Molecular motors are responsible for all long range transport and organization of organelles within cells. However, little is known about the interaction of multiple similar and dissimilar motors. In this thesis I describe experiments to probe the coordination of the motors kinesin and dynein which move towards the opposite ends of microtubules. Cargos they haul show bidirectional movement at short scales yet there is net transport in one direction or the other. Two distinct models for the bidirectional transport exist: regulation and a tug-of-war. In order to differentiate between them, kinesin-specific antibodies are injected into Drosophila embryos and the effect on transport of lipid droplets is quantified and compared to unperturbed motion. The function-blocking antibodies resulted in an increased run length of dynein-mediated transport and a decrease in that of kinesin. Furthermore, reduced velocities in both directions and a trend towards shorter pauses were observed. Comparison of these results to predictions the models provide for this scenario supports a tug-of-war model rather than regulation. / text

Dynamic mitochondrial transport into axons and dendrites of neuronal cells is critical for sustaining neuronal excitability, synaptic transmission, and cell survival. Failure of mitochondrial transport is the direct cause of some neurodegenerative diseases, and an aggravating factor for many others. Mitochondrial transport regulation involves many proteins; factoring prominently among them are the atypical mitochondrial GTPase Miro and the Milton/TRAK adaptor proteins, which link microtubule (MT) motors to mitochondria. Motors of the kinesin family mediate mitochondrial transport towards the plus ends of microtubules, while motors of the dynein family mediate mitochondrial transport towards the minus ends. Selective use of these motors determines the ultimate subcellular distribution of mitochondria, but the underlying control mechanisms remain poorly understood. Drosophila Miro (dMiro) is required for kinesin-driven transport of mitochondria, but its role in dynein-driven transport remains controversial. In Chapter 2 of this study, I show that dMiro is also required for the dynein-driven transport of mitochondria. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to analyze the function of dMiro's N- and C-terminal GTPase domains, respectively. We show that dMiroT25N causes lethality and impairs mitochondrial distribution and transport in a manner indistinguishable from dmiro null mutants. Our analysis suggests that both kinesin- and dynein-driven mitochondrial transport require the activity of Miro's N-terminal GTPase domain, which likely controls the transition from a stationary to a motile state irrespective of the transport direction. dMiroT460N reduced only dynein motility during retrograde axonal transport but had no effect on distribution of mitochondria in neurons, indicating that the C-terminal GTPase domain of Miro most likely has only a small modulatory role on transport. Furthermore, we show that commonly used substitutions in Miro's GTPase domains, based on the constitutively active Ras-G12V mutation, appear to cause neomorphic phenotypic effects which are probably unrelated to the normal function of the protein. In mammalian neurons, kinesin and dynein motors are linked to mitochondria via a Miro complex with the adapter proteins TRAK1 and TRAK2, respectively. Differential linkage of TRAK-motor complexes provides a mechanism for determining the direction of transport and controlling mitochondrial distributions within the cell. Drosophila has only one TRAK gene homolog, Milton, which expresses several protein isoform. Milton has been previously been shown to facilitate mitochondrial transport by binding to kinesin and dMiro, a role analogous to TRAK1. However, the question whether Milton might be able mediate dynein-based transport in a manner similar to TRAK2 has remained unknown. In Chapter 3 of this study, I show that protein isoforms A and B of Milton, generated through alternative mRNA splicing, facilitate differential motor activities analogous to mammalian TRAKs. Specifically, overexpression (OE) of Milton-A caused a mitochondrial redistribution and accumulation at axon terminals, which requires kinesin-driven MT plus end directed transport; while OE of Milton-B caused a redistribution of axonal mitochondria into the soma, which requires dynein-driven MT minus end directed transport. I further show that Milton-motor complex binding to mitochondria requires Miro exclusively, and that transport with either of the motor complexes absolutely requires the activity of Miro's N-terminal GTPase domain. Together, these results suggest that Miro controls the transition of mitochondria from a stationary to a motile phase. Thereafter the direction of transport is likely determined by an alternative binding of opposing Milton/TRAK-motor complexes to Miro, a process which appears to be regulated by a Miro-independent mechanism.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 211-225) Also available in print.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [90]-105).

Leipzig, Universiẗat, Diss., 2005.

In this work the functions of the microtubule motors kinesin-1, -2 and -3 have been analysed in various settings. The reconstitution of microtubule-dependent motor activity in vitro has been primarily used to dissect the contributions of individual motors to cargo motility in two specific scenarios. Initially the regulation of kinesin-1 in a cell cycle-dependent manner has been examined by studying the ability of rat liver endoplasmic reticulum (ER) tubules to move in cytosols prepared from Xenopus laevis egg extracts arrested in interphase, meiosis or mitosis. It was found that kinesin-1-driven ER motility is significantly disrupted during metaphase in vitro. This is likely due to the recruitment or loss of binding partners which has a concomitant influence upon kinesin-1 activity. This work presents the first evidence that kinesin-1-driven ER movement, and not simply network morphology, varies during cell division. Furthermore, it is postulated that the replication of such regulation of kinesin-1 activity in vivo may contribute to the well documented changes in organelle positioning and cargo transit through membrane trafficking pathways which occur during cell division.The fungal metabolite brefeldin A (BFA) induces tubulation of several compartments located within the secretory and endocytic pathways in a microtubule-dependent fashion. The identity of the motor(s) responsible for this motility remains unconfirmed and controversial since several reports with conflicting data have been published. The contributions of kinesin-1, -2 and -3 to these processes have been investigated using in vitro motility assays in which rat liver Golgi membranes were combined with Xenopus laevis egg extract cytosol in the presence of BFA. Function blocking antibodies and dominant negative proteins were used to perturb the activities of various kinesin motors. This data indicates a particular isoform of kinesin-3, KIF1C, is solely responsible for the movement of BFA-induced tubules in vitro. This work was complemented by in vivo immunofluorescence studies using the HeLaM cultured cell line. Transient transfections of dominant negative proteins, or siRNA-mediated depletion, were used to disrupt the activities of various kinesin motors, either in isolation or in combination with each other. This approach revealed a contribution of KIF1C and kinesin-1 to the movement of early endosomal BFA-induced tubules in vivo.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18507号 / 医博第3927号 / 新制||医||1005(附属図書館) / 31393 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 近藤 玄, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les kinésines sont des protéines moteur liées au cytosquelette de microtubules. Elles convertissent l’énergie provenant de l’hydrolyse de l’ATP en un travail mécanique. Leur fonction typique est de se déplacer le long du microtubule pour véhiculer des charges. La plupart des kinésines sont des dimères. Elles comprennent un domaine moteur, qui porte à la fois les sites de liaison du nucléotide et du microtubule, un domaine intermédiaire de dimérisation et une partie dite « queue » qui confère la spécificité des charges à transporter. Mon objectif est d’établir le mécanisme moléculaire à la base de la motilité, avec un intérêt particulier pour la détermination des variations structurales du domaine moteur de la kinésine le long de son cycle mécano-chimique. Au cours de ma thèse, mon objet d’étude principal a été la kinésine-1 humaine, encore appelée kinésine conventionnelle.J’ai étudié plus particulièrement deux aspects du cycle mécano-chimique de la kinésine-1, en combinant des approches de biologie structurale et l’étude de mutants. Les deux aspects concernent l’étude de la fixation de la kinésine-ADP au microtubule, conduisant à l’éjection du nucléotide et à une liaison forte de la kinésine au microtubule. Dans un premier temps, j’ai déterminé la structure du domaine moteur de la kinésine-1, dépourvue de nucléotide, et sous forme d’un complexe avec la tubuline. La tubuline est la protéine constitutive des microtubules. Cette structure était la donnée principale qui nous manquait dans le cycle structural de la kinésine. En comparant cette structure avec celle de la kinésine dans un état ATP, on peut rendre compte des changements de conformation de la kinésine selon le mouvement de trois sous-domaines du domaine moteur. Cette analyse explique notamment le lien entre la fixation de l’ATP et l’ouverture d’une poche hydrophobe distante de 28 Å du site du nucléotide. Cette cavité va accommoder le premier résidu du neck linker, conduisant à la stabilisation de ce peptide situé en partie C-terminale du domaine moteur. En s’ordonnant, le neck linker va faire avancer la charge ainsi que l’autre domaine moteur de la kinésine dimérique. Il lie ainsi la fixation de l’ATP au mouvement. L’étude de l’effet de mutations du neck linker montre aussi comment, réciproquement, le neck linker bloque la kinésine dans la conformation active pour l’hydrolyse de l’ATP. Ceci diminue la probabilité que l’ATP soit hydrolysé avant que l’étape mécanique se soit produite; cet aspect est essentiel pour rendre compte de la processivité de la kinésine-1.Ces données structurales suggèrent également comment la fixation de la kinésine-ADP au microtubule accélère l’éjection de l’ADP. Pour étudier cet aspect plus en détail, j’ai étudié l’effet de mutations sur la vitesse de largage de l’ADP. L’idée était de mimer à l’aide de mutations la fixation au microtubule. J’ai identifié ainsi deux séries de mutants qui présentent une vitesse accélérée de largage spontané de l’ADP, ce qui suggère deux voies pour interférer avec la fixation du nucléotide. J’ai ensuite déterminé la structure de deux de ces mutants dépourvus de nucléotide, ainsi que celle de la kinésine de départ également dans une forme apo, obtenue par digestion de l’ADP. En absence de microtubule, la kinésine dépourvue de nucléotide adopte une conformation soit à l’image de celle de la kinésine-ADP, ou proche de celle de la kinésine-apo liée à la tubuline. Dans un contexte naturel, seule la deuxième conformation est compatible avec la fixation au microtubule. L’ensemble de ces résultats suggère que le microtubule accélère l’éjection du nucléotide par un double mécanisme : en interférant avec la liaison du magnésium et en déstabilisant le motif P-loop de liaison du nucléotide. / Kinesins are a family of microtubule-interacting motor proteins that convert the chemical energy from ATP hydrolysis into mechanical work. Many kinesins are motile, walking along microtubules to fulfill different functions. Most kinesins are dimers, the monomer comprising a motor domain, a dimerizing stalk domain, and a tail domain. The motor domain contains both the nucleotide-binding site and the microtubule-binding site. I am interested in the molecular mechanism of kinesin's motility. In particular I want to establish the structural variations of the kinesin motor domain along with the mechanochemical cycle of this motor protein. During my thesis, I have focused my work on the human kinesin-1, also named conventional kinesin, which is the best characterized kinesin.I have studied two aspects of the kinesin mechanochemical cycle, by combining structural and mutational approaches. Both aspects rely on the binding of ADP-kinesin to a microtubule, which leads to the release of the nucleotide and to a tight kinesin-microtubule association. First I determined the crystal structure of nucleotide-free kinesin-1 motor domain in complex with a tubulin heterodimer, which is the building block of microtubule. This structure represented the main missing piece of the structural cycle of kinesin. Three subdomains in the kinesin motor domain can be identified through the comparison of my structure with ATP-analog kinesin-1-tubulin structure. The relative movements of these subdomains explain how ATP binding to apo-kinesin bound to microtubule triggers the opening of a hydrophobic cavity, 28 Å distant from the nucleotide-binding site. This cavity accommodates the first residue of the “neck linker”, a short peptide that is C-terminal to the motor domain, allowing the neck linker to dock on the motor domain. The docking of the neck linker is proposed to trigger the mechanical step, i.e. the displacement of the cargo and the stepping of the dimeric kinesin. By studying mutants of the neck linker, I have shown that, reciprocally, this peptide locks kinesin in the ATP state, which is also the conformation efficient for ATP hydrolysis. Doing so, it prevents the motor domain from switching back to the apo-state. It prevents also an untimely hydrolysis of ATP, before the mechanical step has occurred. These features are required for movement and processivity.Second, these structural data also suggest how the binding of ADP-kinesin to tubulin enhances nucleotide release from kinesin. To further study this step of the kinesin cycle, I studied the effect of kinesin-1 mutations. These mutations were designed in isolated kinesin to mimic the state when kinesin is bound to a microtubule. I identified two groups of mutations leading to a high spontaneous ADP dissociation rate, suggesting that there are two ways to interfere with ADP binding. Then I determined the crystal structures of the apo form of two mutants as well as that of the nucleotide-depleted wild type kinesin. It showed that apo-kinesin adopts either and ADP-like conformation or a tubulin-bound apo-like one. In the natural context, the second one is stabilized upon microtubule binding. Overall, the mutational and structural data suggest that microtubules accelerate ADP dissociation in kinesin by two main paths, by interfering with magnesium binding and by destabilizing the nucleotide-binding P-loop motif.

In this Ph.D. project, we aim to understand degradation of nanomachines by studying the mechanisms that lead to the in vitro degradation of molecular shuttles, which are nanoscale active systems composed of kinesin motor proteins and cytoskeletal filaments called microtubules. In addition, we aimed to improve learning outcomes by designing a hybrid college-level engineering course combining case-based and lecture-based teaching. The creation of complex active nanosystems integrating cytoskeletal filaments propelled by surface-adhered motor proteins often relies on microtubules’ ability to glide for up to meter-long distances. Even though theoretical considerations support this ability, we show that microtubule detachment (either spontaneous or triggered by a microtubule crossing event) is a non-negligible phenomenon that has been overlooked until now. Furthermore, we show that under our conditions (100, 500, 1000 motors per µm2 and 0.01 or 1 mM ATP), the average gliding distance before spontaneous detachment ranges from 0.3 mm to 8 mm and depends on the gliding velocity of the microtubules, the density of the kinesin motors on the glass surface, and time. Wear, defined as the gradual removal of small amounts of material from moving parts of a machine, as well as breakage, defined as the rupture of a material, are two major causes of machine failure at the macroscale. Since these mechanisms have molecular origins, we expect them to occur at the nanoscale as well. Here, we show that microtubules propelled by surface-adhered kinesin motors are subject to wear and breakage just like macroscale machines. Furthermore, the combined effect of wear, breakage and microtubule detachment from the surface of the flow cell permit to predict how molecular shuttles degrade in vitro. Taking a step back and looking at science in a broader sense, we can say that science does not only consist of acquiring knowledge, but also relies on one’s ability to transmit his/her knowledge. In this regard, one of the biggest challenges in education is to be efficient, that is to say to design a teaching method that would both maximize the student’s retention of information and prepare them to apply their knowledge to real-life situations. We considered this challenge as an integral part of this Ph.D. project, and we tackled it by designing a novel type of engineering course in which the students’ involvement in the learning process plays a central role. To do so, we combined, in a single engineering course, both of the approaches to learning that are used in Engineering education and in Business schools. The final chapter of this manuscript summarizes the findings of the two projects presented here and discusses the future research that can be conducted on the basis of this thesis.

During mitosis, replicated chromosomes are equally distributed among two daughter cells by means of a multi-component machine called the mitotic spindle. Spindle formation and function has been shown to involve numerous microtubule associated proteins and molecular motor proteins, including kinesins and dynein. One such kinesin, the plus-end directed, homotetrameric, Eg5, is involved in centrosome separation during spindle formation. In vitro, Eg5 crosslinks parallel and antiparallel microtubules, and localizes to spindle poles and microtubules in vivo. To further understand the function of Eg5 in mammalian cells, we determined its distribution and dynamics throughout mitosis using novel cloning techniques, fluorescence recovery after photobleaching, and total internal reflection fluorescence microscopy. Eg5-GFP was expressed from a mouse bacterial artificial chromosome to ensure the transgene’s expression was at or near endogenous levels. Our results confirm that Eg5 colocalizes with spindle, but not astral, microtubules and is enhanced at the spindle poles during prometaphase and metaphase. In early anaphase, Eg5 is localized near the poles transitioning to interzone microtubules with the exception of a 1 µm gap during late anaphase. Fluorescence recovery after photobleaching shows that Eg5 is rapidly turning over throughout mitosis with a recovery half time less than 10 s and extent of recovery greater than 85%. TIRF microscopy revealed a population of Eg5 that transiently binds to microtubules with a residency time of less than 6 seconds for all stages of mitosis. Eg5 remained stationary while bound to microtubules with no apparent directional motion. Treatment of cells expressing mEg5-GFP-LAP with the Eg5 inhibitor, STLC, caused Eg5 to no longer bind to microtubules and remain diffuse within the cell. TIRF microscopy also revealed Eg5-decorated tracks during interphase, which were abolished by treatment with STLC or Nocodazole suggesting that Eg5 is present on microtubules in interphase. Taken together, fluorescence recovery after photobleaching and TIRF microscopy reveal that Eg5 is highly dynamic in the mammalian spindle throughout mitosis.