The burden of osteoporosis and low bone mass is unrelenting, affecting over 50% of the U.S. population over the age of 50. In a similar reach but different clinical realm, nearly 40% of all men and women will be diagnosed with cancer at some point during their lifetimes. The impact of both of these diseases is compounded by the limited knowledge of cellular mechanisms and the insufficiency of effective treatment options. At the microscopic level of the cell cytoskeleton, increasing evidence has led researchers to further explore microtubules (MTs) and MT-based structures, such as primary cilia, as potential keys to unlocking improved treatment options. However, the way in which microtubules regulate the processes giving rise to these diseases remains a critical gap in knowledge. The works outlined here aimed to elucidate mechanisms that may be used to combat diseases attacking the skeletal and immune systems. In order to characterize the influence of primary cilia with respect to osteoclast differentiation, we implemented a series of treatments to an immortalized macrophage cell line: cilia lengthening (using Fenoldopam) and mechanical stimulation (using oscillatory fluid flow). The results were analyzed by a combination of immunocytochemistry and quantitative PCR. Our first result showed definitively that while osteoclasts do not possess primary cilia, their macrophage precursors do. We also discovered that these macrophage primary cilia are dynamic and can be modulated; cells whose cilia had been lengthened showed a significant decrease in osteoclast formation, indicating that macrophage cilia resorption may be a necessary step for osteoclast differentiation to occur. Combined with findings from previous studies, there is increasing evidence that the primary cilium, as a therapeutic target for bone diseases, may offer a dual beneficial approach to both promote bone formation and downregulate osteoclast activity. We then explored the possibility of directional MT translocation during T-cell activation being linked to Rho GTPases, which regulate actin polymerization. WASp and WAVE2, known to have functional roles in T-cell activation, were identified as primary candidates. In order to investigate this relationship, we implemented a stepwise micropatterning procedure by which PDMS was used to transfer local areas of activation (presenting fluorescently-tagged antibodies against CD3 and CD28) which, upon T-cell receptor (TCR) triggering, could mimic immune synapse (IS) formation. We showed that, although there was no correlation between the spatial organization of MTs and WASp, MTs and WAVE2 location were highly correlated, providing strong evidence for a link between these two systems. In addition, MT disruption via nocodazole resulted in a significant decrease in T-cell activation and mechanosensing capabilities. Given the role of WAVE2 in promoting cell spreading and adhesion during IS formation, this result provides additional evidence that this cytoskeletal filament is in fact connected to proteins involved in actin nucleation and elongation. We anticipate the work in Aim 1 to help reveal a previously unexplored therapeutic target for osteoporosis, a disease that currently has no clinical manifestations prior to a fracture event. Further investigation has the potential to contribute to diagnosis and prevention techniques, as well as new treatments. Similarly, given the emergence of adoptive T-cell immunotherapy for immune-related disorders, the findings of Aim 2 will advance our understanding of both the biological and mechanical influence of the cytoskeleton and motivate microtubules as one component of a more comprehensive armamentarium of treatment approaches.

No description available.

The organization of molecules within a cell is central to cellular processes ranging from metabolism and damage repair to migration and replication. Uncovering the emergent properties of this biomolecular organization can improve our understanding of how organisms function and reveal ways to repurpose their components outside of the cell. This dissertation focuses on the role of organization in two widely studied systems: enzyme cascades and active cytoskeletal filaments.Part I of this dissertation studies the emergent properties of the spatial organization of enzyme cascades. Enzyme cascades consist of a series of enzymes that catalyze sequential reactions: the product of one enzyme is the substrate of a subsequent enzyme. Enzyme cascades are a fundamental component of cellular reaction pathways, and spatial organization of the cascading enzymes is often essential to their function. For example, cascading enzymes assembled into multi-enzyme complexes can protect unstable cascade intermediates from the environment by forming tunnels between active sites. We use mathematical modeling to investigate the role of spatial organization in three specific systems. First, we examine enzyme cascade reactions occurring in multi-enzyme complexes where active sites are connected by tunnels. Using stochastic simulations and theoretical results from queueing theory, we demonstrate that the fluctuations arising from the small number of molecules involved can cause non-negligible disruptions to cascade throughput. Second, we develop a set of design principles for a compartmentalized cascade reaction with an unstable intermediate and show that there exists a critical kinetics-dependent threshold at which compartments become useful. Third, we investigate enzyme cascades immobilized on a synthetic DNA origami scaffold and show that the scaffold can create a favorable microenvironment for catalysis. Part II of this dissertation focuses on the organization of active cytoskeletal filaments. Many mechanical processes of a cell, such as cell division, cell migration, and intracellular transport, are driven by the ATP-fueled motion of motor proteins (kinesin, dynein, or myosin) along cytoskeletal filaments (microtubules or actin filaments). Over the past two decades, researchers have been repurposing motor protein-driven propulsion outside of the cell to create systems where cytoskeletal filaments glide on surfaces coated with motor proteins. The study of these systems not only elucidates the mechanisms of force production within the cell, but also opens new avenues for applications ranging from molecular detection to computation. We examine how microtubules gliding on surfaces coated with kinesin motor proteins can generate collective behavior in response to mutualistic interactions between the filaments and motors, thereby maximizing the utilization of system components and production. To this end, we used a microtubule-kinesin system where motors reversibly bind to the surface. In experiments, microtubules gliding on these reversibly bound motors were unable to cross each other and at high enough densities began to align and form long, dense bundles. The kinesin motors accumulated in trails surrounding the microtubule bundles and participated in microtubule transport. In conclusion, our study of the emergent properties of the spatial organization of enzyme cascades and the mutualistic interactions within active systems of motor proteins and cytoskeletal filaments provides insight into both how these systems function within cells and how they can be repurposed outside of them.

Etude de l'implication de CRMP4, un partenaire de MAP6, dans la voie de signalisation sémaphorine 3E.Pendant le développement embryonnaire, les neurones établissent des milliards de connexions. Ces connexions ne sont pas aléatoires, mais précisément orientées, dirigées par des molécules de guidage situées dans l’environnement cellulaire. Le branchement inapproprié de ces neurones a de graves conséquences sur les fonctions sensorielles, motrices et cognitives du système nerveux, aboutissant à des pathologies neurologiques et psychiatriques telle que la schizophrénie. Ainsi la mutation de certaines protéines impliquées dans le guidage de ces connexions, comme MAP6 ou CRMP4, peut entraîner des perturbations conduisant à des prédispositions pour le développement de telles pathologies. En effet l'absence de MAP6 (souris KO MAP6) conduit à l'altération de nombreuse connections neuronales associé a différents troubles comportementaux réminiscent avec des symptômes schizoïdes. Parmi les faisceaux d'axones affectés on remarque la disparition du fornix, un faisceau neuronal connu pour son implication dans la schizophrénie. Cette disparition est en partie causée, en l'absence de MAP6, par l'abolition de la signalisation induite par la molécule de guidage sémaphorine 3E (Sema3E). Dans ce projet de thèse, le lien entre MAP6 et CRMP4 dans cette voie de signalisation Sema3E à pu être établi. De plus, l'impact de l'absence de la protéine CRMP4 sur la formation du fornix a pu être caractérisé par l'étude neuroanatomique des souris KO CRMP4. Nous avons par ailleurs pu mettre en évidence de nouvelles altérations causée par l'absence de MAP6. Dans son ensemble ce travail approfondit les connaissances des défauts des connectivités des souris KO MAP6 et identifie CRMP4 comme un nouvel acteur de la signalisation Sema3E et de la formation du fornix. / Study of the involvement of the MAP6 partners, CRMP4, in the semaphorin 3E signaling pathway.During embryonic development, neurons establish billion of connections between them. Those connections are not random. On the contrary, they are precisely targeted thanks to the driving by cellular environment guidance cues. A wrong branching of those neurons can lead to dramatic impairment of sensory, motor and cognitive function of the central nervous system resulting in neurologic or psychiatric disorders such as Schizophrenia. Thus, mutation of proteins implicated on neurons guidance like MAP6 or CRMP4 can lead to susceptibility for those kind of pathology occurrence. In fact, MAP6 deletion ( MAP6 KO mice) leads to diverse neuronal connectivity alterations associated to schizophrenia-like behavior disorders. Among axonal tracts affected we notice the absence of the fornix known for its implication on Schizophrenia. In MAP6 KO mice, this fornix disruption is partly due to the loss of semaphorin 3E (Sema3E) dependant signaling pathway. This project shows the involvement of CRMP4, a partner of MAP6, in the Sema3E signaling pathway. Furthermore, it characterized the impact of the CRMP4 deletion (CRMP4 KO) on fornix formation. Finally, neuroanatomical studies allowed us to identify unknown alteration of MAP6 KO mice connectivity alteration.

Les agents ciblant les microtubules sont des médicaments anticancéreux efficaces. Leur utilisation dans le cadre d’un traitement combiné avec des rayonnements ionisants est également une stratégie prometteuse. Cependant, l’apparition de résistances aux produits chimiques et aux radiations nécessite de rechercher d'autres types de traitements. Nos laboratoires ont récemment décrit deux médicaments qui ciblent directement ou indirectement les microtubules. Premièrement, un analogue du 2-méthoxyestradiol, un poison de fuseau se liant à des microtubules et provoquant la formation de fuseaux mitotiques anormaux. Il s'agit du 2-éthyl-3-O-sulphamoyle-estra-1,3,5 (10) 16-tétraène (ESE-16). Deuxièmement, le 9-benzoyloxy-5,11-diméthyl-2H, 6H-pyrido [4,3-b] carbazol-1-one (LimPyr1), un nouvel inhibiteur des LIM kinases induisant indirectement la stabilisation des microtubules. Il a été démontré récemment que LimPyr1 est actif sur les modèles de cancer du sein résistants au taxol. En tant que médicaments ciblant les microtubules, les deux agents, ESE-16 et LimPyr1, induisent des défauts mitotiques. Nous émettons donc l’hypothèse qu’ils pourraient sensibiliser les cellules aux radiations. Le but de ce projet de thèse était de vérifier cette hypothèse et, plus précisément, de déterminer si de faibles doses de ESE-16 et de LimPyr1 pourraient augmenter l'apoptose et retarder la réparation nucléaire induite par le rayonnement dans les cellules du cancer du sein in vitro.Différentes lignées cellulaires cancéreuses, les cellules MCF-7, MDA-MB-231 et BT-20, ont été exposées à ESE-16 et à LimPyr1 pendant 24 heures avant un rayonnement de 8 Gy. Les effets de ces combinaisons thérapeutiques ont été comparés à ceux obtenus à partir de cellules exposées aux composés seuls ou aux seules radiations. L'activation des voies de survie et des voies apoptotiques intrinsèques a été étudiée. Les résultats ont révélé une augmentation de la signalisation de la survie et de la mort dans les cellules exposées aux traitements individuels. Les traitements combinés ont diminué la survie des cellules alors que la signalisation apoptotique augmentait, entraînant une augmentation de l'apoptose. En outre, les traitements combinés ont augmenté de manière significative la présence de micronoyaux dans les cellules BT-20, indiquant une augmentation des dommages à l'ADN. Les cellules MCF-7 et MDA-MB-231 présentent une formation de micronoyaux similaire lorsqu'elles sont exposées à la combinaison de traitements ou au rayonnement uniquement. La phosphorylation de H2AX (γH2AX) (normalement augmentée lors de dommages à l'ADN) et l'expression de Ku70 (nécessaire pour la réparation de l'ADN) étaient diminuées dans les cellules de cancer du sein prétraitées 2 heures après l'irradiation par rapport aux cellules exposées à l'irradiation uniquement. L'expression de H2AX et Ku70 est cependant significativement accrue 24 heures après irradiation des cellules prétraitées par rapport aux cellules exposées aux traitements individuels. Des expériences portant sur la réponse adaptative ont révélé que LimPyr1 diminuait le développement de la résistance aux radiations en augmentant la perméabilité transmembranaire mitochondriale et en générant des ROS, un mécanisme qui n'est pas observé dans cellules traitées par ESE-16. Nous avons également observé une communication intercellulaire entre les cellules exposées au rayonnement et les cellules non exposées via l'effet induit par le rayonnement.En conclusion, le blocage mitotique partiel induit par ESE-16 et LimPyr1 rend les chromosomes plus exposés aux dommages dus aux radiations, comme l'indique l'augmentation de la présence de micronoyaux. De plus, les deux composés diminuent la signalisation et le trafic des protéines de protection et de dommages à l'ADN. En outre, LimPyr1 empêche le développement de résistances aux radiations dans les cellules exposées aux radiations. / Microtubule targeting agents are effective anti-cancer drugs. Their use as part of a combined treatment modality with ionising radiation is also a promising strategy. However, the emergence of resistance to chemical and radiation requires searching for alternative treatments. Our laboratories have recently described two drugs that directly or indirectly target the microtubules. Firstly, an analogue of 2-methoxyestradiol, a spindle poison binding to microtubules and causing the formation of abnormal mitotic spindles. This is 2-ethyl-3-O-sulphamoyl-estra-1,3,5 (10) 16-tetraene (ESE-16). Secondly, 9-benzoyloxy-5,11-dimethyl-2H, 6H-pyrido [4,3-b] carbazol-1-one (LimPyr1), a novel inhibitor of LIM kinases indirectly inducing microtubule stabilization. It has been recently shown that LimPyr1 is active on taxol-resistant breast cancer models. As microtubule-targeting drugs, both agents, ESE-16 and LimPyr1, induce mitotic defects. We thus hypothesize that they could sensitize cells to radiation. The aim of this PhD project was to test that hypothesis and, more specifically, to investigate whether low-dose ESE-16 and LimPyr1 could increase apoptosis and delay nuclear repair induced by radiation in breast cancer cells in vitro.Various cancer cell lines, MCF-7-, MDA-MB-231- and BT-20 cells, were exposed to ESE-16 and LimPyr1 for 24-hours prior to 8 Gy radiation. The effects of these combination therapies were compared to those obtained from cells exposed to the compounds alone or only to radiation. The activation of the survival and intrinsic apoptotic pathways were investigated. Results revealed an increase in survival and -death signaling in cells exposed to the individual treatments. The combination treatments decreased the cell survival while apoptotic signaling was increased, resulting in increased apoptosis. Furthermore, the combination treatments significantly increased the presence of micronuclei in BT-20 cells, indicating an increase in DNA damage. MCF-7- and MDA-MB-231 cells displayed similar micronuclei formation when exposed to the combination treatments or radiation only. Phosphorylation of H2AX (γH2AX) (normally increased upon DNA damage) and Ku70 expression (required for DNA repair) were decreased in pretreated breast cancer cells 2 hours after irradiation compared to cells exposed to irradiation only. The expression of H2AX and Ku70, however, is significantly increased 24 hours after irradiation of the pretreated cells relative to the cells exposed to the individual treatmentsExperiments investigating the adaptive response revealed that LimPyr1 decreased radiation resistance development by increasing the permeability of the mitochondrial transmembrane (flow cytometry measuring Mitocapture™) and the generation of ROS (flow cytometry employing hydroethidine), a mechanism not observed in ESE-16 pre-treated cells. We also observed an intercellular communication between cells exposed to radiation and non-exposed cells via the radiation induced bystander effect.In conclusion, the anti-mitotic effect of ESE-16 and LimPyr1 renders the chromosomes more exposed to radiation damage, as assessed by the increased occurrence of micronuclei. Moreover, both compounds decrease the signaling and trafficking of DNA damage and repair proteins. Additionally, LimPyr1 prevented the development of radiation resistance in cells exposed to radiation.

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No description available.

No description available.

Bovine Parvovirus (BPV) belongs to the genus Bocavirus, family Parvoviridae. BPV is the leading etiologic agent among the pathogens that cause primary gastroenteritis of cattle. Many of the intracellular events associated with virus replication are unknown. In this research project, we investigated BPV internalization into the host cell and trafficking in the cytosol. Preliminarily, EBTr cells had abundant clathrin, virus attached to purified clathrin, and EM micrographs revealed virus in endocytic vacuoles. Assays detecting virus infectivity (i.e. viral protein synthesis), virus production (completion of the replication cycle), and quantitative PCR (qPCR) to detect viral transcripts were used to evaluate virus uptake and subsequent trafficking events in the presence of selective inhibitors. Cell toxicity mediated by the drugs was evaluated by the MTT test. Virucidal effects of the drugs were assessed. A control virus was used to verify the inhibitor technology. Immunofluoresceinated virus particles were found in clathrin-rich early endosomes. Clathrin-mediated endocytosis (CME) was examined by clathrin polymerization inhibiting agent (chloropromazine), lysosomotropic agents (ammonium chloride and chloroquine), a vacuolar ATPase inhibitor (bafilomycin A1), and a blocker of transition between endosomes (brefeldin A). Caveosome pathway inhibitors included phorbol 12-myristate 13-acetate (a suppressor of caveolae formation), nystatin and methyl-beta-cyclodextrin (lipid raft blockers), and genistein (a tyrosine kinase phosphorylation inhibitor). Trafficking of BPV was investigated using specific inhibitors of proteasomal activity, actin-myosin function, and microtubule-dynein function. The proteasomal protease suppressor (lactacystin), and a proteasomal chymotrypsin inhibitor (epoxomicin) were used. The role of actin was probed by cytochlasin D, latrunculin A, and ML-7. The microtubule inhibitors nocodazole, vanadate, and EHNA were used to probe microtubule function. The inhibitors of CME reduced virus production and reduced infectivity, a result confirmed by qPCR. The blockers of caveolin-mediated entry did not interfere with virus production nor virus infectivity. Proteasome activity blockage did not affect the virus replication. But the virus cycle was affected by actin blockage and by microtubule blockage detected by qPCR. Taken together these data indicate that BPV uptake is mediated by clathrin coated pits and is acid-dependent. Further processing of BPV in the cytosol does not require proteasomal enzymes. Actin-associated vesicular transport appears to be essential to virus replication and trafficking to the nucleus appears to be mediated by microtubules.

Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small(„5% deformation) and large strains(>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules