KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction

Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma‐associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV‐TK, in contrast to HSV1‐TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto‐phosphorylation of tyrosines 65, 85 and 120 in the N‐terminus of KSHV‐TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3‐Kinase, respectively. The interaction of Crk with KSHV‐TK leads to tyrosine phoshorylation of this cellular adaptor. Auto‐phosphorylation of KSHV‐TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA‐ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV‐TK has no effect on focal adhesion integrity or cell morphology. Our observations demonstrate that by acting as a tyrosine kinase, KSHV‐TK modulates signalling and cell morphology.     

Close and focal contact adhesions of fibroblasts to a fibronectin-containing matrix

The fibroblast cell line Balb/c 3T3 makes both close and tight-focal adhesive contacts with the plasma fibronectin (pFn)-coated tissue culture substratum. Detachment of these cells mediated by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid leaves tight-focal contacts and a subset of close contacts as substratum-attached material (SAM). The enrichment of heparan sulfate (HS) proteoglycan (HS-PG) in SAM under specific attachment conditions, as well as the recent demonstration of HS binding to pFn or cellular Fn, has evolved a series of experiments with the selective HS-binding protein platelet factor-4 to analyze the requirement of the HS-binding activity of pFn in the formation of these two types of adhesive contacts. In addition, the cell-binding domain (CBD) of pFn, which recognizes an unidentified cell surface receptor, has been isolated free of HS-binding domains after proteolytic cleavage of pFn. These functional studies indicate that the binding of pFn on the substratum to cell surface HS-PG is necessary and sufficient to generate close contacts with transmembrane signaling of this proteoglycan to reorganize lengthy microfilament bundles in the cytoplasm. Cells spread incompletely on CBD alone, form only close contacts, and reorganize highly concentrated actin filaments only in their spiky projections. Furthermore, the formation of tight-focal contacts with associated stress fibers appears to require both reactions of pFn, binding to cell surface HS-PG and to its unidentified receptor. The importance of HS-PG in these functional studies has led to its better biochemical characterization in SAM. Initially formed close contacts of these cells contain principally a large HS-PG that aggregates into high-molecular-weight complexes by some unknown mechanism. With time, there are two different mechanisms of catabolism of HS-PG, one of which includes the liberation of single-chain HS. The importance of these changes in the HS-PG in SAM are now being analyzed with regard to the formation and disappearance of close and tight-focal adhesions.     

Migration and proliferation of diploid human fibroblasts following "wounding" of confluent monolayers

Six diploid human fibroblast strains were grown in confluent monolayers. Holes were scraped in these monolayers and the number of cells proliferating into these “wounds” with time were determined. The migration and mitotic aspects of the proliferation of fibroblasts into these wounds were analyzed separately. Small amounts of undialysed or dialysed serum were essential for cell division but not migration. Saline extracts of skin could not substitute for serum in the medium. Neither zinc nor cupric ion at tolerable concentrations (10^?5M) increased the rate of cell proliferation. Normal human fibroblasts did not immediately start to divide from confluency into the “wound” space. Their generation time was about 32–39 hours. Fibroblasts from patients with cystic fibrosis began to divide almost immediately into the “wounded” area. Their generation time was about 48 to 56 hours.     

Tyrosine phosphatase SHP-2 regulates IL-1 signaling in fibroblasts through focal adhesions

Interleukin-1beta (IL-1beta) mediates destruction of matrix collagens in diverse inflammatory diseases including arthritis, periodontitis, and pulmonary fibrosis by activating fibroblasts, cells that interact with matrix proteins through integrin-based adhesions. In vitro, IL-1beta signaling is modulated by focal adhesions, supramolecular protein complexes that are enriched with tyrosine kinases and phosphatases. We assessed the importance of tyrosine phosphatases in regulating cell-matrix interactions and IL-1beta signaling. In human gingival fibroblasts plated on fibronectin, IL-1beta enhanced the maturation of focal adhesions as defined by morphology and enrichment with paxillin and alpha-actinin. IL-1beta also induced activation of ERK and recruitment of phospho-ERK to focal complexes/adhesions. Treatment with the potent tyrosine phosphatase inhibitor pervanadate, in the absence of IL-1beta, recapitulated many of these responses indicating the importance of tyrosine phosphatases. Immunoblotting of collagen bead-associated complexes revealed that the tyrosine phosphatase, SHP-2, was also enriched in focal complexes/adhesions. Depletion of SHP-2 by siRNA or by homologous recombination markedly altered IL-1beta-induced ERK activation and maturation of focal adhesions. IL-1beta-induced tyrosine phosphorylation of SHP-2 on residue Y542 promoted focal adhesion maturation. Association of Gab1 with SHP-2 in focal adhesions correlated temporally with activation of ERK and was abrogated in cells expressing mutant (Y542F) SHP-2. We conclude that IL-1beta mediated maturation of focal adhesions is dependent on tyrosine phosphorylation of SHP-2 at Y542, leading to recruitment of Gab1, a process that may influence the downstream activation of ERK.     

FAK dimerization controls its kinase‐dependent functions at focal adhesions

Focal adhesion kinase (FAK) controls adhesion‐dependent cell motility, survival, and proliferation. FAK has kinase‐dependent and kinase‐independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x‐ray crystallography, small angle x‐ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase‐dependent functions—autophosphorylation of tyrosine‐397—requires site‐specific dimerization of FAK. The dimers form via the association of the N‐terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C‐terminal FAT domain. FAT binds to a basic motif on FERM that regulates co‐activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site‐specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.     

Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin‐containing focal adhesions

Actin–myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30–40% higher than serum‐free cultures. Inhibition of myosin light chain kinase or Rho‐kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum‐induced enhancements in strengthening. Blebbistatin‐induced inhibition of myosin II reduced serum‐induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin‐null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin‐dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum‐induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK‐dependent, vinculin‐containing focal adhesion assembly. J. Cell. Physiol. 223:746–756, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 cells and human umbilical vein endothelial cells loaded with GST-Cterm decreased focal adhesion phosphotyrosine content. Neither cell type, however, exhibited a reduction in focal adhesions after GST-Cterm loading. These results indicate that FAK mediates adhesion-associated tyrosine phosphorylation, but not the formation of focal adhesions. We then examined the effect of inhibiting FAK function on other adhesion-dependent cell behavior. Cells microinjected with GST-Cterm exhibited decreased migration. In addition, cells injected with GST-Cterm had decreased DNA synthesis compared with control-injected or noninjected cells. These findings suggest that FAK functions in the regulation of cell migration and cell proliferation.     

Dynamics of focal adhesions and reorganization of F‐actin in VEGF‐stimulated NSCs under varying differentiation states

Precise migration of neural stem/progenitor cells (NSCs) is crucially important for neurogenesis and repair in the nervous system. However, the detailed mechanisms are not clear. Our previous results showed that NSCs in varying differentiation states possess different migratory ability to vascular endothelial growth factor (VEGF). In this study, we demonstrate the different dynamics of focal adhesions (FAs) and reorganization of F‐actin in NSCs during spreading and migration stimulated by VEGF. We found that the migrating NSCs of 0.5 and 1 day differentiation possess more FAs at leading edge than cells of other states. Moreover, the phosphorylation of focal adhesion kinase (FAK) and paxillin in NSCs correlates closely with their differentiation states. VEGF promotes FA formation with broad lamellipodium generation at the leading edge in chemotaxing cells of 0, 0.5, and 1 day differentiation, but not in cells of 3 days differentiation. Furthermore, cells of 1 day differentiation show a maximal asymmetry of FAs between lamella and cell rear, orchestrating cell polarization and directional migration. Time‐lapse video analysis shows that the disassembly of FAs and the cell tail detachment in NSCs of 1 day differentiation are more rapid, along with the concurrent enlarged size of FAs at the leading edge, leading to the most effective chemotactic response to VEGF. Collectively, these results indicate that the dynamics of FAs and reorganization of F‐actin in NSCs that undergo directional migration correlate closely with their differentiation states, contributing to the different chemotactic responses of these cells to VEGF. J. Cell. Biochem. 114: 1744–1759, 2013. © 2013 Wiley Periodicals, Inc.     

Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts

Fibroblast migration involves complex mechanical interactions with the underlying substrate. Although tight substrate contact at focal adhesions has been studied for decades, the role of focal adhesions in force transduction remains unclear. To address this question, we have mapped traction stress generated by fibroblasts expressing green fluorescent protein (GFP)-zyxin. Surprisingly, the overall distribution of focal adhesions only partially resembles the distribution of traction stress. In addition, detailed analysis reveals that the faint, small adhesions near the leading edge transmit strong propulsive tractions, whereas large, bright, mature focal adhesions exert weaker forces. This inverse relationship is unique to the leading edge of motile cells, and is not observed in the trailing edge or in stationary cells. Furthermore, time-lapse analysis indicates that traction forces decrease soon after the appearance of focal adhesions, whereas the size and zyxin concentration increase. As focal adhesions mature, changes in structure, protein content, or phosphorylation may cause the focal adhesion to change its function from the transmission of strong propulsive forces, to a passive anchorage device for maintaining a spread cell morphology.     

Structure and function of focal adhesions

Integrin-dependent cell adhesions come in different shapes and serve in different cell types for tasks ranging from cell-adhesion, migration, and the remodeling of the extracellular matrix to the formation and stabilization of immunological and chemical synapses. A major challenge consists in the identification of adhesion-specific as well as common regulatory mechanisms, motivating the need for a deeper analysis of protein-protein interactions in the context of intact focal adhesions. Specifically, it is critical to understand how small differences in binding of integrins to extracellular ligands and/or cytoplasmic adapter proteins affect the assembly and function of an entire focal adhesion. By using the talin-integrin pair as a starting point, I would like to discuss how specific protein-protein and protein-lipid interactions can control the behavior and function of focal adhesions. By responding to chemical and mechanical cues several allosterically regulated proteins create a dynamic multifunctional protein network that provides both adhesion to the extracellular matrix as well as intracellular signaling in response to mechanical changes in the cellular environment.     

Environmentally‐mediated consumer control of algal proliferation in Florida springs

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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.     

Investigating complexity of protein-protein interactions in focal adhesions

The formation of focal adhesions governs cell shape and function; however, there are few measurements of the binding kinetics of focal adhesion proteins in living cells. Here, we used the fluorescence recovery after photobleaching (FRAP) technique, combined with mathematical modeling and scaling analysis to quantify dissociation kinetics of focal adhesion proteins in capillary endothelial cells. Novel experimental protocols based on mathematical analysis were developed to discern the rate-limiting step during FRAP. Values for the dissociation rate constant k_OFF ranged over an order of magnitude from 0.009 ± 0.001/s for talin to 0.102 ± 0.010/s for FAK, indicating that talin is bound more strongly than other proteins in focal adhesions. Comparisons with in vitro measurements reveal that multiple focal adhesion proteins form a network of bonds, rather than binding in a pair-wise manner in these anchoring structures in living cells.     

Force-induced adsorption and anisotropic growth of focal adhesions

Focal adhesions are micrometer-sized protein aggregates that connect actin stress fibers to the extracellular matrix, a network of macromolecules surrounding tissue cells. The actin fibers are under tension due to actin-myosin contractility. Recent measurements have shown that as the actin force is increased, these adhesions grow in size and in the direction of the force. This is in contrast to the growth of condensed domains of surface-adsorbed molecules in which the dynamics are isotropic. We predict these force-sensitive, anisotropic dynamics of focal adhesions from a model for the adsorption of proteins from the cytoplasm to the adhesion site. Our theory couples the mechanical forces and elasticity to the adsorption dynamics via force-induced conformational changes of molecular-sized mechanosensors located in the focal adhesion. We predict the velocity of both the front and back of the adhesion as a function of the applied force. In addition, our results show that the relative motion of the front and back of the adhesion is asymmetric and in different ranges of forces, the adhesion can either shrink or grow in the direction of the force.     

Dynamics and segregation of cell-matrix adhesions in cultured fibroblasts

Here we use time-lapse microscopy to analyse cell-matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP-paxillin to analyse focal contacts and GFP-tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometers per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain alpha5beta1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometers per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell-matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.     

Identification of β integrin‐like‐ and fibronectin‐like proteins in the bivalve mollusk Mytilus trossulus

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin‐like protein and its presumptive ligand, fibronectin‐like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin‐like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin‐like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin‐like protein was detected firstly at the blastula stage and later, the FN‐LP‐immunoreactive cells were scattered in the trochophore larvae. The fibronectin‐like protein was not expressed in the β integrin‐positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin‐ and fibronectin‐like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.     

Histone mediated changes in morphology and confluent density of cultured cells

Calf thymus histones and histone subfractions were added to media overlying subconfluent mouse fibroblast cells in culture. The histones caused significantly higher cell densities at confluence than control cultures and disruption of the normal ordered arrangement of cells. These changes were seen on application of histones to growing cells but not confluent cells and were reversed when the histones were removed and the cells replated with more growth area. The slightly lysine rich histone fraction had the greatest effect and the lysine rich fraction had the least effect on cell morphology and cell number at confluence. These effects could not be duplicated with other highly charged basic proteins.     

Movement of stress fibers away from focal adhesions identifies focal adhesions as sites of stress fiber assembly in stationary cells

Force generated in contractile actin filament bundles (stress fibers-SFs) is transmitted to the extracellular matrix (ECM) via linker proteins and transmembrane integrins at focal adhesions (FAs). Though it has long been known that actin is rapidly exchanged in FAs, the connection between SFs and FAs has not been studied in detail. We introduced fiduciary marks on SFs by expressing GFP-palladin or GFP-alpha-actinin-1, which are both FA and dense body proteins, and by pattern bleaching of GFP-actin. Following fiduciary marks on SFs over time by time-lapse fluorescence microscopy, we detected assembly of SFs at FAs in stationary cells resulting in movement of SFs away from FAs with a velocity of 0.2-0.4 microm/min. Visualization of FAs in GFP-palladin/DsRed-paxillin double transfected cells showed that SF elongation was not accompanied by a change in FA length. SF elongation at FAs depended on actin polymerization and force as demonstrated by inhibitors of actin polymerization (cytochalasin D, jasplakinolide) and inhibitors of myosin-dependent contraction (blebbistatin, Y-27632), respectively. Our finding of SF assembly at FAs has important implications for SF formation, force transmission, and tension distribution within the actin cytoskeletal network of stationary cells.