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.

<p dir="ltr">The actin cytoskeleton is a dynamic structure that regulates various important cellular processes, such as cell protrusion, migration, transport, and cell shape changes. Cells employ different actin architectures best suited for each of these functions. We have employed an agent-based model to illuminate how the actin cytoskeleton plays such functions in animal and plant cells, via dynamic interactions between molecular players.</p><p dir="ltr">Lamellipodia found in animal cells are two-dimensional actin protrusion formed on the leading edge of cells, playing an important role in sensing surrounding mechanical environments via focal adhesions. Various molecular players, architecture, and dynamics of the lamellipodia have been investigated extensively during recent decades. Nevertheless, it still remains elusive how each component in the lamellipodia mechanically interacts with each other to attain a stable, dynamic steady state characterized by a retrograde flow emerging in the branched actin network. Using the agent-based model, we investigated how the balance between different subcellular processes is achieved for the dynamic steady state. We simulated a branched network found in the lamellipodia, consisting of actin filament (F-actin), myosin motor, Arp2/3 complex, and actin crosslinking protein. We found the importance of a balance between F-actin assembly at the leading edge of cells and F-actin disassembly at the rear end of the lamellipodia. We also found that F-actin severing is crucial to allow for the proper disassembly of an actin bundle formed via network contraction induced by motor activity. In addition, it was found that various dynamic steady states can exist.</p><p dir="ltr">The actin cytoskeleton in plant cells plays a crucial role in intracellular transport and cytoplasmic streaming, and its structure is very different from the actin cytoskeleton in animal cells. The plant actin cytoskeleton is known to show distinct dynamic behaviors with homeostasis. We used the agent-based model to simulate the plant actin cytoskeleton with the consideration of the key governing mechanisms, including F-actin polymerization/depolymerization, different types of F-actin nucleation events, severing, and capping. We succeeded in reproducing experimental observations in terms of F-actin density, length, nucleation frequency, and rates of severing, polymerization, and depolymerization. We found that the removal of nucleators results in lower F-actin density in the network, which supports recent experimental findings.</p>

No description available.

Le complexe actomyosine, formé de l’association de la myosine II avec les filaments d’actine, stabilise le cytosquelette d’actine et génère la contraction cellulaire nécessaire à plusieurs processus comme la motilité et l’apoptose dans les cellules non-musculaires. La myosine II est un hexamère formé d’une paire de chaînes lourdes (MHCs) et de deux paires de chaînes légères MLC20 et MLC17. La régulation de l’activité de la myosine II, c'est-à-dire son interaction avec les filaments d’actine, est directement liée à l’état de phosphorylation des MLC20, mais il reste beaucoup à découvrir sur l’implication des MHCs. Il existe trois isoformes de MHCs de myosine II, MHCIIA, MHCIIB et MHCIIC qui possèdent des fonctions à la fois communes et distinctes. Notre but est de mettre en évidence les différences de fonction entre les isoformes de myosine II, au niveau structurale, dans la stabilisation du cytosquelette d’actine, et au niveau de leur activité contractile, dans la génération des forces de tension. Nous nous sommes intéressés au rôle des isoformes des MHCs dans l’activité du complexe actomyosine qui est sollicité durant le processus de contraction cellulaire de l’apoptose. Dans quatre lignées cellulaires différentes, le traitement conjoint au TNFα et à la cycloheximide causait la contraction et le rétrécissement des cellules suivi de leur détachement du support de culture. Par Western blot, nous avons confirmé que la phosphorylation des MLC20 est augmentée suite au clivage de ROCK1 par la caspase-3, permettant ainsi l’interaction entre la myosine II et les filaments d’actine et par conséquent, la contraction des cellules apoptotiques. Cette contraction est bloquée par l’inhibition des caspases et de ROCK1. MHCIIA est dégradée suite à l’activation de la caspase-3 alors que MHCIIB n’est pas affectée. En utilisant une lignée cellulaire déficiente en MHCIIB, ou MHCIIB (-/-), nous avons observé que la contraction et le détachement cellulaires durant l’induction de l’apoptose se produisaient moins rapidement que dans la lignée de type sauvage (Wt) ce qui suggère que l’isoforme B est impliquée dans la contraction des cellules apoptotiques. Parallèlement, la kinase atypique PKCζ, qui phosphoryle MHCIIB et non MHCIIA, est activée durant l’apoptose. PKCζ joue un rôle important puisque son inhibition bloque la contraction des cellules apoptotiques. Par la suite, nous nous sommes intéressés à la modulation de la morphologie cellulaire par la myosine II. Les fibroblastes MHCIIB (-/-), présentent un large lamellipode dont la formation semble dû uniquement à l’absence de l’isoforme MHCIIB, alors que les fibroblastes Wt ont une morphologie cellulaire étoilée. La formation du lamellipode dans les fibroblastes MHCIIB (-/-) est caractérisée par l’association de la cortactine avec la membrane plasmique. L’observation en microscopie confocale nous indique que MHCIIA interagit avec la cortactine dans les fibroblastes Wt mais très peu dans les fibroblastes MHCIIB (-/-). Le bFGF active la voie des MAP kinases dans les fibroblastes Wt et MHCIIB (-/-) et induit des extensions cellulaires aberrantes dans les fibroblastes MHCIIB (-/-). Nos résultats montrent que l’implication de l’isoforme B de la myosine II dans la modulation de la morphologie cellulaire. L’ensemble de nos résultats participe à distinguer la fonction structurale et contractile de chacune des isoformes de myosine II dans la physiologie cellulaire. / We are interested in studying the modulation of the actomyosin complex which is involved in different cellular processes such as cell locomotion and apoptosis. The actomyosin complex is formed by the association of actin filaments and myosin II. The non-muscle myosin II is a hexamer formed by one pair of heavy chains (MHCs) and two pairs of light chain (MLC20 and MLC17). The actomyosin activity is dependent on MLC20 and MHCs phosphorylation. There are three isoforms of MHCs (MHCIIA, MHCIIB and MHCIIC) which have common but also distinctive roles in several cellular processes. Our aim is to clarify the structural and contractile functions of each isoforme of myosin II in different cellular processes, in particular, cell contraction and cell morphology. First, we studied the implication of myosin II isoforms in cell shrinkage and detachment during apoptosis which are both dependent on actomyosin contractility. We treated four different cell lines with TNFα in combination with cycloheximide (CHX) to trigger apoptosis. We confirmed that TNFα induced caspase-3 activation, ROCK1 cleavage and increased MLC20 phosphorylation. We showed that TNFα/CHX induced the caspase-dependent MHCIIA degradation, whereas MHCIIB levels and association with the actin cytoskeleton remained virtually unchanged. Cell shrinkage and detachment were blocked by caspase and ROCK1 inhibitors. Using the MHCIIB (-/-) cell line, we observed that the absence of MHCIIB did not affect cell death rate. However, MHCIIB (-/-) fibroblasts showed more resistance to TNFα-induced actin disassembly, cell shrinkage and detachment than wild type (Wt) fibroblasts, indicating the participation of MHCIIB in these events. PKCζ, which only phosphorylates MHCIIB, was cleaved during apoptosis, co-immunoprecipitated preferentially with MHCIIB and, interestedly, PKCζ inhibition blocked TNFα-induced shrinkage and detachment. Our results demonstrate that MHCIIB, together with MLC phosphorylation and actin, constitute the actomyosin cytoskeleton that mediates contractility during apoptosis. Second, we studied the involvement of myosin II isoforms in cell shape modulation. Fibroblasts MHCIIB (-/-) spontaneously formed lamellipodia whereas Wt fibroblasts presented a stellate shape. Cortactin was associated with the leading edge of lamellipodia in MHCIIB (-/-) fibroblasts, but it localised diffusely in the cytoplasm or at the end of fine cellular projections in Wt fibroblasts. The levels of cortactin and cortactin phosphorylated in Tyr421 associated with membrane in MHCIIB (-/-) fibroblasts were higher than in Wt fibroblasts. Confocal microscopy showed cortactin/MHCIIA colocalization in wild type but not in MHCIIB (-/-) fibroblasts. bFGF activates Erk1/2 in wild type and MHCIIB (-/-) fibroblasts and induces the formation of aberrant membrane projections in MHCIIB (-/-) fibroblasts. In conclusion, our results contribute to characterize the structural and contractile role of each myosin II isoforms in the physiology of the cell.

No description available.