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"Cell and process migration are critical to the establishment of neural circuitry. The study of these processes is facilitated through use of model organisms with simple nervous systems, such as C. elegans. Research in this nematode has defined the cytoplasmic adaptor MIG-10 as a key regulator of these processes. Mutation of mig-10 disrupts neuronal and axonal migration and outgrowth of the ‘canals’, or processes, of the excretory cell. MIG-10 directs the localization of UNC-34, which remodels actin filaments at the leading edge of a migrating cell or process to modify the direction or rate of its protrusion. An interactor of MIG-10 identified in a yeast two- hybrid analysis, ABI-1, has several roles in actin remodeling, such as targeting Ena/VASP members for phosphorylation by Abl kinase. Mutation of abi-1 in the nematode produces phenotypes that resemble those of mig-10 mutants, including disrupted outgrowth of the excretory canals, a developmental process in which ABI-1 is known to function cell autonomously. To test the hypothesis that the ABI-1/MIG-10 interaction contributes to cell migration and outgrowth, both in vivo and in vitro analyses were performed. Expression of either MIG-10A or MIG-10B exclusively in the excretory cell partially rescued the canal truncation characteristic of mig-10 mutants, suggesting MIG-10 functions autonomously in this cell during canal outgrowth. Physical interaction between MIG-10 and ABI-1 was confirmed using a co-immunoprecipitation system. Both MIG-10A and MIG-10B interact with ABI-1 through a mechanism that likely involves the SH3 domain of ABI-1 and sites in either the central region or C-terminus of MIG-10. These results suggest that MIG-10 and ABI-1 function together in a cell autonomous manner to promote cell or process migration. A possible consequence of this interaction is modulation of the MIG-10 binding to UNC-34 through Abl-mediated phosphorylation of MIG-10."

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed Mar. 23, 2009). Vita. Includes bibliographical references.

The development of fibrosis involves disruption of connective tissue homeostasis that may include inhibition of collagen remodeling pathways such as phagocytosis, as well as the differentiation of myofibroblasts, pro-fibrotic cells. Myofibroblast differentiation is dependent on actin assembly, which can alter cell shape and is required for collagen phagocytosis and remodeling. Cyclosporin A (CsA) is a commonly used drug for prevention of organ transplant rejection that causes marked fibrosis in periodontal tissues by inhibiting collagen phagocytosis. As gelsolin is a Ca2+-dependent actin severing protein that mediates collagen phagocytosis, I determined whether gelsolin is a CsA target. Compared to vehicle-treated controls, CsA-treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. From a series of in vitro experiments, I conclude that CsA-induced accumulation of collagen in the periodontal ECM involves disruption of the actin severing properties of gelsolin. This disruption inhibits the binding step of collagen phagocytosis and promotes fibrosis. During the development of pressure-induced cardiac hypertrophy, collagen accumulates in the interstitium, due to myofibroblasts which express alpha-smooth muscle actin (SMA). As focal adhesion complexes are putative mechanosensing organelles, I examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin, in the regulation of SMA expression. After application of mechanical force to cultured fibroblasts through collagen-coated magnetite beads attached to beta1 integrins, FAK and gelsolin were recruited to beads and there was increased nuclear translocation of MRTF-A, a transcriptional co-activator of SMA. These data suggested a novel pathway in which mechanosensing by FAK regulates actin assembly through gelsolin; actin assembly in turn controls SMA expression through MRTF-A. I also examined a potential role for the actin nucleators, mammalian Diaphanous-related formins (mDia), in the mechanosensing pathway that leads to force-induced expression of SMA. siRNA knockdown of mDia inhibited actin assembly at force-induced focal adhesions. In anchored collagen gels to model myofibroblast-mediated contraction of the matrix, mDia knockdown reduced contraction by 50%. Collectively, these experiments indicate that the regulation of actin assembly plays an important role in the development of force-induced transcriptional activation of SMA, myofibroblast differentiation and collagen phagocytosis.

The development of fibrosis involves disruption of connective tissue homeostasis that may include inhibition of collagen remodeling pathways such as phagocytosis, as well as the differentiation of myofibroblasts, pro-fibrotic cells. Myofibroblast differentiation is dependent on actin assembly, which can alter cell shape and is required for collagen phagocytosis and remodeling. Cyclosporin A (CsA) is a commonly used drug for prevention of organ transplant rejection that causes marked fibrosis in periodontal tissues by inhibiting collagen phagocytosis. As gelsolin is a Ca2+-dependent actin severing protein that mediates collagen phagocytosis, I determined whether gelsolin is a CsA target. Compared to vehicle-treated controls, CsA-treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. From a series of in vitro experiments, I conclude that CsA-induced accumulation of collagen in the periodontal ECM involves disruption of the actin severing properties of gelsolin. This disruption inhibits the binding step of collagen phagocytosis and promotes fibrosis. During the development of pressure-induced cardiac hypertrophy, collagen accumulates in the interstitium, due to myofibroblasts which express alpha-smooth muscle actin (SMA). As focal adhesion complexes are putative mechanosensing organelles, I examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin, in the regulation of SMA expression. After application of mechanical force to cultured fibroblasts through collagen-coated magnetite beads attached to beta1 integrins, FAK and gelsolin were recruited to beads and there was increased nuclear translocation of MRTF-A, a transcriptional co-activator of SMA. These data suggested a novel pathway in which mechanosensing by FAK regulates actin assembly through gelsolin; actin assembly in turn controls SMA expression through MRTF-A. I also examined a potential role for the actin nucleators, mammalian Diaphanous-related formins (mDia), in the mechanosensing pathway that leads to force-induced expression of SMA. siRNA knockdown of mDia inhibited actin assembly at force-induced focal adhesions. In anchored collagen gels to model myofibroblast-mediated contraction of the matrix, mDia knockdown reduced contraction by 50%. Collectively, these experiments indicate that the regulation of actin assembly plays an important role in the development of force-induced transcriptional activation of SMA, myofibroblast differentiation and collagen phagocytosis.

Enterohaemorrhagic Escherichia coli (EHEC) have recently emerged as significant zoonotic pathogens. O157:H7 is one of the most common EHEC serotypes associated with human disease, which is transmitted faeco-orally from a bovine reservoir. EHEC O157:H7 preferentially colonises the bovine terminal rectum (BTR). Injection of virulence factors by type-III secretion is necessary for colonisation of cattle and results in re-modelling of the host cytoskeleton. Flagella machinery is evolutionarily related to the Type III secretion apparatus and O157 strains lacking H7 flagella show reduced adherence to the BTR. Vaccination with FliC, the main component of H7 flagella, has the potential to protect cattle against E. coli O157:H7 infection. The focus of this work was to investigate the molecular basis for H7 flagella binding to the BTR, in order to understand the basis for FliCH7 being an immuno-protective antigen. H7 flagella were shown to adhere across the surface and penetrate into BTR epithelial cells. Both the FliC shaft and the FliD cap components of flagella filaments showed the capacity to adhere to BTR epithelial cells. Preliminary studies indicate that the current FliCH7 vaccination of cattle results in FliD-specific antibodies where oral challenge with O157:H7 does not. FliD is more conserved than FliCH7, which contains a predicted 88aa structural insertion, but variation occurs along the full length of the FliD protein. There was no evidence for post-translational modification of FliCH7. A number of actin binding proteins were identified as potential FliC and FliD binding partners from BTR epithelial cell lysates. From this, a panel of purified galectin-4, cofilin-1 and βγ-actin was used to compare binding of flagella from different pathogens. H7 flagella bound more to cofilin-1 than βγ-actin, whereas phase-1 and phase-2 flagella from Salmonella Typhimurium bound more to βγ-actin, than to cofilin-1. Size-exclusion chromatography indicated that cofilin-1 alters H7 flagella filament polymerisation dynamics. αβ-ctin polymerisation and depolymerisation experiments indicate that H7, phase-1 and phase-2 flagella interactions with actin affect actin dynamics.

<i>Legionella pneumophila,</i> the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates over 330 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK (Lpg0969) as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H₉₅E_XXH₉₉ (x, any amino acid) motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage occurs during <i>L. pneumophila </i>infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, <i>L. pneumophila</i> exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.

Le cytosquelette dactine est une structure dynamique impliquée dans de nombreux processus biologiques tels que les mouvements cellulaires, la phagocytose ou encore la mitose. En plus de son intervention dans ces différents événements essentiels pour lhoméostasie de la cellule, de nombreuses études ont démontré quil était également capable dinfluer sur des voies de transduction notamment en modulant lactivité de protéines kinases ou de facteurs de transcription. Un facteur de transcription important est le facteur de transcription NF-κB. Ce facteur de transcription joue un rôle majeur dans la régulation de nombreux processus cellulaires tels que les réponses immunitaires innée et adaptative, la réponse inflammatoire, lapoptose et la division cellulaire. Il peut être activé en réponse à de nombreux stimuli tels que les cytokines pro-inflammatoires, les produits bactériens ou viraux ou encore suite à un stress oxydant. Malgré les différentes études démontrant que le cytosquelette dactine est capable de moduler certaines voies de signalisation et que certains stimuli capables dactiver le NF-κB, comme le LPS et le TNFα, sont également associés à des modifications du cytosquelette dactine, peu de travaux ont été réalisés afin de déterminer limpact des perturbations du cytosquelette dactine sur lactivation de cet important facteur de transcription. Ces différents arguments nous ont donc poussé à étudier le rôle des perturbations du cytosquelette dactine dans les voies dactivation du NF-κB. Ainsi, dans une première partie, nous avons étudié leffet de plusieurs agents perturbant le cytosquelette dactine [la Cytochalasine D (CytD), le Jasplakinolide (JP) et la Latrunculine B (Lat B)] sur lactivation du NF-κB. Nous avons pu mettre en évidence que ces différentes substances sont capables dinduire la voie classique dactivation du NF-κB uniquement dans des cellules myélomonocytaires. De plus, nous avons également observé que ces agents sont capables dinduire la production despèces réactives à loxygène (ROS) Dans un second temps, nous nous sommes intéressés à leffet de la CytD sur lactivation du NF-κB dans des cellules myélomonocytaires induites par le TNFα ou le LPS. Nous avons pu démontrer que la CytD promouvoit la voie classique du NF-κB dans les cellules induites par le LPS. En effet, il semblerait que la CytD agisse notamment sur cette voie en augmentant le temps de résidence du récepteur à cet inducteur, le TLR4, à la surface de la membrane plasmique. Parallèlement à ces observations, nous avons pu mettre en évidence que la CytD augmente la phosphorylation de certains résidus de la sous-unité RelA du NF-κB induite par les deux inducteurs classiques ce qui permet un meilleur recrutement de la RNA polymérase II sur le promoteur endogène de la chémokine IL-8.

The actin cytoskeleton is critical to the proper function of cells and its misregulation can lead to human disease states. As a consequence, actin dynamics is tightly controlled. To gain further insight into the mechanisms controlling actin dynamics, my studies have focused on two families of proteins implicated in actin regulation. The Nck proteins act as molecular adaptors in signal propagation by linking upstream mediators, which they recognize through the Nck SH2 domain, to downstream effectors, which bind the Nck SH3 domains. I have found that Nck is required in podocyte cells for proper foot process formation, a process involving actin cytoskeletal reorganization, and therefore for proper kidney function. Furthermore, I show that Nck links the podocyte adhesion protein nephrin to actin polymerization. In cell-based assays, nephrin-induced actin polymerization is dependent on an interaction with functional Nck, which occurs through binding of three phosphorylated tyrosine sites within the cytoplasmic tail of nephrin to the Nck SH2 domain. Finally, I demonstrate that the enteropathogenic E.coli protein Tir reorganizes the cytoskeleton by molecular-mimicry of nephrin-like signaling. The srGAP proteins are GTPase activating proteins that attenuate the activity Rho GTPases, proteins directly involved in actin cytoskeletal control. The regulatory mechanisms that control srGAP activity are unclear. I have found that the srGAP family members srGAP1, srGAP2, and srGAP3 interact, through their carboxy-terminal region with 14-3-3 proteins, and that this interaction is dependent on protein kinase C-induced phosphorylation of srGAP. 14-3-3 binding does not affect the activity of srGAP2, as determined using cell-based GAP assays. Further studies are required to clarify the biological significance of this interaction to srGAP regulation. The data presented in this thesis furthers our understanding of signaling networks that control the actin cytoskeleton, and brings us closer to the goal of fully understanding actin dynamics and cellular signaling.

The actin cytoskeleton is critical to the proper function of cells and its misregulation can lead to human disease states. As a consequence, actin dynamics is tightly controlled. To gain further insight into the mechanisms controlling actin dynamics, my studies have focused on two families of proteins implicated in actin regulation. The Nck proteins act as molecular adaptors in signal propagation by linking upstream mediators, which they recognize through the Nck SH2 domain, to downstream effectors, which bind the Nck SH3 domains. I have found that Nck is required in podocyte cells for proper foot process formation, a process involving actin cytoskeletal reorganization, and therefore for proper kidney function. Furthermore, I show that Nck links the podocyte adhesion protein nephrin to actin polymerization. In cell-based assays, nephrin-induced actin polymerization is dependent on an interaction with functional Nck, which occurs through binding of three phosphorylated tyrosine sites within the cytoplasmic tail of nephrin to the Nck SH2 domain. Finally, I demonstrate that the enteropathogenic E.coli protein Tir reorganizes the cytoskeleton by molecular-mimicry of nephrin-like signaling. The srGAP proteins are GTPase activating proteins that attenuate the activity Rho GTPases, proteins directly involved in actin cytoskeletal control. The regulatory mechanisms that control srGAP activity are unclear. I have found that the srGAP family members srGAP1, srGAP2, and srGAP3 interact, through their carboxy-terminal region with 14-3-3 proteins, and that this interaction is dependent on protein kinase C-induced phosphorylation of srGAP. 14-3-3 binding does not affect the activity of srGAP2, as determined using cell-based GAP assays. Further studies are required to clarify the biological significance of this interaction to srGAP regulation. The data presented in this thesis furthers our understanding of signaling networks that control the actin cytoskeleton, and brings us closer to the goal of fully understanding actin dynamics and cellular signaling.