Myosin 10 is a vertebrate specific actin-based motor protein that is expressed in a variety of cell types. Cell biological evidences suggest that myosin 10 plays a role in cargo transport and filopodia extension. In order to fully appreciate these physiological processes, it is crucial to understand the motor property of myosin 10. However, little is known about its mechanoenzymatic characteristics. In vitro biochemical characterization of myosin 10 has been hindered by the low expression level of the protein in most tissues. In this study, we succeeded in obtaining sufficient amount of recombinant mammalian myosin 10 using the baculovirus expression system. The movement directionality of the heterologously expressed myosin 10 was determined to be plus end-directed by the in vitro motility assay with polarity-marked actin filament we developed. The result is consistent with the proposed physiological function of myosin 10 as a plus end-directed transporter inside filopodia. The duty ratio of myosin 10 was determined to be 0.6~0.7 by the enzyme kinetic analysis, suggesting that myosin 10 is a processive motor. Unexpectedly, we were unable to confirm the processive movement of dimeric myosin 10 along actin filaments in a single molecule study. The result does not support the proposed function of myosin 10 as a transporter. One possible explanation for this discrepancy is that the apparent nonprocessive nature of myosin 10 is important for generating sufficient force required for the intrafilopodial transport by working in concert with numbers of other myosin 10 molecules while not interfering with each other. Altogether, the present study provided qualitative and quantitative biochemical evidences for the better understanding of the motor property of myosin 10 and of the biological processes in which it is involved. Finally, a general molecular mechanism of myosin motors behind the movement directionality and the processivity is discussed based on our results together with the currently available experimental evidences. The validity of the widely accepted ‘leverarm hypothesis’ is reexamined.

Assembly and functions of cilia rely on the continuous transport of ciliary components between the cell body and the ciliary tip. This is performed by specialized molecular machines, known as Intraflagellar Transport (IFT) trains. Anterograde IFT trains are powered by kinesin-2 motors and move along the B-tubules of the microtubule doublets. Conversely, retrograde IFT trains are moved by dynein-2 motors along the A-tubules back to the cell body. The segregation of oppositely directed trains on A-tubules or B-tubules is thought to prevent traffic jams in the cilium, but the mechanism by which opposite polarity trains are sorted on either tubule is unknown. It has been reported that A-tubule and B-tubule are differentially enriched in tyrosinated and detyrosinated tubulins, but whether this difference has a role in IFT regula2on is not understood. Here, I show that CRISPR knock out of VASHL, the enzyme that detyrosinates microtubules, causes repeated collisions between oppositely directed trains and reduces the rate of ciliary growth. To test whether this is ascribable to direct interaction between IFT trains and tubulin detyrosination/tyrosination, I developed a method to reconstiute the motility of native IFT trains from Chlamydomonas reinhardtii cilia ex-vivo on synthetically polymerized microtubules enriched for either tubulin post-translational modification. I show that retrograde trains have higher affinity for tyrosinated microtubules (analogous to A-tubules), while anterograde trains for detyrosinated microtubules (analogous to B-tubules). I conclude that tubulin tyrosination/detyrosination is required for the spatial segregation of oppositely directed trains and for their smooth uninterrupted motion. My results provide a model for how IFT motility is governed by the underlying tubulin code.:Table of Contents Abstract ............................................................................................................................. 4 1. Introduc1on .................................................................................................................. 7 1.1 Cilia ............................................................................................................................................... 7 1.1.1 Cilia are ubiquitous and important cell organelle ................................................................................ 7 1.1.2 Pathologies associated with Ciliary dysfunc=on .................................................................................. 7 1.1.3 Axoneme is the structural scaffold of the cilium ................................................................................. 8 1.2 Intraflagellar transport (IFT) ....................................................................................................... 11 1.2.1 IFT complex is large macromolecular protein assembly .................................................................... 11 1.2.2 Microtubule motors drive the IFT trains ............................................................................................ 12 1.3 Bidirec<onal transport by microtubule cytoskeleton motors .................................................... 16 1.3.1 Microtubules ...................................................................................................................................... 16 1.3.2 Kinesin ............................................................................................................................................... 18 1.3.3 Dynein ................................................................................................................................................ 21 1.3.4 Mul=-motor transport ....................................................................................................................... 24 1.3.5 Cargo Logis=cs ................................................................................................................................... 27 1.4 Aims of the thesis ....................................................................................................................... 34 2. Ex-vivo Recons1tu1on of IFT train mo1lity ................................................................... 36 2.1 Capillary micropipeGe can be coupled with TIRFM for IFT train recons<tu<on ........................ 36 2.2 Ex-vivo mo<lity of IFT trains ....................................................................................................... 41 2.3 Ex-vivo trains retain their complex iden<ty ................................................................................ 43 3. Role of tubulin tyrosina1on/detyrosina1on in train sor1ng ......................................... 46 3.1 Chlamydomonas VashL encodes for axonemal tubulin detyrosina<on ac<vity ......................... 47 3.2 Anterograde IFT trains exhibit collisions in Chlamydomonas VashL mutant .............................. 48 3.3 Anterograde trains have a stronger affinity for normal ghost axonemes ................................... 51 3.4 The affinity of anterograde trains reduces for VashL ghost axonemes ...................................... 51 3.5 Retrograde trains are more likely to land on detyrosinated microtubules ................................ 53 4. Sidestepping as a IFT sor1ng mechanism ..................................................................... 57 4.1 2D tracking of IFT trains reveals off-axis stepping component ................................................... 57 4.2 IFT trains do not collide when crossing on microtubules ........................................................... 61 5. Discussion and Outlook ............................................................................................... 63 6. Materials and Methods ............................................................................................... 71 6.1 Chlamydomonas Cell Culture ..................................................................................................... 71 6.2 Crea<on of IFT-46 mNeonGreen::VashL ..................................................................................... 71 6.3 Coverslip Prepara<on ................................................................................................................. 72 6.4 Capillary pipeGe prepara<on and manipula<on ........................................................................ 72 6.5 Microtubule Polymeriza<on and polarity labeling ..................................................................... 73 6.6 Enzyme treatments of Microtubules .......................................................................................... 73 6.7 TIRF Microscopy ......................................................................................................................... 74 6.8 Analysis of TIRFM data ............................................................................................................... 74 6.9 Kymographs of Ghost Axonemes: .............................................................................................. 75 6.10 Flagella Isola<on ...................................................................................................................... 75 6.11 Western Blo_ng ...................................................................................................................... 76 6.12 Par<cle tracking in FIESTA ........................................................................................................ 76 7. Step-by-step protocol for IFT mo1lity recons1tu1on .................................................... 78 7.1 Materials .................................................................................................................................... 78 7.2 Method ...................................................................................................................................... 79 Bibliography .................................................................................................................... 86 Acknowledgements ......................................................................................................... 98 Declara1on according to §5.5 of the doctorate regula1ons ............................................ 100

Many active processes in cells are driven by highly specialised motor proteins, which interact with the cytoskeleton: a network of filamentous structures, e.~g.~ actin filaments and microtubules, which organises intracellular transport and largely determines the cell shape. These motor proteins are able to transduce the chemical energy, stored in ATP molecules, to do mechanical work while interacting with a filament. Certain motor proteins, e.~g.~members of the KIN-13 kinesin subfamily, are able to interact specifically with filament ends and induce depolymerisation of the filament ends. One important role for KIN-13 family members is in the mitotic spindle, a microtubule structure that is formed in the process of cell division and is responsible for separation and distribution of the duplicated genetic material to the forming daughter cells. The aim of this work is to develop a theoretical framework capable of describing experimentally observed behaviour and shed light on underlying principles of motor induced filament depolymerisation. We use two different theoretical approaches to describe motor dynamics in this non- equilibrium situation: On the one hand we use phenomenological continuum equations which themselves are to a large extent independent of the underlying molecular details of the system. Molecular details of the system are incorporated in the equations through the specific values of macroscopic parameters which are determined by the underlying details. On the other hand, we use one- and two-dimensional discrete stochastic descriptions of motors on a filament. These kind of descriptions enable us to investigate the effects of different microscopic mechanisms of filament depolymerisation, and to investigate the role of fluctuations on the dynamic behaviour of motor proteins. We additionally discuss filament depolymerisation in the case where motors are not free to move but are fixed to a common anchoring point and depolymerise filaments under the influence of applied forces, mimicking the situation in the mitotic spindle. Our results can be related to recent experiments on members of the KIN-13 subfamily and predictions made in our theory can be tested by further experiments. Although motivated by experiments involving members of the KIN-13 subfamily, our theory is not restricted to these motors but applies in general to associated proteins which regulate dynamics of filament ends.

<p>Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCF<super>Cdc4</super> ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that in the budding yeast,<italic> Saccharomyces cerevisiae</italic>, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APC<super>Cdh1</super>). I have determined, however, that the Kip1 and Cin8 proteins are actually present at wild-type levels in yeast in the absence of Clb/Cdc28 kinase activity. Here, I show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28, and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's <italic>in vivo</italic> localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, I have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCF<super>Cdc4</super> target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, I propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors. </p><p>In addition to the positive regulation of kinesin-5 function in spindle assembly, I have also found evidence that suggests CDK phosphorylation of kinesin-5 motors at different sites negatively regulates kinesin-5 activity to prevent premature spindle pole separation. I have also begun to characterize a novel putative role for the kinesins-5 in mitochondrial genome inheritance in <italic>S. cerevisiae</italic> that may also be regulated by CDK phosphorylation. </p><p>In the course of my dissertation research, I encountered problems with several established molecular biology tools used by yeast researchers that I have tried to address. I have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in <italic>S. cerevisiae</italic> that can be propagated in the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes <italic>HIS2</italic> and <italic>ADE1</italic>, respectively. My new pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed in order to increase the availability of unique restriction sites within their polylinker regions. Hence, my pRSII plasmids are a complete set of integrating, centromere and 2&#61549; episomal plasmids with the biosynthetic marker genes <italic>ADE2</italic>, <italic>HIS3</italic>, <italic>TRP1</italic>, <italic>LEU2</italic>, <italic>URA3</italic>, <italic>HIS2</italic> and <italic>ADE1</italic> and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, I have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin and bialaphos. Finally, in the process of generating the new plasmids, I have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using updated plasmid sequences, I constructed pRS plasmid backbones with a unique restriction site for inserting new markers in order to facilitate future expansion of the pRS/pRSII series.</p> / Dissertation

Intraflagellar transport (IFT), the bi-directional movement of particles along the length of flagella, is required for flagellar assembly. The IFT particles are moved by kinesin II from the base to the tip of the flagellum, where flagellar assembly occurs. The IFT particles are then moved in the retrograde direction by cytoplasmic dynein 1b/2 to the base of the flagellum. The IFT particles of Chlamydomonas are composed of ~16 proteins, organized into complexes A and B. Alhough IFT is believed to transport cargoes into flagella, few cargoes have been identified and little is known about how the cargos are transported. To study the mechanism of IFT and how IFT is involved in flagellar assembly, this thesis focuses on two questions. 1) In addition to a heavy chain, DHC1b, and a light chain, LC8, what other proteins are responsible for the retrograde movement of IFT particles? 2) What is the specific function of an individual IFT-particle protein? To address these two questions, I screened for Chlamydomonas mutants either defective in retrograde IFT by immunofluorescence microscopy, or defective in IFT-particle proteins and D1bLIC, a dynein light intermediate chain possibly involved in retrograde IFT, by Southern blotting. I identified several mutants defective in retrograde IFT and one of them is defective in the D1bLIC gene. I also identified several mutants defective in several IFT-particle protein genes. I then focused on the mutant defective in D1bLIC and the one defective in IFT46, which was briefly reported as an IFT complex B protein. My results show that as a subunit of the retrograde IFT motor, D1bLIC is required for the stability of DHC1b and is involved in the attachment of IFT particles to the retrograde motor. The P-loop in D1bLIC is not necessary for the function of D1bLIC in retrograde IFT. My results also show that as a complex B protein, IFT46 is necessary for complex B stability and is required for the transport of outer dynein arms into flagella. IFT46 is phosphorylated in vivo and the phosphorylation is not critical for IFT46’s function in flagellar assembly.

No description available.

No description available.

Les microtubules sont de longs polymères cylindriques de la protéine α, β tubuline, utilisés dans les cellules pour construire le cytosquelette, le fuseau mitotique et les axonèmes. Ces polymères creux sont cruciaux pour de nombreuses fonctions cellulaires, y compris le transport intracellulaire et la ségrégation chromosomique pendant la division cellulaire. Au fur et à mesure que les cellules se développent, se divisent et se différencient, les microtubules passent par un processus, appelé instabilité dynamique, ce qui signifie qu’ils basculent constamment entre les états de croissance et de rétrécissement. Cette caractéristique conservée et fondamentale des microtubules est étroitement régulée par des familles de protéines associées aux microtubules. Les protéines de kinésine-13 sont une famille de facteurs régulateurs de microtubules qui dépolymérisent catalytiquement les extrémités des microtubules. Cette thèse traite d’abord des concepts mécanistiques sur le cycle catalytique de la kinésine-13. Afin de mieux comprendre le mécanisme moléculaire par lequel les protéines de kinésine-13 induisent la dépolymérisation des microtubules, nous rapportons la structure cristalline d’un monomère de kinésine-13 catalytiquement actif (Kif2A) en complexe avec deux hétérodimères αβ-tubuline courbés dans un réseau tête-à-queue. Nous démontrons également l’importance du « cou » spécifique à la classe de kinésine-13 dans la dépolymérisation catalytique des microtubules. Ensuite, nous avons cherché à fournir la base moléculaire de l’hydrolyse tubuline-guanosine triphosphate (GTP) et son rôle dans la dynamique des microtubules. Dans le modèle que nous présentons ici, l’hydrolyse tubuline-GTP pourrait être déclenchée par les changements conformationnels induits par les protéines kinésine-13 ou par l’agent chimique stabilisant paclitaxel. Nous fournissons également des preuves biochimiques montrant que les changements conformationnels des dimères de tubuline précèdent le renouvellement de la tubuline-GTP, ce qui indique que ce processus est déclenché mécaniquement. Ensuite, nous avons identifié la kinésine de microtubule Kif2C comme une protéine associée à des modèles d’ADN imitant la rupture double brin (DSB) et à d’autres protéines de réparation DSB connues dans les extraits d’œufs de Xenope et les cellules de mammifères. Les cassures double brin d’ADN (DSB) sont un type majeur de lésions d’ADN ayant les effets les plus cytotoxiques. En raison de leurs graves impacts sur la survie cellulaire et la stabilité génomique, les DSB d’ADN sont liés à de nombreuses maladies humaines, y compris le cancer. Nous avons constaté que les activités PARP et ATM étaient toutes deux nécessaires pour le recrutement de Kif2C sur les sites de réparation de l’ADN. Kif2C knockout ou inhibition de son activité de dépolymérisation des microtubules a conduit à l’hypersensibilité des dommages à l’ADN et à une réduction de la réparation du DSB via la jonction terminale non homologue et la recombinaison homologue. Dans l’ensemble, notre modèle suggère que les protéines de kinésine-13 peuvent interagir avec les dimères de tubuline aux extrémités microtubules et modifier leurs conformations, moduler l’étendue des extrêmités tubuline-GTP dans les cellules et déclencher le désassemblage des microtubules. Ces deux modèles pourraient être des clés pour démêler les mécanismes impliqués dans le nouveau rôle de Kif2C dans la réparation de l’ADN DSB sans s’associer à des polymères de microtubules. / Microtubules are long, cylindrical polymers of the proteins α, β tubulin, used in cells to construct the cytoskeleton, the mitotic spindle and axonemes. These hollow polymers are crucial for many cellular functions including intracellular transport and chromosome segregation during cell division. As cells grow, divide, and differentiate, microtubules go through a process, called dynamic instability, which means they constantly switch between growth and shrinkage states. This conserved and fundamental feature of microtubules is tightly regulated by families of microtubule-associated proteins (MAPs). Kinesin-13 proteins are a family of microtubule regulatory factors that catalytically depolymerize microtubule ends. This thesis first discusses mechanistic insights into the catalytic cycle of kinesin-13. In order to better understand the molecular mechanism by which kinesin-13 proteins induce microtubule depolymerization, we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array. We also demonstrate the importance of the kinesin-13 class-specific “neck” in modulating Adenosine triphosphate (ATP) turnover and catalytic depolymerization of microtubules. Then, we aimed to provide the molecular basis for tubulin-Guanosine triphosphate (GTP) hydrolysis and its role in microtubule dynamics. Although it has been known for decades that tubulin-GTP turnover is linked to microtubule dynamics, its precise role in the process and how it is driven are now well understood. In the model we are presenting here, tubulin-GTP hydrolysis could be triggered via the conformational changes induced by kinesin-13 proteins or by the stabilizing chemical agent paclitaxel. We also provide biochemical evidence showing that conformational changes of tubulin dimers precedes the tubulin-GTP turnover, which indicates that this process is triggered mechanically. Next, we identified microtubule kinesin Kif2C as a protein associated with double strand break (DSB)-mimicking DNA templates and other known DSB repair proteins in Xenopus egg extracts and mammalian cells. DNA double strand breaks (DSBs) are a major type of DNA lesions with the most cytotoxic effects. Due to their sever impacts on cell survival and genomic stability, DNA DSBs are related to many human diseases including cancer. Here we found that PARP and ATM activities were both required for the recruitment of Kif2C to DNA repair sites. Kif2C knockdown/knockout or inhibition of its microtubule depolymerizing activity led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both non-homologous end-joining (NHEJ) and homologous recombination (HR). Interestingly, genetic depletion of KIF2C, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Altogether, our findings shed light on the mechanisms involved in kinesin-13 catalyzed microtubule depolymerization. Our tubulin-GTP hydrolysis model suggests that kinesin-13 proteins may interact with tubulin dimers at microtubules ends and alter their conformations, modulate the extent of the GTP caps in cells and trigger microtubule disassembly. These two models could be keys to unravel the mechanisms involved in the novel role of Kif2C in DNA DSB repair without associating with microtubule polymers.