Early molecular events in the assembly of the focal adhesion‐stress fiber complex during fibroblast spreading

Abstract

Cell adhesion to the extracellular matrix triggers the formation of integrin-mediated contact and reorganization of the actin cytoskeleton. Examination of nascent adhesions, formed during early stages of fibroblast spreading, reveals a variety of forms of actin-associated matrix adhesions. These include: (1). small ( approximately 1 microm), dot-like, integrin-, vinculin-, paxillin-, and phosphotyrosine-rich structures, with an F-actin core, broadly distributed over the ventral surfaces of the cells; (2). integrin-, vinculin-, and paxillin-containing "doublets" interconnected by short actin bundles; (3). arrays of actin-vinculin complexes. Such structures were formed by freshly plated cells, as well as by cells recovering from latrunculin treatment. Time-lapse video microscopy of such cells, expressing GFP-actin, indicated that long actin cables are formed by an end-to-end lining-up and apparent fusion of short actin bundles. All these structures were prominent during cell spreading, and persisted for up to 30-60 min after plating. Upon longer incubation, they were gradually replaced by stress fibers, associated with focal adhesions at the cell periphery. Direct examination of paxillin and actin reorganization in live cells revealed alignment of paxillin doublets, forming long and highly dynamic actin bundles, undergoing translocation, shortening, splitting, and convergence. The mechanisms underlying the assembly and reorganization of actin-associated focal adhesions and the involvement of mechanical forces in regulating their dynamic properties are discussed.

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…by inhibitors of actin polymerization or actomyosin contractility leads to the dissolution of focal adhesions [29,30]. Conversely, increases in cellular contractility have been shown to induce the simultaneous growth of both focal adhesions and the attached actin stress fibres [30].…”

“…Conversely, increases in cellular contractility have been shown to induce the simultaneous growth of both focal adhesions and the attached actin stress fibres [30]. Tension within the cell however, has been shown to be mediated by the clustering of the heterodimeric, transmembrane family of proteins known as integrins [23,31].…”

“…Although paxillin binding is not absolutely necessary for FAK subcellular localization, it is recognized as being the major mechanism by which FAK is not only targeted to sites of focal adhesions, but presented to kinases like Src for full activation [30,51,67]. However, without paxillin binding, FAK cannot be phosphorylated at Y576/Y577 and consequently Y925, which negatively regulates cell migration [66,67].…”

“…Specifically, serine phosphorylation of the LIM domain of paxillin has been found to temporally target and/or regulate the integration of paxillin into focal adhesions while tyrosine phosphorylated paxillin has been shown to recruit the focal adhesion proteins FAK and Src into the adhesion complex [99,110,111]. Actively disassembling adhesions, such as early adhesions and the distal part of late adhesions, are preferential zones of microtubule targeting and catastrophe that have been shown to be enriched in phosphorylated paxillin [22,30,110,140].…”

“…Conversely, larger, more mature adhesions such as fibrillar adhesions, exhibit relatively reduced levels of paxillin phosphorylation [22,30].…”