Developmental processes involving symmetry-breaking of homogeneous cell populations into leaders and followers are found in many important contexts. Cells constrained by culture on two-dimensional scaffolds, as well as in three-dimensional shapes, appear to respond to convex curves with an increasing propensity to protrude, while concave curves in contrast appear to inhibit protrusion. This has interesting implications in terms of a potential positive feedback loop. This feedback may act in symmetry-breaking, through amplification of initial stochastic differences in cell shape, and also in collective migration, through reinforcing and directing the coherent movement of collectives. In this study, epithelial cells were cultured on two-dimensional micropatterns with variable curvatures to examine the effect of edge geometry and other variables on the likelihood of protrusions forming. This platform allowed the quantification of F-actin-based protrusions at the periphery of multicellular epithelial clusters, in segments defined by cluster edge curvature. The initial observations confirmed reports in the literature of preferential localisation of protrusions at more convex regions, and relative inhibition at more concave regions. A previously-published work has postulated a role for secreted modulators of motility, with the shape of a group of cells determining the concentration of diffusing morphogen each individual cell is exposed to. To test this hypothesis, a low-shear flow culture chamber was used to disrupt the putative gradients. Despite theoretical and empirical support for the sufficiency of the flow condition to disrupt autocrine signalling, micropatterned cells cultured under flow showed no significant differences from the control condition. These findings form the basis of a manuscript which has been accepted for publication by the Journal of Anatomy. The results of an Atomic Force Microscopy (AFM) study carried out by collaborators were suggestive of a role for cellular mechanotransduction in sensing and responding to micropattern curvature. Differential calcium channel mechanoactivation was hypothesised as being one potential mechanism underlying the response to curvature, given the known involvement of mechanosensitive ion channels in cellular responses to force and substrate stiffness, and the multiple roles of calcium in cellular motility. Artificially increasing cytosolic calcium levels with Ionomycin reduced protrusion rates at convex curves. However, treatment with BAPTA-AM to sequester intracellular calcium had no effect on protrusion rates. ROCK inhibitor, in contrast, increased protrusion rates at concave curves, and Blebbistatin increased protrusion rates globally. These results together are suggestive of differential control of myosin depending on local curvature: cyclic and driven by calcium-activation of MLCK in the convex regions (with lamellipodia undergoing protrusion-retraction cycles), versus sustained and controlled by ROCK in the concave regions (where lamellipodia are inhibited). The unexpected finding that protrusions at convex regions were resistant to the actin cytoskeleton-disrupting drug Cytochalasin D may point to a role for a tropomyosin isoform in defining the differing mechanical characteristics of the actin cytoskeleton in response to local curvature. In addition, the previously-noted lack of effect of BAPTA-AM treatment (which has been shown to interfere with dynamic microtubules) is suggestive of a role for stabilised microtubules in protrusions at convex regions. These indications of unique characteristics to the protrusions promoted by convex curvature give added support to the curvature-protrusion feedback model, and its relevance to tissue morphogenesis. In summary, this work provides evidence against a previously-published suggested mechanism for the curvature-protrusion feedback loop that is proposed to act during epithelial morphogenesis, and evidence in support of a role for a calcium-based mechanism in driving the initiation and maintenance of leader cells in migrating epithelial sheets. Further work is called for in characterising the protrusions promoted by convex curvature, and the mechanisms controlling them. This area is of significance in gaining greater understanding of tissue morphogenesis in pathogenesis and development, and of potential value in tissue engineering applications.

Pulmonary endothelial cell (EC) barrier dysfunction and recovery is critical to the pathophysiology of acute respiratory distress syndrome. Cytoskeletal and subsequent cell membrane dynamics play a key mechanistic role in determination of EC barrier integrity. Here, we characterizAQe the actin related protein 2/3 (Arp 2/3) complex, a regulator of peripheral branched actin polymerization, in human pulmonary EC barrier function through studies of transendothelial electrical resistance (TER), intercellular gap formation, peripheral cytoskeletal structures and lamellipodia. Compared to control, Arp 2/3 inhibition with the small molecule inhibitor CK-666 results in a reduction of baseline barrier function (1,241 +/- 53 vs 988 +/- 64 ohm; p < 0.01), S1P-induced barrier enhancement and delayed recovery of barrier function after thrombin (143 +/- 14 vs 93 +/- 6 min; p < 0.01). Functional changes of Arp 2/3 inhibition on barrier integrity are associated temporally with increased intercellular gap area at baseline (0.456 +/- 0.02 vs 0.299 +/- 0.02; p < 0.05) and thirty minutes after thrombin (0.885 +/- 0.03 vs 0.754 +/- 0.03; p < 0.05). Immunofluorescent microscopy reveals reduced lamellipodia formation after S1P and during thrombin recovery in Arp 2/3 inhibited cells. Individual lamellipodia demonstrate reduced depth following Arp 2/3 inhibition vs vehicle at baseline (1.83 +/- 0.41 vs 2.55 +/- 0.46 mm; p < 0.05) and thirty minutes after S1P treatment (1.53 +/- 0.37 vs 2.09 +/- 0.36 mm; p < 0.05). These results establish a critical role for Arp 2/3 activity in determination of pulmonary endothelial barrier function and recovery through formation of EC lamellipodia and closure of intercellular gaps.

Les cellules peuvent migrer sous différentes conditions qui dépendent de l’environnement biochimique ou mécanique. Connaître les mécanismes de la migration, les protéines impliquées et leur régulation est essentiel pour comprendre les processus de morphogénèse ou certaines situations pathologiques. Dans ce contexte, la migration collective des cellules est un processus clé qui intervient pendant le développement ainsi que dans la vie adulte. Elle joue un rôle très important pour la formation et l’entretien des couches épithéliales, notamment au cours du développement embryonnaire et pendant la cicatrisation des trous épithéliaux résultant, par exemple, d’une blessure. Lorsque l’épithélium présente une discontinuité, des mécanismes actifs qui impliquent une migration coordonnée des cellules sont nécessaires pour préserver l’intégrité des tissus. Dans ce travail, nous avons étudié les mécanismes impliqués dans la fermeture des trous dans un épithélium. Pour des blessures de faible taille, le mode de fermeture dit de purse string est souvent évoqué, impliquant la contraction d’un anneau contractile d’acto-myosine qui ferme la blessure. Pour des blessures de tailles plus importantes, il est courant d’observer un mécanisme différent conduisant { la migration active des cellules du bord qui couvrent la surface “libre”.Pour étudier ces aspects de manière quantitative et reproductible, nous avons développé une nouvelle méthode basée sur des techniques de microfabrication et de lithographie dite « molle » qui permet de faire une étude quantitative de la fermeture des trous épithéliaux. Nous avons fabriqué des substrats de micropiliers de diamètre et de forme variés dans les quels les cellules sont libres de pousser entre les microstructures. Lorsqu’elles sont parvenues à confluence, on retire le substrat qui laisse apparaître des trous contrôlés.De cette manière, nous avons observé que les cellules épithéliales forment des lamellipodes pour la fermeture de ces trous. Le mécanisme de fermeture dépend de la taille des trous et nous avons pu observer différents régimes en fonction de diamètre des piliers. Les trous petits (de la taille d’une seule cellule) sont fermés par un mécanisme passif alors que la fermeture de trous plus larges nécessite un mécanisme actif de migration conduisant à la formation de lamellipodes et à des modes de migration collective. Par la suite, nous nous sommes intéressés à l’aspect mécanique de la fermeture des trous épithéliaux. Pour cela, nous avons utilisé un système d’ablation laser pour rompre quelques cellules dans une monocouche épithéliale. Nous avons alors mesuré les forces de traction que les cellules exercent au substrat et leur évolution temporelle et spatiale. Nous avons pu mettre en évidence différents modes de traction: au début, les cellules exercent des forces de traction importantes sur leur substrat pour laisser place à des contraintes mécaniques qui sont davantage issues d’un processus collectif au travers de la formation d’un câble multicellulaire qui les relie les cellules de bord entre elles. En conclusion, ce travail nous a permis d’obtenir des informations sur les mécanismes dynamiques de fermeture des tissus épithéliaux qui sont évidemment impliqués dans la cicatrisation des blessures mais aussi dans certains problèmes de malformations congénitales lors l’embryogenèse. / Most cells migrate under the appropriate conditions or stimuli; understanding the mechanisms of migration, the players involved, and their regulation, is pivotal to tackle the pathological situations where migration becomes an undesired effect. While largely overshadowed by the study of single cell migration, collective cell migration is a very relevant process that takes place during development as well as in adult life. Collective migration is very relevant for the formation and maintenance of epithelial layers: extensive migratory processes occur during the shape of the embryo, as well as during the healing of a skin incision in the adult. When openings or discontinuities appear in the epithelia, it is crucial that the appropriate mechanisms are activated.In the present work we attempt at deciphering what are the mechanisms involved in gap closure. Until now, most of the literature concerning the subject has reported contradictory results, mainly arising from the complexity of the process and the lack of systematic analysis. We have designed a novel approach to address epithelial gap closure under well-defined and controlled conditions. By using our gap patterning method, we have observed that epithelial cells extend lamellipodia when exposed to a newly available space. Interestingly, we found that the closure of such gap depends on the size: small gaps are closed by a passive physical mechanism, while large gaps are closed through a Rac-dependent cell crawling mechanism, in a collective migration-like manner. 11Abstract (English)Most cells migrate under the appropriate conditions or stimuli; understanding the mechanisms of migration, the players involved, and their regulation, is pivotal to tackle the pathological situations where migration becomes an undesired effect. While largely overshadowed by the study of single cell migration, collective cell migration is a very relevant process that takes place during development as well as in adult life. Collective migration is very relevant for the formation and maintenance of epithelial layers: extensive migratory processes occur during the shape of the embryo, as well as during the healing of a skin incision in the adult. When openings or discontinuities appear in the epithelia, it is crucial that the appropriate mechanisms are activated.In the present work we attempt at deciphering what are the mechanisms involved in gap closure. Until now, most of the literature concerning the subject has reported contradictory results, mainly arising from the complexity of the process and the lack of systematic analysis. We have designed a novel approach to address epithelial gap closure under well-defined and controlled conditions. By using our gap patterning method, we have observed that epithelial cells extend lamellipodia when exposed to a newly available space. Interestingly, we found that the closure of such gap depends on the size: small gaps are closed by a passive physical mechanism, while large gaps are closed through a Rac-dependent cell crawling mechanism, in a collective migration-like manner. Next, we also addressed the mechanical component of epithelial gap closure. In this study, we took advantage of a laser-ablation system to disrupt some cells within an epithelial monolayer, and study how the remaining cells sealed that gap. By measuring the traction forces that cells exert on the substrate along the closure, we observed that cells first pulled on the substrate to propel themselves. By the last steps of closure, there is a transition in the direction of the force, so that cells are pulled to the center of the gap due to the assembly of a supracellular actin cable. Altogether, this work provides valuable knowledge on the current understanding of the mechanisms accounting for epithelial gap closure. We believe that a better comprehension of these mechanisms can help to shed light in clinically relevant situations where epithelial gap closure is impaired.

Les cellules peuvent migrer sous différentes conditions qui dépendent de l'environnement biochimique ou mécanique. Connaître les mécanismes de la migration, les protéines impliquées et leur régulation est essentiel pour comprendre les processus de morphogénèse ou certaines situations pathologiques. Dans ce contexte, la migration collective des cellules est un processus clé qui intervient pendant le développement ainsi que dans la vie adulte. Elle joue un rôle très important pour la formation et l'entretien des couches épithéliales, notamment au cours du développement embryonnaire et pendant la cicatrisation des trous épithéliaux résultant, par exemple, d'une blessure. Lorsque l'épithélium présente une discontinuité, des mécanismes actifs qui impliquent une migration coordonnée des cellules sont nécessaires pour préserver l'intégrité des tissus. Dans ce travail, nous avons étudié les mécanismes impliqués dans la fermeture des trous dans un épithélium. Pour des blessures de faible taille, le mode de fermeture dit de purse string est souvent évoqué, impliquant la contraction d'un anneau contractile d'acto-myosine qui ferme la blessure. Pour des blessures de tailles plus importantes, il est courant d'observer un mécanisme différent conduisant { la migration active des cellules du bord qui couvrent la surface "libre".Pour étudier ces aspects de manière quantitative et reproductible, nous avons développé une nouvelle méthode basée sur des techniques de microfabrication et de lithographie dite " molle " qui permet de faire une étude quantitative de la fermeture des trous épithéliaux. Nous avons fabriqué des substrats de micropiliers de diamètre et de forme variés dans les quels les cellules sont libres de pousser entre les microstructures. Lorsqu'elles sont parvenues à confluence, on retire le substrat qui laisse apparaître des trous contrôlés.De cette manière, nous avons observé que les cellules épithéliales forment des lamellipodes pour la fermeture de ces trous. Le mécanisme de fermeture dépend de la taille des trous et nous avons pu observer différents régimes en fonction de diamètre des piliers. Les trous petits (de la taille d'une seule cellule) sont fermés par un mécanisme passif alors que la fermeture de trous plus larges nécessite un mécanisme actif de migration conduisant à la formation de lamellipodes et à des modes de migration collective. Par la suite, nous nous sommes intéressés à l'aspect mécanique de la fermeture des trous épithéliaux. Pour cela, nous avons utilisé un système d'ablation laser pour rompre quelques cellules dans une monocouche épithéliale. Nous avons alors mesuré les forces de traction que les cellules exercent au substrat et leur évolution temporelle et spatiale. Nous avons pu mettre en évidence différents modes de traction: au début, les cellules exercent des forces de traction importantes sur leur substrat pour laisser place à des contraintes mécaniques qui sont davantage issues d'un processus collectif au travers de la formation d'un câble multicellulaire qui les relie les cellules de bord entre elles. En conclusion, ce travail nous a permis d'obtenir des informations sur les mécanismes dynamiques de fermeture des tissus épithéliaux qui sont évidemment impliqués dans la cicatrisation des blessures mais aussi dans certains problèmes de malformations congénitales lors l'embryogenèse.

Vascular smooth muscle cell migration is a significant contributor to many aspects of heart disease, and specifically atherosclerosis. Tissue damage in the arteries can result in the formation of a fatty streak. Smooth muscle cells (SMC) can then migrate to this site to form a fibrous cap, stabilizing the fatty plaque. Since cardiovascular disease is the leading cause of death in developed countries, this function of SMC is an essential area of study. The formation of lamellipodia and circular dorsal ruffles were studied in this project as indicators that cell migration is occurring. The roles of the proteins p53, Rac, caldesmon and PTEN were investigated with regards to these actin-based structures. The tumour suppressor p53 is often reported to cause apoptosis, senescence or cell cycle arrest when stress is placed on a cell, but has recently been shown to regulate cell migration as well. It was determined in this project that p53 could inhibit the formation of both lamellipodia and circular dorsal ruffles. It was also shown that this could occur directly through an inhibition of the GTPase Rac. Previous studies have shown that p53 can upregulate caldesmon, a protein which is known to bind to and stabilize actin filaments while inhibiting Arp2/3-mediated branching. It was confirmed that p53 could upregulate caldesmon, and that caldesmon could inhibit the formation of lamellipodia and circular dorsal ruffles. The phosphorylation of caldesmon by p21-associated kinase (PAK) or extracellular signal-related kinase (Erk) was shown to effectively reverse the ability of caldesmon to inhibit these structures. The role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was also studied with regards to this signalling pathway. PTEN was shown to inhibit lamellipodia and circular dorsal ruffles through its lipid phosphatase activity. It was concluded that p53 can inhibit the formation of lamellipodia and circular dorsal ruffles in vascular SMC, and that this occurs through Rac, caldesmon and PTEN. / Thesis (Master, Biochemistry) -- Queen's University, 2011-06-10 13:15:37.081

Adaptor proteins play a vital role in the propagation of cellular signals. Although they lack endogenous catalytic activity, they contain a variety of protein binding modules, which enable them to promote specific and efficient interactions with their binding partners. They form integrative platforms for a variety of molecules (e.g. lipids, tyrosine kinases, cytoskeletal and signaling proteins), and thereby link and coordinate key functions such as cell growth, motility and shape determination. Our laboratory has recently cloned a novel, 130 kDa adaptor protein, named XB130, as a structural homolog of actin-filament-associated-protein (AFAP-110), a stress fiber-binding Src substrate. However, the molecular interactions and functions of this novel adaptor remained to be elucidated. To characterize the function of XB130 we asked two general questions: (1) Is XB130 involved in the signal transduction pathways of tyrosine kinases? And (2) Is XB130 capable of regulating the cytoskeleton and/or is it regulated by the cytoskeleton? To address these questions first we investigated the tissue distribution of XB130 and discovered that it is abundantly expressed in thyroid. Therefore we asked whether it is a target of the thyroid-specific tyrosine kinase, RET/PTC, a genetically rearranged, constitutively active enzyme that plays a pathogenic role in papillary thyroid cancer. We found that XB130 is a RET/PTC substrate that couples RET/PTC signaling to phosphatidylinositol 3-kinase (PI3K) activation through its phosphorylation dependent interaction with the regulatory subunit p85 of PI3K. XB130 plays an important role in PI3K signaling, as downregulation of XB130 in TPC1 papillary thyroid cancer cells, harboring the RET/PTC1 kinase, strongly reduced Akt activity and concomitantly inhibited cell cycle progression and survival in suspension. In the second part we demonstrate that XB130 is a novel Rac- and cytoskeleton-regulated protein that exhibits high affinity to lamellipodial (branched) F-actin and impacts motility and invasiveness of tumor cells. In conclusion, my work characterized a novel adaptor protein and assigned two well-defined pathophysiological functions to it in the context of thyroid cancer cells.

Adaptor proteins play a vital role in the propagation of cellular signals. Although they lack endogenous catalytic activity, they contain a variety of protein binding modules, which enable them to promote specific and efficient interactions with their binding partners. They form integrative platforms for a variety of molecules (e.g. lipids, tyrosine kinases, cytoskeletal and signaling proteins), and thereby link and coordinate key functions such as cell growth, motility and shape determination. Our laboratory has recently cloned a novel, 130 kDa adaptor protein, named XB130, as a structural homolog of actin-filament-associated-protein (AFAP-110), a stress fiber-binding Src substrate. However, the molecular interactions and functions of this novel adaptor remained to be elucidated. To characterize the function of XB130 we asked two general questions: (1) Is XB130 involved in the signal transduction pathways of tyrosine kinases? And (2) Is XB130 capable of regulating the cytoskeleton and/or is it regulated by the cytoskeleton? To address these questions first we investigated the tissue distribution of XB130 and discovered that it is abundantly expressed in thyroid. Therefore we asked whether it is a target of the thyroid-specific tyrosine kinase, RET/PTC, a genetically rearranged, constitutively active enzyme that plays a pathogenic role in papillary thyroid cancer. We found that XB130 is a RET/PTC substrate that couples RET/PTC signaling to phosphatidylinositol 3-kinase (PI3K) activation through its phosphorylation dependent interaction with the regulatory subunit p85 of PI3K. XB130 plays an important role in PI3K signaling, as downregulation of XB130 in TPC1 papillary thyroid cancer cells, harboring the RET/PTC1 kinase, strongly reduced Akt activity and concomitantly inhibited cell cycle progression and survival in suspension. In the second part we demonstrate that XB130 is a novel Rac- and cytoskeleton-regulated protein that exhibits high affinity to lamellipodial (branched) F-actin and impacts motility and invasiveness of tumor cells. In conclusion, my work characterized a novel adaptor protein and assigned two well-defined pathophysiological functions to it in the context of thyroid cancer cells.

The endothelial cells lining the inner surface of the tissue capillaries and post-capillary venules form a semi-permeable barrier between the blood circulation and interstitial compartments. The semi-permeable barrier in these vessels is the major site of blood-tissue exchange. A compromised endothelial barrier contributes to the pathological process such as edema, acute respiratory distress syndrome (ARDS) and tumor metastasis. Sphingosine-1-phosphate (S1P), an endogenous, bioactive lipid present in all cells, is a potential therapeutic agent that can restore compromised endothelial barrier function. On the other hand, S1P also has pleotropic effects and can either increase or decrease arterial tone and tissue perfusion under different conditions. The detailed mechanisms underlining S1P’s endothelial barrier protective effect are still largely unknown, but are suggested to depend on cell spreading termed “lamellipodia”. Therefore, to fully take advantage of the beneficial properties of S1P, it is important to first understand how S1P-induced lamellipodia protrusions correlate with its effect on endothelial barrier function. It is also important to know the underlining mechanisms that S1P enhances endothelial barrier function, including intracellular signaling and receptor signaling. To study local lamellipodia activities, we acquired time-lapse images of live endothelial cells expressing GFP-actin, and subsequently analyzed different lamellipodia parameters. Experiments were performed under baseline conditions, and during endothelial barrier disruption or enhancement. The compounds used in these experiments included thrombin and S1P. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function for in vitro studies. Changes of local lamellipodia dynamics and endothelial barrier function within the same time frame were studied. For mechanistic studies, we combined biochemical, immunological and pharmacological approaches. Rho family small GTPase activities were measured with an ELISA pull-down assay. Fluorescence Resonance Energy Transfer (FRET) was also used to study the localization of RhoA activation. Pharmacological compounds targeting intracellular signaling messengers were used to test the involvement of Rac1, RhoA, MLC-2 in endothelial local lamellipodia activity and S1P-mediated endothelial barrier enhancement. Receptor agonists and antagonists were used to study the involvement of S1P receptor signaling. Finally, for cell behavior and cytoskeleton studies, we utilized immunofluorescence labeling that enables direct visualization of changes in cytoskeleton, cell-cell junction and focal adhesions. We found that S1P increases both local lamellipodia protrusions and TER. The rapid increase in local lamellipodia protrusion frequencies also corresponded to the rapid increase in TER seen within the same time frame. Under the microscope, local lamellipodia protrusions from adjacent cells overlapped with each other and extended beyond junctional cell-cell contacts. Strikingly, S1P-induced lamellipodia protrusions carry VE-cadherin molecules to the cell-cell contact, established junctional adhesions. Combined with our previous published studies on thrombin induced lamellipodia activity changes, we think lamellipodia protrusions are a major component that regulates endothelial barrier function. Combined, our imaging studies revealed the mechanisms on how lamellipodia regulates endothelial barrier function: 1) lamellipodia overlap and increase the apical to basal diffusion distance, which in turn decreases permeability and upregulates endothelial barrier function. 2) Local lamellipodia protrusions contain VE-cadherin, which is delivered the to the cell-cell contact by the lamellipodia to increase junctional stability. S1P is effective for rescuing thrombin-induced endothelial barrier dysfunction. The known barrier disruptor thrombin, decreased local lamellipodia protrusions, disrupted VE-cadherin integrity, and caused a drop in TER. S1P increased local lamellipodia protrusions after thrombin challenge, and resulted in faster recovery towards baseline TER compared with vehicle controls. Interestingly, we also found that both thrombin and S1P increased MLC-2 phosphorylation at Thr18/Ser19. We subsequently accessed Rho family GTPase activity after thrombin and S1P. As expected, thrombin rapidly increased GTP-bound RhoA levels, and decreased GTP-bound Rac1 levels. Unexpectedly, S1P not only increased GTP-bound Rac1, but also increased GTP-bound RhoA to a more prominently levels (4-fold). Since Rac1 has been implicated in promoting lamellipodia protrusions, we tested the role of Rac1 on the local lamellipodia activities first. We found that Wild-Type (WT) Rac1 group had the highest local lamellipodia protrusion frequencies, protrusion distances, withdraw time and highest percentage of protrusions that lasted more than 5 min. WT Rac1 overexpression had greatest protrusion frequencies and lowest monolayer permeability to FITC-albumin compared to GFP and DN-Rac1 overexpression monolayers. These results suggest that Rac1 is important for baseline endothelial barrier function. This is also confirmed by the finding that pharmacological inhibition of Rac1 significantly decreased baseline TER. Although Rac1 is important for baseline endothelial barrier function, we noticed that it is dispensable in S1P-mediated endothelial barrier enhancement. Rac1 inhibitors, DN-Rac1 overexpression, and Rac1 siRNA knockdown all failed to abolish the S1P-mediated increase in TER. This is partially explained by the findings that S1P-induced Rac1 activation is short-lived and less pronounced in contrast to RhoA activation. We subsequently tested the role of RhoA in S1P-mediated endothelial barrier enhancement, based on our findings that both S1P and thrombin significantly activated RhoA and induced MLC-2 phosphorylation. Significant RhoA activation was found to be mainly at cell periphery and lamellipodia protrusions in HUVEC on FRET, after S1P was given. In addition, RhoA inhibitors significantly decreased the amplitude of S1P-induced MLC-2 phosphorylation, vinculin redistribution and barrier enhancement. The data suggest that the mechanisms involved in S1P-mediated endothelial barrier enhancement depend on RhoA activation and subsequent cytoskeletal rearrangement. We next investigated which receptor is responsible for the endothelial barrier enhancement of S1P. However, antagonism of S1P1, S1P2 or S1P3 alone with W146, JTE-013 or TY-52156 respectively all failed to attenuate S1P-mediated increase in TER. While agonism of S1P1 with CYM-5442 hydrochloride alone produced significant increase in TER, neither S1P2 nor S1P3 activation (CYM 5520 & CYM 5541) produced any change on TER. Interestingly, S1P1 antagonist failed to block the effect of S1P1 agonist on TER. This could be due to that the S1P1 agonist may not be very selective at concentrations tested. We also identified that S1P4 and S1P5 are present on endothelial cells. Further studies would be necessary to elucidate the roles of newly identified S1P4 or S1P5 alone on endothelial barrier function. It is also worth investigating in the future if multiple S1P receptors are involved in its endothelial barrier enhancing effect. In conclusion, we found that lamellipodia protrusions contribute to the endothelial barrier enhancement of S1P. While Rac1 is important for the maintenance of endothelial barrier function, it is dispensable in S1P-mediated endothelial barrier enhancement. On the other hand, RhoA activation appears to be, at least in part, responsible for the endothelial barrier enhancement of S1P. It is currently still unclear if S1P’s endothelial barrier enhancing effect is through one single receptor activation or activation of multiple receptors. Future studies are needed to elucidate the receptor signaling that contributes to S1P-mediated endothelial barrier enhancement.

Cell migration is an essential element in the immune response on the one hand and in cancer metastasis on the other hand. The architecture of the actin network in lamellipodia determines the elasticity of the leading edge and contributes to the regulation of migration. We have implemented a new method for the analysis of actin network morphology in the lamellipodia of B16F1 mouse melanoma cells. This method is based on fitting multilayer geometrical models to electron microscopy images of lamellipodial actin networks. The chosen model and F-actin concentrations are thereby deterministic parameters. Using this approach, we identified distinct structural features of actin networks in lamellipodia. The mesh size which defines the elasticity of the lamellipodium was determined as 34 and 78 nm for a two-layer network

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.