FAK promotes recruitment of talin to nascent adhesions to control cell motility

Cell migration is a dynamic process that involves the continuous formation, maturation, and turnover of matrix-cell adhesion sites. New (nascent) adhesions form at the protruding cell edge in a tension-independent manner and are comprised of integrin receptors, signaling, and cytoskeletal-associated proteins. Integrins recruit focal adhesion kinase (FAK) and the cytoskeletal protein talin to nascent adhesions. Canonical models support a role for talin in mediating FAK localization and activation at adhesions. Here, alternatively, we show that FAK promotes talin recruitment to nascent adhesions occurring independently of talin binding to β1 integrins. The direct binding site for talin on FAK was identified, and a point mutation in FAK (E1015A) prevented talin association and talin localization to nascent adhesions but did not alter integrin-mediated FAK recruitment and activation at adhesions. Moreover, FAK E1015A inhibited cell motility and proteolytic talin cleavage needed for efficient adhesion dynamics. These results support an alternative linkage for FAK-talin interactions within nascent adhesions essential for the control of cell migration.     

Calpain-mediated proteolysis of paxillin negatively regulates focal adhesion dynamics and cell migration

The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.     

Coronin 1C negatively regulates cell-matrix adhesion and motility of intestinal epithelial cells

Coronins, WD-repeat actin-binding proteins, are known to regulate cell motility by coordinating actin filament turnover in lamellipodia of migrating cell. Here we report a novel mechanism of Coronin 1C-mediated cell motility that involves regulation of cell-matrix adhesion. RNAi silencing of Coronin 1C in intestinal epithelial cells enhanced cell migration and modulated lamellipodia dynamics by increasing the persistence of lamellipodial protrusion. Coronin 1C-depleted cells showed increased cell-matrix adhesions and enhanced cell spreading compared to control cells, while over-expression of Coronin 1C antagonized cell adhesion and spreading. Enhanced cell-matrix adhesion of coronin-deficient cells correlated with hyperphosphorylation of focal adhesion kinase (FAK) and paxillin, and an increase in number of focal adhesions and their redistribution at the cell periphery. siRNA depletion of FAK in coronin-deficient cells rescued the effects of Coronin 1C depletion on motility, cell-matrix adhesion, and spreading. Thus, our findings provide the first evidence that Coronin 1C negatively regulates epithelial cell migration via FAK-mediated inhibition of cell-matrix adhesion.     

Tetraspanin CD9 in cell migration

Tetraspanin CD9 is associated with integrin adhesion receptors and it was reported that CD9 regulates integrin-dependent cell migration and invasion. Pro- and anti-migratory effects of CD9 have been linked to adhesion-dependent signalling pathways, including phosphorylation of FAK (focal adhesion kinase) and activation of phosphoinositide 3-kinase, p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). In the present paper, we describe a novel mechanism whereby CD9 specifically controls localization of talin1, one of the critical regulators of integrin activation, to focal adhesions: CD9-deficiency leads to impaired localization of talin1 to focal adhesions and correlates with increased motility of breast cancer cells.     

PIPKIγ regulates focal adhesion dynamics and colon cancer cell invasion

Focal adhesion assembly and disassembly are essential for cell migration and cancer invasion, but the detailed molecular mechanisms regulating these processes remain to be elucidated. Phosphatidylinositol phosphate kinase type Iγ (PIPKIγ) binds talin and is required for focal adhesion formation in EGF-stimulated cells, but its role in regulating focal adhesion dynamics and cancer invasion is poorly understood. We show here that overexpression of PIPKIγ promoted focal adhesion formation, whereas cells expressing either PIPKIγ^K188,200R or PIPKIγ^D316K, two kinase-dead mutants, had much fewer focal adhesions than those expressing WT PIPKIγ in CHO-K1 cells and HCT116 colon cancer cells. Furthermore, overexpression of PIPKIγ, but not PIPKIγ^K188,200R, resulted in an increase in both focal adhesion assembly and disassembly rates. Depletion of PIPKIγ by using shRNA strongly inhibited formation of focal adhesions in HCT116 cells. Overexpression of PIPKIγ^K188,200R or depletion of PIPKIγ reduced the strength of HCT116 cell adhesion to fibronection and inhibited the invasive capacities of HCT116 cells. PIPKIγ depletion reduced PIP₂ levels to ∼40% of control and PIP₃ to undetectable levels, and inhibited vinculin localizing to focal adhesions. Taken together, PIPKIγ positively regulates focal adhesion dynamics and cancer invasion, most probably through PIP₂-mediated vinculin activation.     

CaMK-II promotes focal adhesion turnover and cell motility by inducing tyrosine dephosphorylation of FAK and paxillin

Transient elevations in Ca2+ have previously been shown to promote focal adhesion disassembly and cell motility through an unknown mechanism. In this study, evidence is provided to show that CaMK-II, a Ca2+/calmodulin dependent protein kinase, influences fibroblast adhesion and motility. TIRF microscopy reveals a dynamic population of CaMK-II at the cell surface in migrating cells. Inhibition of CaMK-II with two mechanistically distinct, membrane permeant inhibitors (KN-93 and myr-AIP) freezes lamellipodial dynamics, accelerates spreading on fibronectin, enlarges paxillin-containing focal adhesions and blocks cell motility. In contrast, constitutively active CaMK-II is not found at the cell surface, reduces cell attachment, eliminates paxillin from focal adhesions and decreases the phospho-tyrosine levels of both FAK and paxillin; all of these events can be reversed with myr-AIP. Thus, both CaMK-II inhibition and constitutive activation block cell motility through over-stabilization or destabilization of focal adhesions, respectively. Coupled with the existence of transient Ca2+ elevations and a dynamic CaMK-II population, these findings provide the first direct evidence that CaMK-II enables cell motility by transiently and locally stimulating tyrosine dephosphorylation of focal adhesion proteins to promote focal adhesion turnover.     

Integrin adhesions: Who’s on first? What's on second?

Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.     

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.     

A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions

Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin-cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or alpha-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd approximately 10(-7) is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.     

Integrin adhesions

Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.     

Focal adhesion kinase‐dependent regulation of adhesive forces involves vinculin recruitment to focal adhesions

Background information. FAK (focal adhesion kinase), an essential non‐receptor tyrosine kinase, plays pivotal roles in migratory responses, adhesive signalling and mechanotransduction. FAK‐dependent regulation of cell migration involves focal adhesion turnover dynamics as well as actin cytoskeleton polymerization and lamellipodia protrusion. Whereas roles for FAK in migratory and mechanosensing responses have been established, the contribution of FAK to the generation of adhesive forces is not well understood. Results. Using FAK‐null cells expressing wild‐type and mutant FAK under an inducible tetracycline promoter, we analysed the role of FAK in the generation of steady‐state adhesive forces using micropatterned substrates and a hydrodynamic adhesion assay. FAK expression reduced steady‐state strength by 30% compared with FAK‐null cells. FAK expression reduced VCL (vinculin) localization to focal adhesions by 35% independently of changes in integrin binding and localization of talin and paxillin. RNAi (RNA interference) knock‐down of VCL abrogated the FAK‐dependent differences in adhesive forces. FAK‐dependent changes in VCL localization and adhesive forces were confirmed in human primary fibroblasts with FAK knocked down by RNAi. The autophosphorylation Tyr‐397 and kinase domain Tyr‐576/Tyr‐577 sites were differentially required for FAK‐mediated adhesive responses. Conclusions. We demonstrate that FAK reduces steady‐state adhesion strength by modulating VCL recruitment to focal adhesions. These findings provide insights into the role of FAK in mechanical interactions between a cell and the extracellular matrix.     

The C terminus of talin links integrins to cell cycle progression

Integrins are cell adhesion receptors that sense the extracellular matrix (ECM) environment. One of their functions is to regulate cell fate decisions, although the question of how integrins initiate intracellular signaling is not fully resolved. In this paper, we examine the role of talin, an adapter protein at cell-matrix attachment sites, in outside-in signaling. We used lentiviral small hairpin ribonucleic acid to deplete talin in mammary epithelial cells. These cells still attached to the ECM in an integrin-dependent manner and spread. They had a normal actin cytoskeleton, but vinculin, paxillin, focal adhesion kinase (FAK), and integrin-linked kinase were not recruited to adhesion sites. Talin-deficient cells showed proliferation defects, and reexpressing a tail portion of the talin rod, but not its head domain, restored integrin-mediated FAK phosphorylation, suppressed p21 expression, and rescued cell cycle. Thus, talin recruits and activates focal adhesion proteins required for proliferation via the C terminus of its rod domain. Our study reveals a new function for talin, which is to link integrin adhesions with cell cycle progression.     

FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion

Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells. We show that depletion of FAK induces the formation of active invadopodia but impairs invasive cell migration. FAK-deficient MTLn3 breast cancer cells display enhanced assembly and dynamics of invadopodia that are rescued by expression of wild-type FAK but not by FAK that cannot be phosphorylated at tyrosine 397. Moreover, our findings demonstrate that FAK depletion switches phosphotyrosine-containing proteins from focal adhesions to invadopodia through the temporal and spatial regulation of c-Src activity. Collectively, our findings provide novel insight into the interplay between FAK and Src to promote invasion.     

Beta2-Adaptin Binds Actopaxin and Regulates Cell Spreading,Migration and Matrix Degradation

Cell adhesion to the extracellular matrix is a key event in cell migration and invasion and endocytic trafficking of adhesion receptors and signaling proteins plays a major role in regulating these processes. Beta2-adaptin is a subunit of the AP-2 complex and is involved in clathrin-mediated endocytosis. Herein, β2-adaptin is shown to bind to the focal adhesion protein actopaxin and localize to focal adhesions during cells spreading in an actopaxin dependent manner. Furthermore, β2-adaptin is enriched in adhesions at the leading edge of migrating cells and depletion of β2-adaptin by RNAi increases cell spreading and inhibits directional cell migration via a loss of cellular polarity. Knockdown of β2-adaptin in both U2OS osteosarcoma cells and MCF10A normal breast epithelial cells promotes the formation of matrix degrading invadopodia, adhesion structures linked to invasive migration in cancer cells. These data therefore suggest that actopaxin-dependent recruitment of the AP-2 complex, via an interaction with β2-adaptin, to focal adhesions mediates cell polarity and migration and that β2-adaptin may control the balance between the formation of normal cell adhesions and invasive adhesion structures.     

The mechanisms and dynamics of (alpha)v(beta)3 integrin clustering in living cells

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated alphavbeta3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin-independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn(2+)-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.     

Evidence that talin alternative splice variants from Ciona intestinalis have different roles in cell adhesion

Background  

Talins are large, modular cytoskeletal proteins found in animals and amoebozoans such as Dictyostelium discoideum. Since the identification of a second talin gene in vertebrates, it has become increasingly clear that vertebrate Talin1 and Talin2 have non-redundant roles as essential links between integrins and the actin cytoskeleton in distinct plasma membrane-associated adhesion complexes. The conserved C-terminal I/LWEQ module is important for talin function. This structural element mediates the interaction of talins with F-actin. The I/LWEQ module also targets mammalian Talin1 to focal adhesion complexes, which are dynamic multicomponent assemblies required for cell adhesion and cell motility. Although Talin1 is essential for focal adhesion function, Talin2 is not targeted to focal adhesions. The nonvertebrate chordate Ciona intestinalis has only one talin gene, but alternative splicing of the talin mRNA produces two proteins with different C-terminal I/LWEQ modules. Thus, C. intestinalis contains two talins, Talin-a and Talin-b, with potentially different activities, despite having only one talin gene.     

FAK and talin: who is taking whom to the integrin engagement party?

In this issue, Lawson et al. provide new insight into the relationship between FAK and talin during assembly of integrin adhesions on fibronectin. They show that FAK is upstream of talin, and that talin is not required for FAK recruitment or for integrin activation at nascent adhesions. However, FAK-talin binding is required for adhesion turnover and cell motility. The findings question the view that talin is always upstream of focal adhesion protein recruitment to clustered integrin sites.     

The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity

Chemical agents which activate specific kinases were employed to disrupt the stress fiber and focal adhesion organization of cells spread on a substratum. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted a rapid loss of stress fibers and focal adhesions from African green monkey kidney (BSC-1) cells. This was paralleled by an increase in the level of talin phosphorylation suggesting that this may play a role in the removal of talin from focal adhesions. Similar morphological changes were produced in the rat embryo fibroblast line (REF 52) by dibutyryl-cAMP, which stimulates protein kinase A. In contrast, however, the phosphorylation of talin was reduced in REF 52 cells when treated with dibutyryl cAMP. In untreated cells we found that the levels of vinculin phosphorylation were very low relative to the levels of talin phosphorylation and did not change following drug treatment in either cell line. Although limited proteolytic cleavage of cytoskeletal proteins represents a potential mechanism for focal adhesion disruption, we observed no proteolysis of talin or vinculin in response to either drug treatment.     

αV-Integrins Are Required for Mechanotransduction in MDCK Epithelial Cells

The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood. Here, we have studied the functions of RGD-binding αV-integrins in a Madin Darby Canine Kidney (MDCK) cell model and found that αV-integrins regulate the maturation of focal adhesions (FAs) and cell spreading. αV-integrin-deficient MDCK cells bound collagen I (Col I) substrate via α2β1-integrins but failed to efficiently recruit FA components such as talin, focal adhesion kinase (FAK), vinculin and integrin-linked kinase (ILK). The apparent inability to mature α2β1-integrin-mediated FAs and link them to cellular actin cytoskeleton led to disrupted mechanotransduction in αV-integrin deficient cells seeded onto Col I substrate.