Endosomal-sorting complexes required for transport (ESCRT) pathway-dependent endosomal traffic regulates the localization of active Src at focal adhesions

Abstract

Active Src localization at focal adhesions (FAs) is essential for cell migration. How this pool is linked mechanistically to the large pool of Src at late endosomes (LEs)/lysosomes (LY) is not well understood. Here, we used inducible Tsg101 gene deletion, TSG101 knockdown, and dominant-negative VPS4 expression to demonstrate that the localization of activated cellular Src and viral Src at FAs requires the endosomal-sorting complexes required for transport (ESCRT) pathway. Tsg101 deletion also led to impaired Src-dependent activation of STAT3 and focal adhesion kinase and reduced cell migration. Impairment of the ESCRT pathway or Rab7 function led to the accumulation of active Src at aberrant LE/LY compartments followed by its loss. Analyses using fluorescence recovery after photo-bleaching show that dynamic mobility of Src in endosomes is ESCRT pathwaydependent. These results reveal a critical role for an ESCRT pathwaydependent LE/LY trafficking step in Src function by promoting localization of active Src to FAs.cell migration | endosomal sorting complexes required for transport | focal adhesion | late endosomal trafficking

The ESCRT machinery mediates polarization of fibroblasts through regulation of myosin light chain

“…1F), because cells unable to orient are presumably incapable of undergoing directed cell migration. These results are consistent with recent evidence implicating TSG101 in the regulation of cell migration in mouse embryonic fibroblasts (Tu et al, 2010).…”

“…1E; supplementary material Fig. S1) (Tu et al, 2010)] are consistent with ESCRTs being positive regulators of cell migration. Increased cell migration in cells derived from vinculin knockout mice has been attributed to an increase in FA turnover (Saunders et al, 2006), as vinculin is thought to stabilize FAs.…”

“…1E;supplementary material Fig. S1) (Tu et al, 2010)], and is accompanied by an accumulation of focal adhesions ( Fig. 2A).…”

“…Phosphorylation of MLCK at Y471 is mediated by Src (Huveneers and Danen, 2009), which was recently found to accumulate at late endosomes and lysosomes upon TSG101 depletion in mouse embryonic fibroblasts (Tu et al, 2010). Integrin engagement induces autophosphorylation of focal adhesion kinase (FAK) at Y397, creating a binding site for Src, which subsequently phosphorylates and fully activates FAK (Mitra et al, 2005).…”

“…However, the mechanisms that orchestrate cell polarization and migration are still not fully understood. The endosomal sorting complex required for transport (ESCRT) has been associated with the regulation of cell migration (Chanut-Delalande et al, 2010;ChanutDelalande et al, 2007;Lobert et al, 2010;Sevrioukov et al, 2005;Tu et al, 2010). This machinery recognizes ubiquitylated receptors and attenuates their signaling by sorting them into multivesicular endosomes, thereby committing them to lysosomal degradation.…”