Trypanosoma brucei, un parasite flagellé unicellulaire, est responsable de la trypanosomiase humaine africaine aussi connue comme la maladie du sommeil.Les microtubules (MTs) sous-pelliculaires, le quartet de MTs (MTQ), le flagelle (F) et le collier de la poche flagellaire (CPF) sont les principaux composants du cytosquelette dutrypanosome. À ce jour, une seule protéine du CPF, BILBO1, a été identifiée et caractérisée.Dans cette étude, nous montrons in vivo que BILBO1 forme des polymères capables deconstruire un échafaudage qui permet l’ancrage de protéines partenaires. Ainsi, un crible en double hybride chez la levure a identifié plusieurs protéines partenaires de BILBO1,notamment une nouvelle protéine appelée FPC4. Nous démontrons que FPC4 est une protéine spécifique des kinétoplastides, localisée au CPF mais aussi au hook-complex, une structure proche du CPF. L’interaction FPC4 – BILBO1 est démontrée in vitro et in vivo, etles domaines d'interaction identifiés. En outre, nous démontrons in vivo et in vitro que FPC4est une protéine associée aux microtubules. Nos données suggèrent fortement que FPC4est impliquée dans le processus de séparation des CPFs au cours du cycle cellulaire. Nos résultats mettent en évidence un lien étroit entre le MtQ et le CPF et l'implication probable duhook-complex. Enfin, nous mettons en évidence une structure analogue au hook-complex chez les Leishmanies. L’interaction BILBO1 – FPC4 représente une nouvelle cible thérapeutique et sera caractérisée plus avant. / Trypanosoma brucei, a unicellular flagellated parasite, is responsible for the human African trypanosomiasis also known as sleeping sickness. Sub-pellicular microtubules (MT), the MT quartet (MtQ), the flagellum (F) and the Flagellar Pocket Collar (FPC) are the main components of the T. brucei cytoskeleton. To date, only a single FPC protein, BILBO1, has been identified and characterized. In this study we demonstrate in vivo that BILBO1 forms polymers able to build a scaffold structure that anchors partner proteins. As such, a yeast-2-hybrid screen identified several BILBO1 interacting protein partners. We demonstrate that FPC4 is a kinetoplastid-specific protein, which is localized at the FPC and at the hook complex. Its specific interaction with BILBO1 has been demonstrated in vitro and in vivo, and the interacting domains identified. Furthermore, we demonstrate that FPC4 is a microtubule binding protein. Our data strongly suggest that FPC4 is involved in the separation of the old and the newly formed FPC during the cell cycle. Altogether, our results demonstrate a tight connection and interplay between the MtQ and the FPC and the likely involvement of an adjacent third structure, the hook complex. Finally, we highlight a structure similar to the hook-complex in Leishmania. The BILBO1 – FPC4 interaction represents a new therapeutic target and will be characterized further.

The main structural protein VP1 is the product of late polyomaviral genes and it is the largest and the most abundant protein of the whole polyomaviral capsid. Because of the low coding capacity of the polyomaviral genomes, it is considered that in addition to its structural role the VP1 protein might have some additional functions in the late phase of the infectious cycle. This diploma thesis is exactly on these additional functions. In the case of the VP1 protein of mouse polyomavirus, it was observed that the protein is capable of binding to the structure of cellular microtubules. The first objective of this work was to test whether pentamers of the VP1 protein are able of this binding without the participation of other cellular (or viral) proteins. Based on an in vitro experiment, we showed that protein VP1 binds to the structure of microtubules very inefficiently. The second objective of this work was to prepare a detection system that would allow an identification of potential interaction partners of BK polyomavirus VP1 protein. Therefore, expression plasmids producing the N and C-terminally tagged VP1 protein were prepared. These tagged proteins had the property of being biotinylated whilst being produced in the transfected cells. By using affinity chromatography, the entire protein complexes...

Optical tweezers are a sensitive position and force transducer widely employed in physics and biology. In a focussed laser, forces due to radiation pressure enable to trap and manipulate small dielectric particles used as probes for various experiments. For sensitive biophysical measurements, microspheres are often used as a handle for the molecule of interest. The force range of optical traps well covers the piconewton forces generated by individual biomolecules such as kinesin molecular motors. However, cellular processes are often driven by ensembles of molecular machines generating forces exceeding a nanonewton and thus the capabilities of optical tweezers. In this thesis I focused, fifirst, on extending the force range of optical tweezers by improving the trapping e fficiency of the probes and, second, on applying the optical tweezers technology to understand the mechanics of molecular motors. I designed and fabricated photonically-structured probes: Anti-reflection-coated, high-refractive-index, core-shell particles composed of titania. With these probes, I significantly increased the maximum optical force beyond a nanonewton. These particles open up new research possibilities in both biology and physics, for example, to measure hydrodynamic resonances associated with the colored nature of the noise of Brownian motion. With respect to biophysical applications, I used the optical tweezers to study the mechanics of single kinesin-8. Kinesin-8 has been shown to be a very processive, plus-end directed microtubule depolymerase. The underlying mechanism for the high processivity and how stepping is affected by force is unclear. Therefore, I tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads. We found that kinesin-8 is a low-force motor protein, which stalled at loads of only 1 pN. In addition, we discovered a force-induced stick-slip motion, which may be an adaptation for the high processivity. Further improvement in optical tweezers probes and the instrument will broaden the scope of feasible optical trapping experiments in the future.

La migration des cellules tumorales à travers la matrice extracellulaire dépend de l'activité d'une métalloprotéase matricielle, MT1-MMP, ancrée à la membrane plasmique. MT1-MMP accumule aux invadopodes, des protrusions membranaires à base d'actine responsables de la dégradation de la matrice. La petite protéine G ARF6 est impliquée dans la régulation du trafic membranaire et dans le remodelage du cytosquelette d'actine. Dans mon travail de thèse, j'ai montré qu'ARF6 et deux de ses protéines effectrices JIP3 et JIP4, sont nécessaires à l'exocytose de MT1-MMP au niveau des invadopodes et, par conséquent, à la capacité des cellules tumorales à remodeler la matrice extracellulaire et migrer à travers un environnement matriciel tridimensionnel. ARF6, à travers son interaction avec JIP3/4, contrôle négativement l'activité du complexe dynactine/dynéine, un moteur moléculaire qui se déplace en direction du bout (-) des microtubules, et donc la clairance des endosomes MT1-MMP à partir de la périphérie cellulaire. En plus dans des échantillons humaines ARF6 est accumulée au niveau de la membrane plasmique, avec MT1-MMP, dans un sous-groupe de carcinomes mammaires agressifs, en confirmant donc l'implication d'un axe ARF6-JIP3/JIP4-MT1-MMP dans le processus invasif du cancer du sein. Dans une deuxième étude, j'ai montré que l'hyperactivation d'ARF6 induit un réarrangement important du cytosquelette d'actine à la surface ventrale des cellules tumorales mammaires et contribue à l'activation et au ciblage de Rac1 au front cellulaire. Mon travail a permis d'identifier de nouveaux mécanismes moléculaires par lesquels ARF6 contribue au programme invasif des cellules tumorales mammaires. / The ability of cancer cells to traffic through the extracellular matrix relies on the action of the membrane-anchored matrix metalloprotease MT1-MMP. MT1-MMP is exocytosed to invadopodia, the actin-based membrane protrusions responsible for matrix degradation. The small GTP-binding protein ARF6 is known to coordinate post-endocytic recycling and actin cytoskeletal organization at the plasma membrane and was shown to be up-regulated in breast cancer cells. In my PhD work I showed that ARF6 and two of its effectors JIP3 and JIP4 are required for MT1-MMP endosomes intracellular positioning and exocytosis at invadopodia and consequently for tumor cells ability to remodel the matrix and invade through a three-dimensional matrix environment. ARF6, through the interaction with JIP3/4, negatively controls the activity of the minus-end-directed microtubule motor dynactin/dynein, thus negatively regulating the clearance and inward movement of MT1-MMP endosomes from the cell periphery. In human samples ARF6 is accumulated at the plasma membrane, together with MT1-MMP, in a subset of highly aggressive breast carcinomas, thus corroborating the ARF6-JIP3/JIP4-MT1-MMP axis in breast cancer invasion. In a second study I addressed the contribution of ARF6 activation on actin cytoskeleton remodeling in breast cancer cells. ARF6 links epidermal growth factor receptor signaling to Rac1 activation and targeting to the leading edge where it activates the SCAR/WAVE complex and regulates ventral actin polymerization during lamellipodia extension. Collectively my work identifies novel molecular mechanisms through which ARF6 contributes to the invasive program of breast tumor cells

Muitas evidências indicam que a tumorigênese em humanos é um processo com várias etapas. A progressão de um tumor em direção a malignidade ocorre de maneiras muito distintas entre os diferentes tipos de câncer. Entender os processos celulares que levam a tumorigênese é importante para se delinear tratamentos mais adequados contra os diversos tipos de câncer. Drogas antimitóticas (ou venenos de fuso) são utilizadas no tratamento de alguns cânceres, e entre eles está o câncer de mama. A ação dessas drogas reside sobre a mitose, e têm como alvo os fusos mitóticos, estruturas essenciais que dirigem o ciclo celular na divisão das células. Recentemente, pesquisadores têm delineado possíveis respostas da célula aos venenos de fuso, e essas respostas são dependentes da concentração da droga. Baixas concentrações de venenos de fuso provocam o aparecimento de populações celulares aneuplóides, que ocorrem quando a célula sai do bloqueio mitótico. O presente trabalho utilizou as drogas vincristina e paclitaxel sobre linhagens celulares de mama humana (células normais e tumorais) para pesquisar se as drogas são capazes de induzir células aneuplóides. Os resultados obtidos no presente trabalho indicam que baixa concentração de vincristina e paclitaxel pode induzir o aparecimento de população aneuplóide através de mitoses aberrantes. Os venenos de fuso induzem a formação de mitoses contendo múltiplos fusos mitóticos, e essas mitoses não ficam bloqueadas, dando origem a células aneuplóides. O papel da aneuploidia na tumorigênese não foi ainda estabelecido, mas indiferente da sua importância no desenvolvimento do tumor, encontrar maneiras de inibir sua formação ou de super induzir seu aparecimento podem ter implicações significativas para as terapias contra o câncer. / Several lines of evidence indicate that tumorigenesis in humans is a multistep process and that these steps reflect genetic alterations that drive the progressive transformation of normal cells into highly malignant derivatives. Understanding the cellular processes that lead to tumorigenesis is important to devise more appropriate treatments against various types of cancer. Antimitotic drugs are used in the treatment of some cancers, and among them is breast cancer. The action of these drugs lies on the mitotic spindles, essential structures that drive the cell cycle in the division of cells. Recently, researchers have outlined possible responses to the antimitotic drugs on cells, and these responses are dependent on the concentration of the drug. Low concentrations of drugs can cause the appearance of aneuploid population, which occur when a cell leaves the mitotic block. This study used the drug vincristine and paclitaxel on human breast cancer cell lines (normal and tumoral cells) to find if the drugs are capable of inducing cell aneuploidy. The results of this study indicate that low concentration of vincristine and paclitaxel can induce the emergence of aneuploidy population through aberrant mitosis. The drugs induce the formation of mitosis containing multiple mitotic spindles, resulting in aneuploidy population of cells. The role of aneuploidy in tumorigenesis has not yet been established, but indifferent to this is important find ways to inhibit its formation or the super-induce their appearance. These questions may have significant implications for therapies against cancer.

Taxol™, isolated from the bark of Taxus brevifolia in the late 1960s, and the semisynthetic analog Taxotere™ have proven clinical importance for the treatment of ovarian and breast cancer. Taxol™ exerts its biological effect by binding to polymerized tubulin and stabilizing the resulting microtubules. Studies aimed at understanding the biologically active conformation of taxol and its binding environment on β-tubulin are described. This knowledge is important because it could lead to the design of structurally less complicated drugs with better efficacy and better bioavailability. Moreover, the information can be extended to other natural products that possess microtubule-stabilizing properties similar to Taxol™. In this work, the synthesis of a triply labeled taxol analog is described as well as REDOR studies of this compound complexed to tubulin are in progress. Macrocyclic analogs of taxol have been prepared and their biological activities were evaluated. Chemical modeling of these analogs and their activities agrees with the hypothesis that Taxol™ adopts T-shaped conformation. Difficulties were encountered with the key ring-closing metathesis strategy, suggesting that a more flexible and efficient macrocyclization method will be needed to synthesize additional macrocyclic analogs. / Ph. D.

Mikrotubuli sind filamentöse intrazelluläre Polymere, die als grundlegende Bestandteile subzellulärer Strukturen in Eukaryoten dienen. Diese Studie verwendet einen vergleichenden Ansatz, um zu untersuchen, wie sich die intrinsischen dynamischen und biochemischen Eigenschaften von Tubulin zwischen verschiedenen Spezies unterscheiden, und zeigt ihre Konsequenzen in zwei verschiedenen physiologischen Kontexten: 1) Bestimmung der Spindelgröße bei Fröschen der Gattung Xenopus und 2) Spezifität von Mikrotubuli-Inhibitoren für Plasmodium falciparum-Mikrotubuli über denen ihres menschlichen Wirts. In den Eiern der Froschgattung Xenopus wird die Länge der meiotischen Spindel biochemisch festgelegt und erreicht unabhängig von räumlichen Einschränkungen eine Obergrenze. Messungen der Dynamik von Xenopus-Mikrotubuli zeigen, dass X. laevis-Mikrotubuli sowohl schneller wachsen als auch länger leben als die von X. tropicalis. Darüber hinaus spielt die Quantifizierung der Länge und Massenverteilung der Xenopus-Mikrotubuli zusammen mit den Reaktionen der Eiextrakt-Spindelanordnung eine Rolle für die intrinsische Dynamik der Mikrotubuli bei der Modulation der Spindellänge. Mikrotubuli sind auch Wirkstofftargets bei Pilz- und parasitären Helmintheninfektionen und haben in den letzten Jahrzehnten die Aufmerksamkeit als potenzielles Wirkstoffziel beim Malariaparasiten Plasmodium falciparum auf sich gezogen. Um die Dynamik und Medikamentspezifität von Mikrotubuli von P. falciparum zu charakterisieren, haben wir Tubulin direkt von den Parasiten gereinigt. Zum ersten Mal wurden hier dynamische P. falciparum-Mikrotubuli in vitro rekonstituiert und eine parasitenspezifische Unterdrückung der Dynamik von Mikrotubuli durch Oryzalin und Amiprofos-Methyl direkt nachgewiesen. Diese Studie legt einen experimentellen Rahmen fest, um direkt auf parasitenspezifische Hemmung von Mikrotubuli zu testen, die bisher unter Verwendung bestehender in-vitro-Ansätze nicht beobachtet wurden. / Microtubules are filamentous intracellular polymers that are fundamental components of subcellular structures including the spindle, the cytoskeleton, and flagella in eukaryotes. This study uses a comparative approach to investigate how the intrinsic dynamic and biochemical characteristics of tubulin vary between species and demonstrates their consequences in two different physiological contexts: 1) Spindle size control in Xenopus frogs, and 2) The specificity of microtubule inhibitors for Plasmodium falciparum microtubules over those of their human host. In Xenopus frog eggs, the length of the spindle is biochemically controlled and reaches an upper limit independent of spatial constraints. In this study, in vitro measurements of Xenopus microtubule dynamics show that X. laevis microtubules are both faster-growing and longer-lived X. tropicalis, independent of the influence of microtubule-associated proteins. Furthermore, quantification of Xenopus microtubule length and mass distributions, combined with egg extract spindle assembly reactions, establishes a role for intrinsic microtubule dynamics in modulating spindle length. Microtubules are also established drug targets in fungal and parasitic helminth infections and have in the past decades drawn attention as a potential drug target in the malaria parasite Plasmodium falciparum. In order to characterize P. falciparum microtubule dynamics, structure, and drug specificity, we have used an affinity chromatography-based approach to purify tubulin directly from blood-stage parasites. For the first time, dynamic P. falciparum microtubules have been reconstituted in vitro and parasite-specific suppression of microtubule dynamics by oryzalin and amiprofos methyl has been directly demonstrated. This study establishes an experimental framework to directly test for parasite-specific microtubule inhibition, microtubule structure, and interactions with MAPs that previously have not observed using existing in vitro approaches.

During embryonal development, axons of newly differentiated neurons need to properly interconnect and create a functional neuronal network. To achieve this, the cell requires a growth cone. The growth cone is a highly dynamic structure at the end of growing axons that serves both as the navigator and the propeller. Crosstalk between actin and microtubules is vital for proper axonal pathfinding. But the exact mechanism of this cooperation remains unknown. This diploma thesis investigates the possible role of a candidate scaffolding protein called anillin in this process. Anillin has been studied in two human cell lines. SH-SY5Y neuroblastoma cell line was used for overexpression and siRNA knock-down experiments. Anillin overexpression led to perturbed neurite morphology and growth cone dynamics in SH-SY5Y cells, whereas cells with lower anillin expression had fewer neurites. Next, neurons differentiated from human iPSC (induced pluripotent stem cells) expressing endogenous fluorescently tagged anillin were studied. Local dynamic high concentration spots of anillin have been observed at the base of cell protrusions of differentiating neurons. These anillin flares appeared during cell migration, early neurite initiation, and in newly created growth cones. These results suggest that anillin plays a...

Die vorliegende Doktorarbeit stammt aus (ist in) dem Bereich der NanoBioTechnologie. Ihr Ziel ist es, das Motorprotein Kinesin und Mikrotubuli einzusetzen, um DNS-Moleküle in einem synthetischen Umgebung zu manipulieren. Diese Doktorarbeit setzt sich aus fünf Kapiteln zusammen. In der Einführung wird die makromolekulare Struktur der Zelle beschrieben, z.B. das Zytoskelett und Kinesin, eins der Motorproteine, die auf Mikrotubuli entlang laufen können. Der Schwerpunkt dieses Kapitels liegt auf der Nützlichkeit biologischer Motoren für den Aufbau und die Organisation von Strukturen im technischen Umfeld. Das zweite Kapitel zeigt, wie Kinesin und Mikrotubulis in einem synthetischen Umfeld für den Transport verschiedener Frachten, z.B. Streptavidin, Quantum dots oder DNS-Molekülen, genutzt werden können. Hier liegt der Schwerpunkt auf der Manipulation der DNS-Moleküle durch motor-gesteuerte Mikrotubulis und wie dieser Fracht-Transport-Mechanismus prinzipiell als Basis für die Entwicklung neuer Konzepte im Bereich des Bioingenieurwesens dienen kann. Ein Beispiel für ein solches Konzept ist die auf DNS basierende Molekularelektronik, bei der die Bindung und Streckung von DNS-Molekülen zwischen leitfähigen Oberflächen notwendig ist. Das dritte Kapitel beschreibt den Einfluß der Oberflächeneigenschaften auf die DNS-Anbindung. Es bietet Antworten darauf, wie diese Eigenschaften erforscht, spezifisch gestaltet und vorbereitet werden können, so daß sie der wissenschaftlichen Zielsetzung angemessen sind. Auf die Betrachtung von komplexen Musteranordnungen, wie sie in der Nanoelektronik genutzt werden können, wird im vierten Kapitel eingegangen. Hier wird auf praktische Art und Weise deutlich gemacht, wie DNS-Moleküle an leitfähige Oberflächen gebunden und dort durch Motorproteine und Mikrotubulis manipuliert werden können. Die Vorteile der motor-basierten Manipulation gegenüber den konventionellen Methoden wie AFM oder der optischen Pinzette werden diskutiert. Das fünfte und letzte Kapitel zeigt, wie man das Kinesin-Mikrotubuli-System nutzen kann, um daraus Informationen über DNS-Moleküle abzuleiten. Dafür wurde das Verhalten der Mikrotubulis in Beziehung auf die von gebundenen DNS-Molekülen ausgeübten Kräfte untersucht. Zusammenfassend habe ich experimentelle Untersuchungen und Färbeprotokolle entwickelt, um den gesamten Manipulationsprozeß zu detektieren, visualisieren und kontrollieren. Weiterhin untersuchte ich seine Implikationen auf theoretische Analysen, sowie auf praktische Anwendungen im Nano-Ingenieurwesen. Meine Daten demonstrieren, das DNS-Moleküle im synthetischen Umfeld so manipuliert werden können, daß kontrollierte DNS-Bioschnittstellen entstehen; Schnittstellen, die sowohl für weitere nanoelektronische Anwendungen als auch für topologische DNS-Studien genutzt werden können. Es wird weiterhin erwartet, daß das Kinesin-Mikrotubuli-System für die 3D-Anordnung auf biomolekularer Ebene im technischen Umfeld eine ebenso wichtige Rolle spielen wird. Die Fähigkeit, Vorlagen von Biomolekülen und/oder Anordungen mit definierten Eigenschaften zu schaffen und gleichzeitig ihre biologische Aktivität zu erhalten, kann als Beweis dienen, daß biologische Motoren für die molekulare Fertigung genutzt werden können. - (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 86 MB)- Übersicht über Inhalte siehe Dissertation S. IX - XIII) / The work described in this thesis is in the field of NanoBioTechnology. Its goal is to leverage the motor protein kinesin and its microtubule track to manipulate DNA molecules in synthetic environment. This thesis contains five chapters. The first chapter describes macromolecular structures of the cell: i. e. the cytoskeleton and one of the motor proteins that move along it, kinesin. Emphasized is how biological motors might prove useful for organizing structures in engineered environments. The second chapter demonstrates how kinesin and microtubules can be used in synthetic environments to transport different cargos: i.e. streptavidin, quantum dots and DNA molecules. Special emphasis is placed on the manipulation of DNA molecules by the motor-driven microtubules. This cargo transport mechanism serves as a proof-of-principle for new bioengineering concepts such as DNA-based molecular electronics. The third chapter describes the influences of the surface properties on the DNA attachment and offers answers as how surface characteristics can be investigated, specifically designed and prepared so that they can serve the desired scientific purpose. The fourth chapter describes the manner in which DNA molecules can be attached to conductive surfaces and manipulated with motor proteins and microtubules. The complex DNA pattern formation that can be used for nanoelectronics is demonstrated. The advantages of motor-based manipulation over the conventional "one-by-one" methods (AFM, optical tweezers etc.) are discussed. The fifth and last chapter shows how one can use the kinesin-microtubule system to derive information about DNA molecules. For this, the response of the microtubules to forces exerted by attached DNA molecules has been studied. In summary, I have generated experimental assays and staining procedures to detect, visualize and control the entire manipulation process and to investigate its implications for theoretical analysis as well as for practical nano-engineered applications. My data demonstrated that DNA molecules can be manipulated in synthetic environment by kinesin and microtubules in such a way that controlled DNA biointerfaces can be generated. These biointerfaces can then be used for nanoelectronical application as well as for DNA topological studies. The kinesin-microtubule system is also expected to be equally important for 3D biomolecular assembly in engineered environments. The ability to generate templates of biomolecules and/or bioassemblies with well-defined features while maintaining their bioactivity, serves as proof-of-principle that biological motors can be used for molecular manufacturing. - (The pressure copies contain in each case a CD-ROM as component: QuickTimeMovies (ca. 86 MB)- To overview of contents see thesis P. IX - XIII)

The spatio-temporal organization and dynamic behavior of microtubules accurately react to cellular needs during intracellular transport, signal transduction, growth, division, and differentiation. The cell generates centrosomal microtubules de novo with the help of γ-tubulin complexes (γTuRCs). The post-translational modifications fine-tune microtubule nucleation by targeting the proteins, interacting with γTuRCs. However, the exact signaling pathways, regulating centrosomal microtubule nucleation, remain mostly unknown. In the presented thesis, we functionally characterized protein tyrosine phosphatase SHP-1 and E3 UFM-protein ligase 1 (UFL1) with its interacting protein CDK5RAP3 (C53) in the regulation of centrosomal microtubule nucleation. We also elucidated the role of actin regulatory protein profilin 1 in this process. We found that SHP-1 formed complexes with γTuRC proteins and negatively regulated microtubule nucleation by modulating the amount of γ-tubulin/γTuRC at the centrosomes in bone marrow-derived mast cells (BMMCs). We suggested a novel mechanism with centrosomal tyrosine-phosphorylated Syk kinase, targeted by SHP-1 during Ag-induced BMMCs activation, regulating microtubules. We showed for the first time that UFL1/C53 protein complex is involved in the regulation of microtubule...