Bidirectional control of the inner dynamics of focal adhesions promotes cell migration

Focal adhesions (FA) are bidirectional mechanical biosensors that allow cells to integrate intracellular and extracellular cues. Their function is tightly regulated by changes in molecular composition and also by variation in the spatio-temporal dynamics of FA components within this structure. A closely regulated turnover of FA proteins within FA sites allows cells to respond appropriately to their environment, thereby impacting on cell shape and function. FA protein dynamics are linked to FA maturation and rates of assembly and disassembly, and have a significant influence on tumor cell migration. Using the FRAP technique to investigate the hidden internal dynamics of FA, we identified two new regulators of FA dynamics and cell migration: the Mgat5/galectin-3 lattice and tyrosine phosphorylated caveolin-1 (pY14Cav1). In this short review we first introduce FA and their complex dynamic behavior. We then present the Mgat5/galectin-3 lattice and caveolin-1 and discuss their concerted role in FA dynamics, which defines previously unknown, interdependent roles in tumor cell migration. We conclude with a discussion of interesting unexplored avenues that might lead to a better understanding of the complex mechanism of FA dynamics.Key words: focal adhesion, migration, caveolin-1, tyrosine 14, galectin-3, Mgat5, turnover, dynamics     

The inner lives of focal adhesions

In focal adhesions of eukaryotic cells, transmembrane receptors of the integrin family and a large set of adaptor proteins form the physical link between the extracellular substrate and the actin cytoskeleton. During cell migration, nascent focal adhesions within filopodia and lamellipodia make the initial exploratory contacts with the cellular environment, whereas maturing focal adhesions pull the cell forward against the resistance of 'sliding' focal adhesions at the cell rear. Experimental approaches are now available for analysing the dynamics and interior structure of these different focal adhesions. Analysing focal-adhesion dynamics using green-fluorescent-protein-linked integrin leads us to propose that the acto-myosin-controlled density and turnover of integrins in focal adhesions is used to sense the elasticity and spacing of extracellular ligands, regulating cell migration by mechanically transduced signaling.     

Targeting focal adhesions:Helicobacter pylori-host communication in cell migration

Highly dynamic integrin-based focal adhesions provide an important structural basis for anchoring the cellular actin cytoskeleton to the surrounding extracellular matrix. The human pathogen Helicobacter pylori (H. pylori) directly targets integrins with drastic consequences on the epithelial cell morphology and migration, which might contribute to the disruption of the gastric epithelium in vivo. In this review, we summarize the recent findings concerning the complex mechanism through which H. pylori interferes with host integrin signaling thereby deregulating focal adhesions and the actin cytoskeleton of motile epithelial cells.     

A computational model of cell migration coupling the growth of focal adhesions with oscillatory cell protrusions

Cell migration is a highly integrated process where actin turnover, actomyosin contractility, and adhesion dynamics are all closely linked. In this paper, we propose a computational model investigating the coupling of these fundamental processes within the context of spontaneous (i.e. unstimulated) cell migration. In the unstimulated cell, membrane oscillations originating from the interaction between passive hydrostatic pressure and contractility are sufficient to lead to the formation of adhesion spots. Cell contractility then leads to the maturation of these adhesion spots into focal adhesions. Due to active actin polymerization, which reinforces protrusion at the leading edge, the traction force required for cell translocation can be generated. Computational simulations first show that the model hypotheses allow one to reproduce the main features of fibroblast cell migration and established results on the biphasic aspect of the cell speed as a function of adhesion strength. The model also demonstrates that certain temporal parameters, such as the adhesion proteins recycling time and adhesion lifetimes, influence cell motion patterns, particularly cell speed and persistence of the direction of migration. This study provides some elements, which allow a better understanding of spontaneous cell migration and enables a first glance at how an individual cell would potentially react once exposed to a stimulus.     

Endoglin controls cell migration and composition of focal adhesions: function of the cytosolic domain

Mutations in the human endoglin gene result in hereditary hemorrhagic telangiectasia type 1, a vascular disorder characterized by multisystemic vascular dysplasia, arteriovenous malformations, and focal dilatation of postcapillary venules. Previous studies have implicated endoglin in the inhibition of cell migration in vivo and in vitro. In the course of studies to address the relationship of the conserved cytosolic domain to endoglin function, we identified zyxin, a LIM domain protein that is concentrated at focal adhesions, as an interactor with endoglin in human umbilical vein vascular endothelial cells. This interaction is localized within the 47-amino acid carboxyl-terminal cytosolic domain of endoglin, and maps within zyxin residues 326-572. The endoglin-zyxin interaction was found to be largely mediated by the third LIM domain of zyxin, and is specific for endoglin because the homologous cytosolic domain of the transforming growth factor-beta type III receptor, betaglycan, fails to interact with zyxin. Expression of endoglin is associated with reduction of zyxin, as well as its interacting proteins p130(cas) and CrkII, from a focal adhesion protein fraction, and this reduction is correlated with inhibition of cell migration. We also show that endoglin-dependent: (i) inhibition of cell migration, (ii) reduction of focal adhesion-associated p130(cas)/CrkII protein levels, (iii) tyrosine phosphorylation of p130(cas), and (iv) focal adhesion-associated endoglin levels are mediated by the cytosolic domain of endoglin. These results suggest a novel mechanism of endoglin function involving its interaction with LIM domain-containing proteins, and associated adapter proteins, affecting sites of focal adhesion.     

Glycogen synthase kinase 3 and h-prune regulate cell migration by modulating focal adhesions

h-prune, which has been suggested to be involved in cell migration, was identified as a glycogen synthase kinase 3 (GSK-3)-binding protein. Treatment of cultured cells with GSK-3 inhibitors or small interfering RNA (siRNA) for GSK-3 and h-prune inhibited their motility. The kinase activity of GSK-3 was required for the interaction of GSK-3 with h-prune. h-prune was localized to focal adhesions, and the siRNA for GSK-3 or h-prune delayed the disassembly of paxillin. The tyrosine phosphorylation of focal adhesion kinase (FAK) and the activation of Rac were suppressed in GSK-3 or h-prune knocked-down cells. GSK-3 inhibitors suppressed the disassembly of paxillin and the activation of FAK and Rac. Furthermore, h-prune was highly expressed in colorectal and pancreatic cancers, and the positivity of the h-prune expression was correlated with tumor invasion. These results suggest that GSK-3 and h-prune cooperatively regulate the disassembly of focal adhesions to promote cell migration and that h-prune is useful as a marker for tumor aggressiveness.     

RhoB regulates cell migration through altered focal adhesion dynamics

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.     

Integrin Cytoplasmic domain-Associated Protein-1 (ICAP-1) promotes migration of myoblasts and affects focal adhesions

Integrin Cytoplasmic domain-Associated Protein-1 (ICAP-1) binds specifically to the beta1 integrin subunit cytoplasmic domain. We observed that RNAi-induced knockdown of ICAP-1 reduced migration of C2C12 myoblasts on the beta1 integrin ligand laminin and that overexpression of ICAP-1 increased this migration. In contrast, migration on the beta3 integrin ligand vitronectin was not affected. ICAP-1 knockdown also greatly diminished migration of microvascular endothelial cells on collagen. The number of central focal adhesions in C2C12 cells on laminin was reduced by ICAP-1 knockdown and increased by ICAP-1 overexpression. Previously, we demonstrated that ICAP-1 binds to the ROCK-I kinase and translocates ROCK-I to the plasma membrane. We show here that the ROCK kinase inhibitor Y27362 reduces migration on laminin and causes a loss of central focal adhesions, similarly as ICAP-1 knockdown. ICAP-1 and ROCK were co-immune-precipitated from C2C12 cells, and in cells that overexpressed ICAP-1, YFP-ROCK was translocated to membrane ruffles. These results indicate that ICAP-1 regulates beta1 integrin-dependent cell migration by affecting the pattern of focal adhesion formation. This is likely due to ICAP-1-induced translocation of ROCK to beta1 integrin attachment sites.     

Actin,microtubules and focal adhesion dynamics during cell migration

Cell migration is a complex cellular behavior that results from the coordinated changes in the actin cytoskeleton and the controlled formation and dispersal of cell-substrate adhesion sites. While the actin cytoskeleton provides the driving force at the cell front, the microtubule network assumes a regulatory function in coordinating rear retraction. The polarity within migrating cells is further highlighted by the stationary behavior of focal adhesions in the front and their sliding in trailing ends. We discuss here the cross-talk of the actin cytoskeleton with the microtubule network and the potential mechanisms that control the differential behavior of focal adhesions sites during cell migration.     

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.     

Spatial regulation of MCAK promotes cell polarization and focal adhesion turnover to drive robust cell migration

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK (mitotic centromere-associated kinesin), in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared with the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream of or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning, and focal adhesion dynamics, which all help facilitate robust directional migration.     

Angiogenin enhances cell migration by regulating stress fiber assembly and focal adhesion dynamics

Angiogenin (ANG) acts on both vascular endothelial cells and cancer cells, but the underlying mechanism remains elusive. In this study, we carried out a co-immunoprecipitation assay in HeLa cells and identified 14 potential ANG-interacting proteins. Among these proteins, β-actin, α-actinin 4, and non-muscle myosin heavy chain 9 are stress fiber components and involved in cytoskeleton organization and movement, which prompted us to investigate the mechanism of action of ANG in cell migration. Upon confirmation of the interactions between ANG and the three proteins, further studies revealed that ANG co-localized with β-actin and α-actinin 4 at the leading edge of migrating cells. Down-regulation of ANG resulted in fewer but thicker stress fibers with less dynamics, which was associated with the enlargements of focal adhesions. The focal adhesion kinase activity and cell migration capacity were significantly decreased in ANG-deficient cells. Taken together, our data demonstrated that the existence of ANG in the cytoplasm optimizes stress fiber assembly and focal adhesion formation to accommodate cell migration. The finding that ANG promoted cancer cell migration might provide new clues for tumor metastasis research.     

Lipid rafts control human melanoma cell migration by regulating focal adhesion disassembly

Tumor cell migration is a crucial step in the metastatic cascade, and interruption of this step is considered to be logically effective in preventing tumor metastasis. Lipid rafts, distinct liquid ordered plasma membrane microdomains, have been shown to influence cancer cell migration, but the underlying mechanisms are still not well understood. Here, we report that lipid rafts regulate the dynamics of actin cytoskeleton and focal adhesion in human melanoma cell migration. Disrupting the integrity of lipid rafts with methyl-β cyclodextrin enhances actin stress fiber formation and inhibits focal adhesion disassembly, accompanied with alterations in cell morphology. Furthermore, actin cytoskeleton, rather than microtubules, mediates the lipid raft-dependent focal adhesion disassembly by regulating the dephosphorylation of focal adhesion proteins and the internalization of β3 integrin. We also show that Src–RhoA–Rho kinase signaling pathway is responsible for lipid raft disruption-induced stress fiber formation. Taken together, these observations provide a new mechanism to further explain how lipid rafts regulate the migration of melanoma cell and suggest that lipid rafts may be novel and attractive targets for cancer therapy.     

MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation

Over the past several years, it has become clear that the Rho family of GTPases plays an important role in various aspects of neuronal development including cytoskeleton dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated Rac1 and Cdc42 can rescue the formation of these structures. We have also established quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes.     

Translocation of CrkL to focal adhesions mediates integrin-induced migration downstream of Src family kinases

The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130(Cas) (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas. Forced localization of CRKL to FAs (FA-CRKL) by itself was sufficient to induce activation of Rac1 and Cdc42 and rescued haptotaxis defects of mouse embryonic fibroblasts (MEFs) lacking Src, Yes, and Fyn, three broadly expressed Src family members required for integrin-induced migration. Consistent with Rac1 activation, FA-CRKL induced cotranslocation of a Rac1 activator, Dock1, to focal adhesions. These results therefore indicate a role for CrkL in mediating Src signaling by activating small G proteins at focal adhesions. Furthermore, MEFs lacking CrkL show impaired integrin-induced migration despite expression of a closely related protein, Crk-II, in these cells. These results therefore provide formal evidence that CrkL plays a specific role in integrin-induced migration as a downstream mediator of Src.     

Modeling the size distribution of focal adhesions

We calculate the size distribution of two-dimensional aggregates, for different simple dynamical growth models. The resulting size distributions of these domains, at steady state, are shown to depend strongly on the mode of domain growth. We then compare to the measured size-distribution of focal-adhesion domains. Using our calculation and the measured exponential distribution of focal-adhesion domain lengths can be used to test the validity of recent models proposed to describe the dynamics of these complexes in adhering cells.     

Mitogenesis, cell migration, and loss of focal adhesions induced by tenascin-C interacting with its cell surface receptor, annexin II.

In a previous study we demonstrated that the alternatively spliced region of tenascin-C, TNfnA-D, bound with high affinity to a cell surface receptor, annexin II. In the present study we demonstrate three changes in cellular activity that are produced by adding intact tenascin-C or TNfnA-D to cells, and we show that all three activities are blocked by antibodies against annexin II. 1) TNfnA-D added to confluent endothelial cells induced loss of focal adhesions. 2) TNfnA-D produced a mitogenic response of confluent, growth-arrested endothelial cells in 1% serum. TNfnA-D stimulated mitogenesis only when it was added to cells before or during exposure to other mitogens, such as basic fibroblast growth factor or serum. Thus the effect of TNfnA-D seems to be to facilitate the subsequent response to growth factors. 3) TNfnA-D enhanced cell migration in a cell culture wound assay. Antibodies to annexin II blocked all three cellular responses to TNfnA-D. These data show that annexin II receptors on endothelial cells mediate several cell regulatory functions attributed to tenascin-C, potentially through modulation of intracellular signalling pathways.     

cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edge dynamics

Epithelial cell migration is a complex process crucial for embryonic development, wound healing and tumor metastasis. It depends on alterations in cell–cell adhesion and integrin–extracellular matrix interactions and on actomyosin-driven, polarized leading edge protrusion. The small GTPase Rap is a known regulator of integrins and cadherins that has also been implicated in the regulation of actin and myosin, but a direct role in cell migration has not been investigated. Here, we report that activation of endogenous Rap by cAMP results in an inhibition of HGF- and TGFβ-induced epithelial cell migration in several model systems, irrespective of the presence of E-cadherin adhesion. We show that Rap activation slows the dynamics of focal adhesions and inhibits polarized membrane protrusion. Importantly, forced integrin activation by antibodies does not mimic these effects of Rap on cell motility, even though it does mimic Rap effects in short-term cell adhesion assays. From these results, we conclude that Rap inhibits epithelial cell migration, by modulating focal adhesion dynamics and leading edge activity. This extends beyond the effect of integrin affinity modulation and argues for an additional function of Rap in controlling the migration machinery of epithelial cells.