Interactions between the Eph family of receptor tyrosine kinases, and their ligands, the ephrins, are required for the normal development and maintenance of many patterns of connectivity within the nervous system. Eph receptors and ephrins are expressed widely throughout both the developing and mature nervous system where they function as important regulators of cell migration and axon guidance. The studies presented in this thesis examine the role of one particular member of the Eph receptor family, EphA4, in regulating mechanisms that underlie the development and maintenance of certain neural connections within the nervous system. This thesis demonstrates that the EphA4 receptor is expressed within specific regions of the developing and mature nervous system, some of which are associated with the control of locomotor activity. Consistent with these observations are the locomotor defects exhibited by animals with a targeted disruption of the EphA4 gene. These animals exhibit abnormal bilateral limb movements and have severe disruptions of a number of major axonal pathways. One of these disrupted axonal pathways, the corticospinal tract (CST), is a particularly important mediator of locomotor activity. This thesis reveals that EphA4 is expressed on the axons that comprise the CST. It demonstrates that although EphA4 is not required for the initial development of the CST, repulsive interactions between EphA4-bearing CST axons and ephrinB3, a ligand for EphA4 that is expressed at the midline of the spinal cord, appear to prevent CST axons from aberrantly recrossing the spinal midline during development.

In the lung, epithelial cell migration plays a key role in both physiological and pathophysiological conditions. When the respiratory epithelium is injured, the epithelial lining in the respiratory system can be seriously damaged. Spreading and migrating of the surviving cells neighboring a wound are essential for airway epithelial repair. When the repair process is affected, aberrant remodeling may occur, which is important in the pathogenesis of lung diseases. However, in comparison with other cellular and molecular functions in the respiratory system, our understanding on lung epithelial cell migration and invasion is limited. To gain insight into the molecular mechanisms that govern these cellular processes, I asked whether normal (non-cancerous) human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls cell migration and invasion. I found that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased the invasiveness of these epithelial cells. I further demonstrated that PDBu-induced podosome formation was mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes, whereas atypical PKCζ played a dominant role in the proteolytic activity of podosomes through recruitment of MMP-9 to podosomes, and MMP-9 secretion and activiation. I also found that that PDBu can activate PI3K/Akt/Src and ERK1/2 and JNK but not p38. PI3K, Akt and Src were critical for podosome formation, whereas ERK1/2 and JNK mediated the proteolytic activity of podosomes via MMP-9 recruitment, gene expression, release and activation without affecting podosome assembly. Podosomes are important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration. My thesis work unveils the molecular mechanisms that regulate podosomal formation and proteolytic function in normal human bronchial epithelial cells. These novel findings may enhance our understanding of cell migration and invasion in lung development and repair. Similar mechanisms may be also applicable to other cell types in distinct organs.

In the lung, epithelial cell migration plays a key role in both physiological and pathophysiological conditions. When the respiratory epithelium is injured, the epithelial lining in the respiratory system can be seriously damaged. Spreading and migrating of the surviving cells neighboring a wound are essential for airway epithelial repair. When the repair process is affected, aberrant remodeling may occur, which is important in the pathogenesis of lung diseases. However, in comparison with other cellular and molecular functions in the respiratory system, our understanding on lung epithelial cell migration and invasion is limited. To gain insight into the molecular mechanisms that govern these cellular processes, I asked whether normal (non-cancerous) human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls cell migration and invasion. I found that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased the invasiveness of these epithelial cells. I further demonstrated that PDBu-induced podosome formation was mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes, whereas atypical PKCζ played a dominant role in the proteolytic activity of podosomes through recruitment of MMP-9 to podosomes, and MMP-9 secretion and activiation. I also found that that PDBu can activate PI3K/Akt/Src and ERK1/2 and JNK but not p38. PI3K, Akt and Src were critical for podosome formation, whereas ERK1/2 and JNK mediated the proteolytic activity of podosomes via MMP-9 recruitment, gene expression, release and activation without affecting podosome assembly. Podosomes are important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration. My thesis work unveils the molecular mechanisms that regulate podosomal formation and proteolytic function in normal human bronchial epithelial cells. These novel findings may enhance our understanding of cell migration and invasion in lung development and repair. Similar mechanisms may be also applicable to other cell types in distinct organs.

In cellular MRI, micrometer-sized iron oxide particles (MPIO) are a more sensitive contrast agent for tracking inflammatory-cell migration compared to ultra-small superparamagnetic iron oxide particles (USPIO). Inflammation, which promotes adverse tissue remodeling, is known to occur in the viable myocardium adjacent to the necrosed area after a myocardial infarction (MI). This study investigated the temporal relationship between inflammatory cell infiltration and cardiac function during tissue remodeling post-MI using MPIO-enhanced MRI. The MPIO were injected into 7 C57Bl/6 mice (MI+MPIO group) via intravenous administration. The MI was induced 7 days post-MPIO injection. As control groups, 7 mice (Sham+MPIO group) underwent sham-operated surgery without myocardial injury post-MPIO injection and another 6 mice (MI-MPIO group) underwent MI surgery without MPIO injection. MRIs performed post-MI showed a significant signal attenuation at the MI zone in the MI+MPIO group compared to the control groups. The findings suggested that the inflammatory cells containing MPIO infiltrated into the myocardial injury site. Cardiac function was also measured and correlated with the labeled-cell infiltration at the MI site. This study demonstrated a noninvasive technique for monitoring inflammatory cell migration using the MPIO contrast agent. This MPIO-enhanced MRI technique could provide additional insight concerning cardiac disease progression that would improve therapeutic treatment for MI patients.

Embryonic epiboly has become an important developmental model for studying the mechanisms underlying collective movements of epithelial cells. In the last couple of decades, most studies of epiboly have utilized Xenopus or zebrafish as genetically tractable model organisms, while the avian epiboly model has received virtually no attention. Here, we re-visit epiboly in quail embryos and characterize several molecular markers of epithelial-to-mesenchymal transition (EMT) in the inner zone of the extraembryonic Area Opaca and at the blastoderm edge. Our results show that the intermediate filament vimentin, a widely-used marker of the mesenchymal phenotype, is strongly expressed in the edge cells compared to the cells in the inner zone, and that epiboly is inhibited when embryos are treated with Withaferin-A, a vimentin-targeting drug. Laminin, an extracellular matrix protein that is a major structural and adhesive component of the epiblast basement membrane, is notably absent from the blastoderm edge, and shows three distinct morphological regions approaching the leading edge. While these expression profiles are consistent with a mesenchymal phenotype, several other epithelial markers, including cytokeratin, β-catenin, and E-cadherin, were present in the blastoderm edge cells. Moreover, the results of a BrDU proliferation assay suggest that expansion of the edge cell population is primarily due to recruitment of cells from the inner zone, and not proliferation. Taken together, our data suggest that the edge cells of the avian blastoderm have characteristics of both epithelial and mesenchymal cells, and could serve as an in-vivo model for cancer and wound healing studies.

No description available.

Blood vessels are essential for the delivery of nutrients and oxygen to tissues, as well as for the removal of waste products. Patients with tumors, wounds or diabetes all have active angiogenesis, formation and remodeling of blood vessels, a process that is initiated and manipulated by gradients of secreted signaling proteins. This thesis describes the development of new microfluidic in vitro assays where directed migration of single endothelial cells and three dimensional vascular structures can be monitored in real time. Combining these assays with live imaging microscopy we have studied the behavior of endothelial cells in gradients of proangiogenic factors as well as directed sprouting in embryonic kidneys and stem cell cultures. With the 2D assay we have quantified endothelial cell chemotaxis towards FGF2, VEGFA165 and VEGFA121 and we also demonstrate that constant levels of VEGFA165, but not of FGF2, are able to reduce chemokinesis of endothelial cells. In the 3D migration chamber we have studied directed endothelial cell sprouting in mouse embryonic kidneys and embryoid bodies in response to VEGFA gradients. In both models directed angiogenesis is detected towards increasing levels of growth factor. Using the microarray technique on differentiating embryonic stem cells we have been able to identify the gene exoc3l2 as potentially involved in angiogenesis and endothelial cell migration and we present evidence that ExoC3l2 is associated with the exocyst complex; an important regulator of cell polarity. We have also shown that siRNA mediated gene silencing of exoc3l2 results in impaired VEGFR2 phosphorylation as well as loss of directionality in response to a VEGFA gradient. / (Faculty of Medicine)

No description available.

Metastasis is one of the critical hallmarks of malignancy tumor and the principal cause of death in patients with cancer. Cell migration is the basic and essential step in cancer metastasis process. To systematically investigate functional genes regulating cell migration and cancer metastasis on large scale, we developed a novel on-chip method, SAMcell (self-assembled cell microarray). This method was demonstrated to be particularly suitable for loss-of-function high-throughput screening because of its unique advantages. The first application of SAMcell was to screen human genome miRNAs, considering that more and more miRNAs had been proved to govern cancer metastasis. We found that over 20 % of miRNAs have migratory regulation activity in diverse cell types, indicating a general involvement of miRNAs in migratory regulation. Through triple-round screenings, we discovered miR-23b, which is down-regulated in human colon cancer samples, potently mediates the multiple steps of metastasis, including cell motility, cell growth and cell survival. In parallel, the second application of SAMcell was to screen human genome kinase genes, considering that more and more kinase genes had become successful diagnostic marker or drug targets. We found over 11% migratory kinase genes, suggesting the important role of kinase group in metastasis regulation. Through both functional screening and bioinformatics analysis, we discovered and validated 6 prospective metastasis-related kinase genes, which can be new potential targets in cancer therapy. These findings allow the understanding of regulation mechanism in human cancer progression, especially metastasis and provide the new insight into the biological and therapeutical importance of miRNAs or kinases in cancer.

Les protéines DOCK180 et ELMO coopèrent ensemble biochimiquement et génétiquement afin d’activer la GTPase Rac1 lors de plusieurs évènements biologiques. Toutefois, le rôle que jouent ces protéines dans la signalisation par Rac est encore mal compris. Nous émettons l’hypothèse que Dock180 agit comme activateur de Rac, alors que ELMO est requis pour l’intégration de la signalisation de Rac plutôt que son activation per se. Nous postulons que ELMO agit comme signal de localisation intracellulaire afin de restreindre de façon spatio-temporelle la signalisation de Rac en aval de Dock180, et/ou que ELMO agit comme protéine d’échafaudage entre Rac et ses effecteurs pour amplifier la migration cellulaire. Dans l’objectif nº 1, nous démontrons que le domaine PH atypique de ELMO1 est le site d’interaction principal entre cette protéine et DOCK180. De plus, nous démontrons que la liaison entre ELMO et DOCK180 n’est pas nécessaire pour l’activation de Rac, mais est plutôt essentielle pour faciliter la réorganisation du cytosquelette induite par l’activation de Rac en aval de Dock180. Ces résultats impliquent que ELMO pourrait jouer des rôles additionnels dans la signalisation par Rac. Dans l’objectif nº 2, nous avons découvert l’existence d’une homologie structurelle entre ELMO et un module d’autorégulation de la formine Dia1, et avons identifié trois nouveaux domaines dans la protéine ELMO : les domaines RBD, EID et EAD. De façon analogue à Dia1, nous avons découvert que ELMO à l’état basal est autoinhibé grâce à des intéractions intramoléculaires. Nous proposons que l’état d’activation des protéines ELMO est régulé de façon similaire aux formines de la famille Dia, c’est-à-dire grâce à des interactions avec d’autres protéines. Dans l’objectif nº 3, nous identifions un domaine RBD polyvalent chez ELMO. Ce domaine possède une double spécificité pour les GTPases de la famille Rho et Arf. Nous avons découvert que Arl4A agit comme signal de recrutement membranaire pour le module ELMO/DOCK180/Rac. Nos résultats nous permettent de supposer que d’autres GTPases pourraient être impliquées dans l’activation et la localisation de cette voie de signalisation. Nous concluons qu’à l’état basal, ELMO et DOCK180 forment un complexe dans lequel ELMO est dans sa conformation autoinhibée. Bien que le mécanisme d’activation de ELMO ne soit pas encore bien compris, nous avons découvert que, lorsqu’il y a stimulation cellulaire, certaines GTPases liées au GTP peuvent intéragir avec le domaine RBD de ELMO pour relâcher les contacts intramoléculaires et/ou localiser le complexe à la membrane. Ainsi, les GTPases peuvent servir d’ancrage au complexe ELMO/DOCK180 pour assurer une regulation spatiotemporelle adequate de l’activation et de la signalisation de Rac. / DOCK180 and ELMO cooperate biochemically and genetically to activate Rac in several biological events. However, the role of these proteins in Rac signaling is still poorly understood. We hypothesize that DOCK180 functions as a RacGEF, with ELMO binding to DOCK180 being required for integration of proper Rac signaling rather than Rac activation per se. We postulate that ELMO acts as a subcellular targeting signal for spatio-temporal restriction of DOCK180-mediated Rac signaling and/or as a scaffold for Rac effectors to enforce cell migration. In Aim #1, we elucidate that the atypical ELMO1 PH is the major DOCK180 binding site. We demonstrate that the binding of ELMO1 to DOCK180 is not necessary for Rac GTP-loading, but is instead required to facilitate Rac-GTP induced cytoskeletal changes following DOCK180 activation. These results imply additional roles for ELMO in mediating Rac signaling. In Aim #2, we reveal structural homology between ELMO and an autoregulatory module in the formin, Dia1, and identify three novel domains in ELMOs: the RBD, EID and EAD. Analogous to Dia1, we uncovered that ELMO is autoinhibited via intramolecular interactions at basal state. We propose that the activation state of ELMO proteins is regulated, much like in Dia-family formins, via interaction with other proteins. Aim #3 identifies a polyvalent RBD in ELMO with dual specificity for Rho and Arf family GTPases. We found Arl4A as a novel membrane recruitment signal for the ELMO/DOCK180/Rac module. Our results may have broad implications in the activation and localization of this pathway by additional GTPases. We conclude that, at basal levels, ELMO/DOCK180 is complexed, with ELMO in an autoinhibited state in the cytosol. Through cell stimulation, certain GTPases will be activated that now bind the ELMO RBD and alleviate the intramolecular contacts. In this way, the GTPase anchors the activated ELMO/DOCK180 module in place for proper spatio-temporal regulation of Rac activation and signaling.