Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of many studies, the effects that environmental changes have on this form of movement are poorly understood. In certain pathologies where the cornea exists in a chronic hypoxic state, wound healing is delayed. The goal of this work is to examine the changes in corneal structure in hypoxic corneas that may affect migration and to determine the effects that changes in basement membrane stiffness and focal adhesion turnover have on epithelial cell migration. We analyzed migration after injury in organ cultures and determined that hypoxic corneas exhibited alterations in leading edge morphology. Under hypoxia, fibronectin localization to the apical epithelium and anterior stroma was reduced. Investigators have suggested that alterations in basal lamina composition may increase the stiffness of the membrane. To examine the effect that increased stiffness has on collective migration we performed traction force microscopy. Using multi-layered corneal epithelial sheets, we developed a novel method to analyze the generation of cellular traction forces and the directionality of sheet movement on polyacrylamide gels. We determined that the leading edges of corneal epithelial sheets undergo contraction prior to migration. Alterations in stiffness affected the amount of force exerted by cells at the leading edge. On stiffer surfaces, individual cells within sheets exhibited greater movement compared to cells on softer substrates. To further assess sheet dynamics, we examined the activation of focal adhesion proteins in hypoxic corneas and in human corneal limbal epithelial (HCLE) cells seeded onto soft and rigid substrates. Wounded hypoxic corneas displayed alterations in the localization of the focal adhesion proteins paxillin and vinculin. In HCLE cells cultured on stiff substrates, there was an elevation in vinculin pY1065 phosphorylation after injury, a reduction in vinculin-positive focal adhesions, and decreased actin bundle thickness. Our results demonstrate that changes in membrane stiffness may affect cellular tractions and vinculin dynamics, possibly contributing to the delayed healing response associated with certain corneal pathologies.

Myxococcus Xanthus possède un cycle developpemental multicellulaire entièrement sous la dépendance de la capacité des cellules à se déplacer sur des surfaces solides. M. xanthus possède deux systèmes de motilité génétiquement séparé, une motilité Sociale dépendant des pili de Type IV et une motilité Aventurière dont le mécanisme est encore peu compris. Notre hypothèse de travail est que la motilité Aventurière est qu’en des points régulièrement répartis le long du corps cellulaire soient couplés adhésion et traction de ce corps par une interaction entre des moteurs moléculaire et le cytosquelette d’Actine bactérienne MreB. Mon projet est de caractériser la relation qu’il pourrait y avoir entre le cytosquelette et les points d’adhésion durant la motilité. Pour étudier l’implication du cytosquelette MreB durant le mouvement, nous avons utilisé une approche pharmaceutique utilisant l’A22, une drogue permettant la dépolymérisation rapide et spécifique du cytosquelette sans affecter la viabilité des cellules à court terme. De plus j’ai aussi étudier les interactions possible entre MreB et différentes protéines de motilité comme la petite GTPase MglA, qui est connue pour est essentielle au recrutement des machineries de motilité. / Myxococcus xanthus has a multicellular developmental cycle which is dependent on the capacity of the cells to move accross solid surfaces. M. xanthus uses two motility systems: Social motility system is dependent on Type-IV pili, and the Adventurous motility system, the mechanism of which is poorly understood. Our working hypothesis is that Adventurous motility is performed by adhesion points localized along the cell body where a molecular machinery pulls the cell body by interacting with the MreB cytoskeleton. My project aims to characterize the relationship between the adhesion points and the cytoskeleton during movement. To study the involvement of MreB during motility we use A22, a drug known to rapidly and specifically depolymerise in live microscopy assays. Furthermore, I have study also the interactions between MreB and differents proteins like MglA a small GTPase, which we belive is essential for the recruitment of the machineries.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis in cells. It has been implicated as a therapeutic target for various metabolic diseases like type II diabetes and obesity. However, its role in cancer is context-dependent and therefore warrants further studies to explore its possible use as a therapeutic target. AMPK can either promote or retard the growth of cancer cells depending on other cues and stresses in the milieu of the cancer cells. This study aims to understand AMPK signalling in response to extracellular cues of matrix deprivation and matrix stiffness that are important determinants of metastasis. 1) Calcium-Oxidant Signalling Network Regulates AMPK Activation upon Matrix Deprivation. Recent work from our lab, as well as others, has identified a novel role for the cellular energy sensor AMP-activated protein kinase in epithelial cancer cell survival under matrix deprivation. However, the molecular mechanisms that activate AMPK upon matrix-detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, while re-attachment to the matrix leads to its dephosphorylating and inactivation. Since matrix-detachment leads to loss of integrin signalling, we investigate whether integrin signalling negatively regulates AMPK activation. However, modulation of FAK or Src, the major downstream components of integrin signalling, fails to cause a corresponding change in AMPK signalling. Further investigations reveal that the upstream AMPK kinases, LKB1 and CaMKKβ, contribute to AMPK activation upon detachment. Additionally, we show LKB1 phosphorylation and cytosolic translocation upon matrix deprivation, which might also contribute to AMPK activation. In LKB1-deficient cells, we find AMPK activation to be predominantly dependent on Caskβ. We observe no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment is not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signalling and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that ER calcium release induced store-operated calcium entry (SOCE) contributes to intracellular calcium increase, leading to ROS production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. We find a significant increase in LKB1 as well as pACC levels in breast tumour tissues in comparison to normal tissues. Further, we observe a significant correlation between LKB1 and pACC levels in breast tumour tissues suggesting that LKB1-AMPK signaling pathway is active in vivo in breast cancers. Thus, the Ca2+/ROS triggered LKB1/CaMKK-AMPK signalling cascade may provide a quick, adaptable switch to promote survival of metastasising cancer cells. 2) Extracellular Matrix Stiffness Regulates Stemless through AMPK. Cancer cells experience changes in extracellular matrix stiffness during cancer progression. However, the signalling pathways utilised in sensing matrix stiffness are poorly understood. In this study, we identify AMPK pathway as a possible mechanosensory pathway in response to matrix stiffness. AMPK activity, as measured by downstream target phosphorylation, is found to be higher in soft matrix conditions. We additionally show that compared to stiff matrices, soft matrices increase stemless properties, as evidenced by the increased expression of stemless markers, which is dependent on AMPK activity. Thus, we elucidate a novel mechanotransduction pathway triggered by matrix stiffness that contributes to stemness of cancer cells by regulating AMPK activity. Taken together, our study identifies a novel calcium-oxidant signaling network in the rapid modulation of AMPK signaling in the context of matrix detachment. This pathway is especially relevant in the context of metastasising cancer cells that may not face energy stress in the blood stream but are matrix-deprived. Inhibition of AMPK might compromise the viability of these circulating cells thereby reducing the metastatic spread of cancer. Our study further suggests that varying stiffnesses experienced by cancer cells can modulate AMPK activity and this, in turn, regulates stem-like properties. Thus our study provides novel insights into various extracellular cues that regulate this kinase and contribute to cell survival and metastasis. This knowledge can be utilised in the stage-specific use of AMPK inhibitors in the treatment of breast cancer patients.