Highlights d Microvilli exhibit persistent, active motility driven by actin assembly d Microvillar F-actin cores treadmill during motility d Barbed-end binding factors regulate microvillar motility d Motility promotes intermicrovillar collisions, adhesion, and cluster formation

The docking protein p130Cas is a major Src substrate involved in integrin signaling and mechanotransduction. Tyrosine phosphorylation of p130Cas in focal adhesions (FAs) has been linked to enhanced cell migration, invasion, proliferation, and survival. However, the mechanism of p130Cas targeting to FAs is uncertain, and dynamic aspects of its localization have not been explored. Using live cell microscopy, we show that fluorophore-tagged p130Cas is a component of FAs throughout the FA assembly and disassembly stages, although it resides transiently in FAs with a high mobile fraction. Deletion of either the N-terminal Src homology 3 (SH3) domain or the Cas-family C-terminal homology (CCH) domain significantly impaired p130Cas FA localization, and deletion of both domains resulted in full exclusion. Focal adhesion kinase was implicated in the FA targeting function of the p130Cas SH3 domain. Consistent with their roles in FA targeting, both the SH3 and CCH domains were found necessary for p130Cas to fully undergo tyrosine phosphorylation and promote cell migration. By revealing the capacity of p130Cas to function in FAs throughout their lifetime, clarifying FA targeting mechanism, and demonstrating the functional importance of the highly conserved CCH domain, our results advance the understanding of an important aspect of integrin signaling.p130Cas (Crk-associated substrate) is a Src substrate that functions in integrin signaling to promote cell motility, invasion, proliferation, and survival (1, 2). p130Cas was first recognized as a prominent tyrosine-phosphorylated protein in cells transformed by v-crk (3) and v-src (4). The observation that p130Cas interacts directly with the Src homology 2 (SH2) 2 domain of the v-Crk protein (5, 6) contributed to the recognition of SH2 domains as phosphotyrosine-binding modules in signal transduction. The primary structure of p130Cas (7) indicated a function as a docking/scaffolding protein, lacking domains indicative of intrinsic enzymatic activity but having various domains and motifs for mediating interactions with other proteins.p130Cas was independently identified in a screen for proteins that interact with focal adhesion kinase (FAK) (8), a tyrosine kinase named for its prominent localization to sites of integrin-mediated cell adhesion. A Src homology 3 (SH3) domain at the N terminus of p130Cas mediates the FAK interaction. Like FAK, p130Cas localizes to focal adhesions (FAs) and undergoes tyrosine phosphorylation in response to adhesion (9 -11). Thus, p130Cas is a signaling component of the FA protein complex ("adhesome") that assembles to bring about cellular responses to integrin engagement. A primary role for p130Cas in integrin signaling is consistent with the phenotype of p130Cas-deficient mice, which die during embryonic development due to defects associated with a disorganized actin cytoskeleton (12). Despite the direct interaction with FAK, tyrosine phosphorylation of p130Cas is attributed to Src-family kinases (13-15). However, FAK can act as a scaffold to recru...

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas −/− mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.

Highlights d EPS8 and IRTKS puncta mark sites of new microvillus growth d EPS8 and IRTKS puncta remain enriched at the distal tips of nearly all microvilli d Existing microvilli also serve as mothers that give rise to daughter protrusions d Microvilli collapse when membrane wrapping and EPS8/ IRTKS tip enrichment are lost