ABSTRACT
Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for direct lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER transmembrane contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4)P and PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, is essential to stabilize and anchor ventral ER-PM contact sites to FA, and mediates microtubule-dependent FA disassembly. To conclude, our results reveal unknown functions for VAPA-mediated membrane contact sites during cell motility and provide a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.
Subject(s)
Focal Adhesions , Golgi Apparatus , Humans , Caco-2 Cells , Actin Cytoskeleton , Cell Movement , Vesicular Transport ProteinsABSTRACT
Reactive oxygen species (ROS) are important regulators of intracellular signaling pathways in health and disease. It is implicated that ROS may play critical roles in pathogenesis of a number of kidney diseases including diabetic nephropathy. However, due to the lack of tools for in vivo detection of redox status, our knowledge of redox dynamics is still fragmentary. In this study, we present novel zebrafish UAS transgenic lines expressing mitochondrial and cytoplasmic targeted redox fluorescent biosensors, Grx1-roGFP2 and mitoGrx1-roGFP2. As the zebrafish is an ideal animal model for intravital imaging, these transgenic zebrafish provide useful tools to analyze renal redox dynamics in vivo.
