Super‐resolution Imaging Shows Spatial Regulation of p130Cas Phosporylation within Focal Adhesions

Abstract

Cell motility is critical for physiological processes such as tissue development and remodeling as well as pathologies such as cancer metastasis. Cell motility is influenced by its surrounding matrix through biochemical and mechanical stimuli. p130Cas is a signaling molecule that has been proposed to be a mechanosensor, which responds to mechanical stimuli and mediates cell motility during the early stage of lamellipodial protrusions. p130Cas localizes to focal adhesions (FAs) and binds with its effectors via tyrosine phosphorylation of its substrate domain. Previous studies have suggested that mechanical stimuli increase p130Cas tyrosine phosphorylation and these phosphotyrosines recruit SH2‐domain containing effectors like Crk, that initiate a signaling cascade activating the small Rho‐GTPase Rac1 leading to lamellipodial protrusions.Due to spatial heterogeneity of stresses within FAs, we hypothesized that p130Cas phosphorylation is not uniform across a single FA. Spatial differences across such small structures as FAs cannot be resolved using biochemical assays or standard microscopy. We used a super resolution imaging technique, PALM (photo‐activated localization microscopy) to study the differences in p130Cas phosphorylation within individual FAs using SH2 domains of Crk as probes. Our results show punctate localization of SH2 probes at single FAs compared to uniform distribution of p130Cas.Our data suggest that p130Cas activity is spatially regulated. In the future we will look at correlations between mechanical stress within FAs and p130Cas activity. Our results will help elucidate the mechanism of mechanical regulation of p130Cas phosphorylation, and may help understand how cells sense and respond to external mechanical stimuli.