Le morphogène Sonic hedgehog (Shh) est requis pour le guidage axonal des neurones commissuraux lors du développement de la moelle épinière, phénomène impliquant des événements de réorganisation du cytosquelette d’actine. Bien qu’il soit généralement admis que le cytosquelette d’actine soit régulé via les petites GTPases de la famille Rho, un effet de Shh sur ces protéines n’a jamais été observé dans aucun contexte physiologique. Nous démontrons que Shh active les petites GTPases Rac1 et Cdc42 et que cette activation est rapide et donc, compatible avec les effets de guidage induits par Shh sur les neurones commissuraux. En parallèle, nous avons étudié l’activation de la protéine Boc, qui est un récepteur de Shh requis pour le guidage axonal des neurones commissuraux. Ces résultats contribuent à raffiner notre compréhension de la transduction cellulaire induite par Shh lors du guidage axonal des neurones commissuraux. / Sonic hedgehog (Shh) is required for axon guidance of commissural neurons during spinal cord development, which involves reorganization of the actin cytoskeleton. Even if it is known that this process is regulated by small Rho GTPases, an effect of Shh on these proteins has not been clearly demonstrated. In this study, we show that Shh activates the small GTPases Rac1 and Cdc42. This activation occurs rapidly, which is compatible with the guidance effects of Shh on commissural neurons. In parallel, we characterized the Shh-dependent activation of Boc, which is a Shh receptor required for commissural axon guidance. Taken together, these results help refine our understanding of the signal transduction mediated by Shh during axon guidance of commissural neurons.

Cell polarisation is a key biological process crucial for the functioning of essentially all cells. Regulation of cell polarity is achieved through various processes determined by both internal and external factors. An example of the latter is that cell polarity can be disrupted or lost as a consequence of a variety of external stresses. When facing such stresses, cells adapt to unfavourable conditions by activating a range of molecular signalling pathways, collectively termed ‘stress response’. Despite the connections between external stress and cell polarity, whether stress-response signalling regulates cell polarisation and what the molecular basis for such regulation remains an open question. The fission yeast Schizosaccharomyces pombe presents an excellent biological platform to study the complexity of cell polarity regulation on a systematic level. This study is aimed at understanding the functional relationship between stress-response signalling and maintenance of cell polarity in this model organism. The findings presented in this thesis set the basis for establishing a functional link between the activation of the S.pombe stress-response pathway and the activity of the master regulator of cell polarity- the Rho GTPase Cdc42. Here, I describe experiments that identify an active involvement of the stress-response mitogen-activated kinase (MAPK) Sty1 in the dispersal of active Cdc42 from the sites of growth. This new role for Sty1 occurs independently from its involvement in transcription regulation and other previously identified signalling pathways involving Sty1. Furthermore, I also find that Sty1’s involvement in Cdc42 regulation has direct implications for fission yeast physiology as it is essential for the maintenance of cellular quiescence upon nitrogen starvation. This thesis also focuses on identifying the targets of Sty1 orchestrating the active Cdc42 disruption. Here, I describe a candidate-based approach, where I investigate the role of proteins from the Cdc42 regulatory network during Sty1 activation. Additionally, I present a global phospho-proteomics approach to identify novel targets of Sty1 and offer preliminary findings which might explain Sty1’s involvement in Cdc42 regulation.

B cells can be activated by T cell-dependent stimuli, such as CD40 ligation and cytokines, which induce extensive proliferation, class switch recombination and somatic hypermutation. Epstein-Barr virus (EBV) can also induce B cell activation by mimicking T cell help through its main oncoprotein, latent membrane protein 1 (LMP-1). It is regulated by another EBV-encoded protein, EBV nuclear antigen 2 (EBNA-2), which is absent in Hodgkin and Burkitt lymphomas. We have studied LMP-1 induction by cytokines in vitro and shown that LMP-1 is induced through the transcription factor signal transducer and activator of transcription (STAT6) and a newly defined high-affinity STAT6-binding site. When IL-4 is added together with lipopolysaccharide (LPS) or α-CD40 to B cells, it induces homotypic round and tight aggregates in vitro, whereas LPS alone does not induce such morphological changes. I describe here attempts to identify the molecules that regulate these responses. I have shown that the Rho GTPase Cdc42 controls the spreading of B cells, whereas two other molecules in the same family, Rac1 and Rac2, control homotypic adhesion. Further, I have shown by conditional deletion of Cdc42 in B cells that it is important in the humoral immune response.  Dock10 is a guanosine nucleotide exchange factor (GEF) for Cdc42. It is expressed through all differentiation stages of B cell development. However, targeted deletion of Dock10 in B cells does not result in an aberrant phenotype. Furthermore, by studying conditional knockout mice for Dock10, Cdc42, Rac1 and Rac2, I have elucidated the mechanism of cytoskeletal changes during B cell activation, leading to adhesion and motility. My results may lead to a better understanding of normal B cell activation and of EBV infection, which is associated with many human tumours and may help to understand cancer development and progression in B cells. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p>

The common type of myxoid soft tissue sarcomas is myxofibrosarcoma. Clinically, increased tumor grading and staging are frequently observed in myxofibrosarcomas after relentless local recurrences, which may eventually lead to metastatic diseases. However, metastatic myxofibrosarcomas are often refractory to current treatment strategies and constitute the primary cause of sarcoma-related death. Immunohistochemistry staining was applied to analyze myxoid tumors of soft tissue in our previous studies, and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) was identified to be significantly upregulated in myxoid soft tissue sarcomas. The PAK1 is a pivotal serine/threonine kinase, which integrates stimuli from various signaling pathways to regulate cell survival, mitosis and cytoskeletal remodeling, etc. We first examined the endogenous PAK1 mRNA and total PAK1 protein levels in various myxoid sarcoma cell lines, including OH931, NMFH1 and NMFH2. This initial screening detected that upregulated PAK1 expression in OH931 and NMFH1, whereas downregulated PAK1 in NMFH2 cells. By wound healing and matrigel transwell assay, we further found that transfection of the expression plasmid carrying wild-type PAK1 gene or PAK1 T423E mutant promoted cell migration and invasion abilities in NMFH2 cells. On the other hand, knockdown of the PAK1 gene by short hairpin RNA interference inhibited the migration rate and invasion ability in NMFH1 cells. By 5-bromo-2-deoxyuridine assay and colony formation assay, we found that either exogenous expression of PAK1 protein or knockdown of PAK1 gene affected cell proliferation and transformation. Interestingly, immunofluorescence demonstrated that treatment with hepatocyte growth factor induced phosphorylation of PAK1 (Thr212) and promoted its nuclear import in NMFH2 cells. In summary, PAK1 plays oncogenic roles in myxoid sarcoma carcinogenesis.

All eukaryotic cells rely on endocytic events to satisfy a constant need for nutrient and fluid uptake from their surroundings. Endocytosis-dependent turnover of cell surface constituents also serves to control signal transduction and establish morphological changes in response to extracellular stimuli. During endocytosis, distinct protein machineries re-sculpt the plasma membrane into vesicular carriers that enclose molecules that are to be taken up into the cell. Besides those produced from the canonical clathrin-mediated endocytic machinery, it is becoming increasingly clear that other membrane carriers exist. The indisputable connection between the function of these uptake systems and various disease states, highlights why it is so important to increase our knowledge about the underlying molecular machineries. The aim of this thesis was therefore to characterise the function of GRAF1, a protein suggested to be a tumour suppressor due to that the gene has been found to be mutated in certain cancer patients. My work focused on understanding how this protein operates during formation of clathrin-independent carriers, with possible implications for disease development. Previous in vitro studies showed that GRAF1 harbours a GTPase activating domain to inactivate Rho GTPase Cdc42, a major actin cytoskeleton regulator. Herein, microscopy based approaches used to analyse HeLa cells demonstrated the importance of a transient interaction between GRAF1 and Cdc42 for proper processing of GRAF1-decorated carriers. Although GRAF1-mediated inactivation of Cdc42 was not vital for the budding of carriers from the plasma membrane, it was important for carrier maturation. In addition, studies of purified GRAF1 and its association with lipid bilayers identified a membrane scaffolding-dependent oligomerisation mechanism, with the ability to sculpt membranes. This was consistent with the assumption that GRAF1 possesses an inherent banana shaped membrane binding domain. Remarkably, this function was autoinhibited and in direct competition with the Cdc42 interaction domain. Finally, other novel GRAF1 interaction partners were identified in this study. Interestingly, many of these partners are known to be associated with protein complexes involved in cell adherence, spreading and migration. Although never actually seen localising to mature focal adhesions that anchor cells to their growth surface, dynamic GRAF1 carriers were captured travelling to and from such locations. Moreover, GRAF1 was recruited specifically to smaller podosome-like structures. Consistent with this, the tracking of GRAF1 in live cells uncovered a clear pattern of dynamic carrier formation at sites of active membrane turnover – notably protrusions at the cell periphery. Furthermore, the silencing of GRAF1 gave rise to cells defective in spreading and migration, indicating a targeting of GRAF1-mediated endocytosis to aid in rapid plasma membrane turnover needed for morphological changes that are a prerequisite for cell movement. Since these cells exhibited an increase in active Rab8, a GTPase responsible for polarised vesicle transport, the phenotype could also be explained by a defect in Rab8 trafficking that results in hyperpolarisation. Taken together, the spatial and temporal regulation of GRAF1 membrane sculpting function is likely to be accomplished via its membrane binding propensity, in concert with various protein interactions. The importance of GRAF1 in aiding membrane turnover during cell movement spans different functional levels – from its local coordination of membrane and actin dynamics by interacting with Cdc42, to its global role in membrane lipid trafficking.

Morphological transitions in Wangiella dermatitidis, a causative agent of human phaeohyphomycosis, influence virulence processes in this polymorphic fungus. My project first involved the cloning and characterizion of the β(1→3) glucan synthase gene WdFKS1, which encodes the enzyme's catalytic subunit, followed by cloning and characterizing the WdRHO1 gene, which encodes its regulatory subunit. To better understand the Rho-type GTPase-mediated regulation of cell polarity and its role in fungal morphological transitions, a homologue of WdRAC1 from a W. dermatitidis was subsequently identified by degenerate PCR and gene walking. Gene deletions of WdFKS1 and WdRHO1 in haploid W. dermatitidis were lethal, whereas the deletion of WdRAC1 was not. RNA interference on WdFKS1 mRNA expression resulted in incomplete septa and damaged cell wall integrity, as well as slow growth rate in W. dermatitidis. Overexpression studies, after site-specific integrations of WdRHO1 and WdRAC1 alleles under control of the glaA promoter into the nonessential WdPKS1 locus, showed the different alleles had different effects on the cell morphological development. For example, whereas overexpression of the wdrho1⁺ allele did not affect the growth rate of W. dermatitidis, the overexpression of wdrho1[superscript G14V], a constitutively active mutation, slowed growth and repressed true filamentous hyphal growth by promoting pseudohyphal growth. While the deletion of WdRAC1 did not affect growth, its loss retarded polarized hyphal growth in a hyphal-inducing minimal medium. Moreover, three new phenotypes of a previously derived WdCDC42 deletion mutant were discovered during this study: in the first, the wdcdc42[Delta] mutant displayed cell lysis when incubated in YPMaltose medium at 37°C; in the second, a dark budding neck abnormality was found after Calcoflour staining; and in the third, the wdcdc42[Delta] mutant displayed no branching during true hyphal growth. Interestingly, the overexpression of wdrac1[superscript G16V] complemented the second and the third phenotypes caused by the WdCDC42 deletion. In addition, the wdcdc42[Delta]/wdrac1[superscript G16V] double mutant unexpectedly displayed an interrupted carotenogenesis pathway. These results support that in W. dermatitidis, Rho-type GTPases play essential roles in growth rate determination and cellular morphogenesis, especially while producing polarized hyphal growth during its many morphological transitions. / text

Le morphogène Sonic hedgehog (Shh) est requis pour le guidage axonal des neurones commissuraux lors du développement de la moelle épinière, phénomène impliquant des événements de réorganisation du cytosquelette d’actine. Bien qu’il soit généralement admis que le cytosquelette d’actine soit régulé via les petites GTPases de la famille Rho, un effet de Shh sur ces protéines n’a jamais été observé dans aucun contexte physiologique. Nous démontrons que Shh active les petites GTPases Rac1 et Cdc42 et que cette activation est rapide et donc, compatible avec les effets de guidage induits par Shh sur les neurones commissuraux. En parallèle, nous avons étudié l’activation de la protéine Boc, qui est un récepteur de Shh requis pour le guidage axonal des neurones commissuraux. Ces résultats contribuent à raffiner notre compréhension de la transduction cellulaire induite par Shh lors du guidage axonal des neurones commissuraux. / Sonic hedgehog (Shh) is required for axon guidance of commissural neurons during spinal cord development, which involves reorganization of the actin cytoskeleton. Even if it is known that this process is regulated by small Rho GTPases, an effect of Shh on these proteins has not been clearly demonstrated. In this study, we show that Shh activates the small GTPases Rac1 and Cdc42. This activation occurs rapidly, which is compatible with the guidance effects of Shh on commissural neurons. In parallel, we characterized the Shh-dependent activation of Boc, which is a Shh receptor required for commissural axon guidance. Taken together, these results help refine our understanding of the signal transduction mediated by Shh during axon guidance of commissural neurons.

INTRODUCTION: Persistent Pulmonary Hypertension of the Newborn (PPHN) is defined as the failure of normal circulatory relaxation in the lungs at birth. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes. Actin polymerization (APM), regulated by Rho GTPases, stabilizes force generation. We studied basal and thromboxane (TP)-induced APM and contraction in normoxic and hypoxic PA myocytes and rings. We also examined the downstream signaling pathways regulating hypoxia and TP-induced APM, and the role that actin plays in TP receptor internalization. METHODS: Smooth muscle myocytes from 2nd to 6th generation PAs of newborn piglets were cultured and exposed to hypoxia (10% O2) or normoxia (21% O2) for 72 hrs, then challenged with 10-6M TP-agonist U46619. APM was quantified by laser-scanning cytometry and stress fiber isolation. Downstream signaling pathways of TP receptor were studied by immunoprecipitation, Rhotekin-RBD and PAK-PBD affinity precipitation, Western blot, immunofluoresence and ELISA. Isometric force to serial concentrations of U46619 was measured in resistant PAs from PPHN and 3-day control swine. RESULTS: Hypoxia induced 2-fold APM via alpha- and gamma-actin isoforms, which contributed to increase U46619-induced contraction. Hypoxia decreased TP association with G12/13 in favor of Gαq. Basal RhoA and Cdc42 activity increased in hypoxia, while Rac activity decreased. U46619-challenge did not further alter RhoA activity in hypoxic cells, but increased Cdc42 and Rac activity. Hypoxia increased phosphorylation of LIMK and PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia, but increased after U46619 exposure. Jasplakinolide significantly stabilized gamma filaments, increasing force generation; cytochalasin D depolymerized all actin isoforms, which attenuated contractile force. Both actin-modifying agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. CONCLUSIONS: PA myocytes exhibit marked RhoA- and Rac-dependent APM in hypoxia. The additional APM response to U46619 challenge is independent of RhoA, but requires Cdc42 signaling. Hypoxia induces APM in PA myocytes, particularly causing an increase in filamentous alpha- and gamma-actin that contributes to increased U46619-induced force generation, a characteristic of PPHN. Dynamic actin also facilitates internalization of the TP receptor. Determining the mechanism that controls TP-mediated APM maybe beneficial as a potential target for PPHN.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2002. / Vita. Bibliography: 116-128.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: References located at the end of each chapter.