tubulin subunit partitioning - MAP4 and Op18 counteractive and activities (Fig. 47B) - Op18 phosphorylation is both necessary and sufficient for T-lymphocyte activation mediated microtubule-stabilization, which is a prerequisite for polarization (Fig. 47C) - Aberrant Op18 activity during mitosis results in chromosomal instability in tumors Fig. 46. Chromosomal loss caused by destabilization of kinetochore-microtubules during anaphase 15.3 The significance of life-span of animals for the demand for increased chromosomal instability during tumor-progression It is notable that Op18 overexpression is a common phenomenon in human malignancies, while few studies have indicated overexpression in naturally occurring or artificially induced tumors in mice model systems (concluded by exhaustive PubMed searches). An obvious difference between mice and humans is that tumor progression in mice is a matter of weeks, while it in general is a matter of decades in humans. This is despite that the cell cycle times are similar in mammalian cells. It seems therefore reasonable to assume that the demand for chromosomal stability of somatic cells depends on the life-span and time-span of the reproductive cycle of a metazoan. This assumption is indeed supported by the fact that the extremely short reproductive cycle of mice (~7 weeks) is associated with a less stringent spindle assembly checkpoint as compared to humans. Thus, while microtubule poisoning drugs cause a persistent mitotic arrest of non-transformed human cells, cultured mouse cells have an intrinsically high propensity to undergo “mitotic slippage” and thereby enter a tetraploid pseudo G1 state (shown in e.g. Cross et al., but incorrectly interpreted as evidence for a p53dependent mouse spindle checkpoint, (Cross et al., 1995)) As outlined in section 8.2, most tumors appear more or less chromosomally unstable, which precludes correlative studies on the clinical relevance of our finding that both the Q18ÆE mutation and overexpression of wild type Op18 contribute to chromosomal instability. However, I believe that the arguments presented above concerning the time perspective of tumors originating from either mice or humans are relevant for the understanding of how dysregulated Op18 may contribute to tumor progression. Thus, I interpret the apparent absence of convincing reports on Op18 overexpression in mice in favor of a model in which the main Mitosis have interphase-specific, phosphorylation-inactivated Interphase Signal transducing kinases A) C) T-cell receptor B) PAR-1/MARK family kinases MCAK TOGp Op18 MAP4 CaMKIIγ Fig. 47. Main findings of the thesis 17. ACKNOWLEDGEMENTS After these nine fantastic years, possible the best years of my life yet, I wish to thank all of you concerned – in private. 18. REFERENCES Ahmad FJ, Yu W, McNally FJ, Baas PW (1999) An essential role for katanin in severing microtubules in the neuron. 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