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.     

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.     

JNK-mediated phosphorylation of paxillin in adhesion assembly and tension-induced cell death by the adenovirus death factor E4orf4

The adenovirus type 2 Early Region 4 ORF4 (E4orf4) protein induces a caspase-independent death program in tumor cells involving changes in actin dynamics that are functionally linked to cell killing. Because an increase in myosin II-based contractility is needed for the death of E4orf4-expressing cells, we have proposed that alteration of cytoskeletal tension is part of the signals engaging the death pathway. Yet the mechanisms involved are poorly defined. Herein, we show that the Jun N-terminal kinase JNK is activated in part through a pathway involving Src, Rho, and ROCK (Rho kinase) and contributes to dysregulate adhesion dynamics and to kill cells in response to E4orf4. JNK supports the formation of atypically robust focal adhesions, which are bound to the assembly of the peculiar actomyosin network typifying E4orf4-induced cell death and which are required for driving nuclear condensation. Remarkably, the dramatic enlargement of focal adhesions, actin remodeling, and cell death all rely on paxillin phosphorylation at Ser-178, which is induced by E4orf4 in a JNK-dependent way. Furthermore, we found that Ser-178-paxillin phosphorylation is necessary to decrease adhesion turnover and to enhance the time residency of paxillin at focal adhesions, promoting its recruitment from an internal pool. Our results indicate that perturbation of tensional homeostasis by E4orf4 involves JNK-regulated changes in paxillin adhesion dynamics that are required to engage the death pathway. Moreover, our findings support a role for JNK-mediated paxillin phosphorylation in adhesion growth and stabilization during tension signaling.     

Selective decrease of membrane-associated PKC-α and PKC-ε in response to elevated intracellular O-GlcNAc levels in transformed human glial cells

Increased flux through the hexosamine biosynthetic pathway (HBP) has been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. It has been suggested that this effect is due to increases in the β-O-linked N-acetylglucosamine (O-GlcNAc) modification. Herein, we demonstrate the effect of increasing the O-GlcNAc modification on the translocation of select PKC isozymes in a human astroglial cell line. Treating cells with either 8 mM d-glucosamine (GlcN), 5 mM streptozotocin (STZ), or 80 μM O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) produced a significant increase in the O-GlcNAc modification on both cytosolic and membrane proteins; however, both the level and rate of O-GlcNAc increase varied with the compound. GlcN treatment resulted in a rapid, transient translocation of PKC-βII that was maximal after 3 h (73±8%) and also produced a 48±15% decrease in membrane-associated PKC-ε after 9 h of treatment. Similar to GlcN treatment, STZ and PUGNAc treatment also resulted in decreased levels of PKC-ε in the membrane fraction. Significant decreases were seen as early as 5 h and, by 9 h of treatment, had decreased by 87±6% with STZ and 73±7% with PUGNAc. Unlike GlcN, both STZ and PUGNAc produced a decrease in PKC-α membrane levels by 9 h posttreatment (78±10% with STZ and 66±8% with PUGNAc) while neither compound produced any changes in PKC-βII translocation. In addition, none of the three compounds affected membrane levels of PKC-ι. Altogether, these results demonstrate a novel link between increased levels of the O-GlcNAc modification and the regulation of specific PKC isoforms.     

O-GlcNAcylation of Cofilin Promotes Breast Cancer Cell Invasion

O-GlcNAcylation is a post-translational modification that regulates a broad range of nuclear and cytoplasmic proteins and is emerging as a key regulator of various biological processes. Previous studies have shown that increased levels of global O-GlcNAcylation and O-GlcNAc transferase (OGT) are linked to the incidence of metastasis in breast cancer patients, but the molecular basis behind this is not fully known. In this study, we have determined that the actin-binding protein cofilin is O-GlcNAcylated by OGT and mainly, if not completely, mediates OGT modulation of cell mobility. O-GlcNAcylation at Ser-108 of cofilin is required for its proper localization in invadopodia at the leading edge of breast cancer cells during three-dimensional cell invasion. Loss of O-GlcNAcylation of cofilin leads to destabilization of invadopodia and impairs cell invasion, although the actin-severing activity or lamellipodial localization is not affected. Our study provides insights into the mechanism of post-translational modification in fine-tuning the regulation of cofilin activity and suggests its important implications in cancer metastasis.     

Upregulation of paxillin and focal adhesion signaling follows Dystroglycan Complex deletions and promotes a hypertensive state of differentiation

Anchorage to matrix is mediated for many cells not only by integrin-based focal adhesions but also by a parallel assembly of integral and peripheral membrane proteins known as the Dystroglycan Complex. Deficiencies in either dystrophin (mdx mice) or γ-sarcoglycan (γSG^?/? mice) components of the Dystroglycan Complex lead to upregulation of numerous focal adhesion proteins, and the phosphoprotein paxillin proves to be among the most prominent. In mdx muscle, paxillin-Y31 and Y118 are both hyper-phosphorylated as are key sites in focal adhesion kinase (FAK) and the stretch-stimulatable pro-survival MAPK pathway, whereas γSG^?/? muscle exhibits more erratic hyper-phosphorylation. In cultured myotubes, cell tension generated by myosin-II appears required for localization of paxillin to adhesions while vinculin appears more stably integrated. Overexpression of wild-type (WT) paxillin has no obvious effect on focal adhesion density or the physical strength of adhesion, but WT and a Y118F mutant promote contractile sarcomere formation whereas a Y31F mutant shows no effect, implicating Y31 in striation. Self-peeling of cells as well as Atomic Force Microscopy (AFM) probing of cells with or without myosin-II inhibition indicate an increase in cell tension within paxillin-overexpressing cells. However, prednisolone, a first-line glucocorticoid for muscular dystrophies, decreases cell tension without affecting paxillin at adhesions, suggesting a non-linear relationship between paxillin and cell tension. Hypertension that results from upregulation of integrin adhesions is thus a natural and treatable outcome of Dystroglycan Complex down-regulation.     

Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC-12 cells

Cell adhesions play an important role in neurite extension. Paxillin, a focal adhesion adaptor protein involved in focal adhesion dynamics, has been demonstrated to be required for neurite outgrowth. However, the molecular mechanism by which paxillin regulates neurite outgrowth is unknown. Here, we show that paxillin is phosphorylated by p38MAPK in vitro and in nerve growth factor (NGF)-induced PC-12 cells. Ser 85 (Ser 83 for endogenous paxillin) is identified as one of major phosphorylation sites by phosphopeptide mapping and mass spectrometry. Moreover, expression of the Ser 85 --> Ala mutant of paxillin (paxS85A) significantly inhibits NGF-induced neurite extension of PC-12 cells, whereas expression of wild-type (wt) paxillin does not influence neurite outgrowth. Further experiments indicate that cells expressing paxS85A exhibit small, clustered focal adhesions which are not normally seen in cells expressing wt paxillin. Although wt paxillin and paxS85A have the same ability to bind vinculin and focal adhesion kinase, wt paxillin more efficiently associates with Pyk2 than paxS85A. Thus, phosphorylation of paxillin is involved in NGF-induced neurite extension of PC-12 cells, probably through regulating focal adhesion organization.     

ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPγS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPγS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion–like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH₂-terminally truncated Δ17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPγS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Δ17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.     

Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.     

Tyrosine phosphorylation of paxillin alpha is involved in temporospatial regulation of paxillin-containing focal adhesion formation and F-actin organization in motile cells

Temporal and spatial regulation of actin-based cytoskeletal organization and focal adhesion formation play an essential role in cell migration. Here, we show that tyrosine phosphorylation of a focal adhesion protein, paxillin, crucially participates in these regulations. We found that tyrosine phosphorylation of paxillin was a prominent event upon integrin activation during epithelial-mesenchymal trans-differentiation and cell migration. Four major tyrosine phosphorylation sites were identified, and two of them were highly inducible upon integrin activation. Paxillin exhibits three distinct subcellular localizations as follows: localization along the cell periphery colocalized with circumferential actin meshworks, macroaggregation at focal adhesions connected to actin stress fibers, and diffuse cytoplasmic distribution. Tyrosine phosphorylation of paxillin localized at the cell periphery and focal adhesions was shown using phosphorylation site-specific antibodies. Mutations in the phosphorylation sites affected the peripheral localization of paxillin and paxillin-containing focal adhesion formation during cell migration and cell-cell collision, accompanied by altered actin organizations. Our analysis indicates that phosphorylation of multiple tyrosines in paxillin alpha is necessary for the proper function of paxillin and is involved in the temporospatial regulation of focal adhesion formation and actin cytoskeletal organization in motile cells.     

O-Linked N-Acetylglucosamine Cycling Regulates Mitotic Spindle Organization

Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events.     

Flow-induced focal adhesion remodeling mediated by local cytoskeletal stresses and reorganization

Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm² for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35-40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.     

Mapping <Emphasis Type="Italic">O</Emphasis>-GlcNAc modification sites on tau and generation of a site-specific <Emphasis Type="Italic">O</Emphasis>-GlcNAc tau antibody

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-d-glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.     

Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.     

Flow-induced focal adhesion remodeling mediated by local cytoskeletal stresses and reorganization

Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm² for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35-40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.     

Nudel and FAK as Antagonizing Strength Modulators of Nascent Adhesions through Paxillin

Adhesion and detachment are coordinated critical steps during cell migration. Conceptually, efficient migration requires both effective stabilization of membrane protrusions at the leading edge via nascent adhesions and their successful persistence during retraction of the trailing side via disruption of focal adhesions. As nascent adhesions are much smaller in size than focal adhesions, they are expected to exhibit a stronger adhesivity in order to achieve the coordination between cell front and back. Here, we show that Nudel knockdown by interference RNA (RNAi) resulted in cell edge shrinkage due to poor adhesions of membrane protrusions. Nudel bound to paxillin, a scaffold protein of focal contacts, and colocalized with it in areas of active membrane protrusions, presumably at nascent adhesions. The Nudel-paxillin interaction was disrupted by focal adhesion kinase (FAK) in a paxillin-binding–dependent manner. Forced localization of Nudel in all focal contacts by fusing it to paxillin markedly strengthened their adhesivity, whereas overexpression of structurally activated FAK or any paxillin-binding FAK mutant lacking the N-terminal autoinhibitory domain caused cell edge shrinkage. These results suggest a novel mechanism for selective reinforcement of nascent adhesions via interplays of Nudel and FAK with paxillin to facilitate cell migration.     

O-Linked β-N-Acetylglucosamine (O-GlcNAc) Regulates Emerin Binding to Barrier to Autointegration Factor (BAF) in a Chromatin- and Lamin B-enriched “Niche”

Emerin, a membrane component of nuclear “lamina” networks with lamins and barrier to autointegration factor (BAF), is highly O-GlcNAc-modified (“O-GlcNAcylated”) in mammalian cells. Mass spectrometry analysis revealed eight sites of O-GlcNAcylation, including Ser-53, Ser-54, Ser-87, Ser-171, and Ser-173. Emerin O-GlcNAcylation was reduced ∼50% by S53A or S54A mutation in vitro and in vivo. O-GlcNAcylation was reduced ∼66% by the triple S52A/S53A/S54A mutant, and S173A reduced O-GlcNAcylation of the S52A/S53A/S54A mutant by ∼30%, in vivo. We separated two populations of emerin, A-type lamins and BAF; one population solubilized easily, and the other required sonication and included histones and B-type lamins. Emerin and BAF associated only in histone- and lamin-B-containing fractions. The S173D mutation specifically and selectively reduced GFP-emerin association with BAF by 58% and also increased GFP-emerin hyper-phosphorylation. We conclude that β-N-acetylglucosaminyltransferase, an essential enzyme, controls two regions in emerin. The first region, defined by residues Ser-53 and Ser-54, flanks the LEM domain. O-GlcNAc modification at Ser-173, in the second region, is proposed to promote emerin association with BAF in the chromatin/lamin B “niche.” These results reveal direct control of a conserved LEM domain nuclear lamina component by β-N-acetylglucosaminyltransferase, a nutrient sensor that regulates cell stress responses, mitosis, and epigenetics.     

Direct observation of α-actinin tension and recruitment at focal adhesions during contact growth

Adherent cells interact with extracellular matrix via cell–substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive α-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we have simultaneously observed the time-dependent changes in tension in α-actinin and the dynamics of focal adhesion during cell migration. We show that increase in tension in α-actinin at the focal adhesion coincides with elongation of the adhesion in its growth phase. The enlargement of focal adhesion is through a force sensitive recruitment of α-actinin and paxillin to the adhesion sites. Changes in α-actinin tension and correlated relocation of α-actinin in an active adhesion also guide the growth direction of the adhesion. The results support the model that cytoskeletal tension is coupled to focal adhesion via the linking protein, α-actinin at the adhesion complex. Lysophosphatidic acid caused an immediate increase in α-actinin tension followed by drastic focal adhesion formation and elongation. Application of Rho-ROCK inhibitor, Y27632, resulted in reversible reduction in tension in α-actinin and disassociation of focal adhesion, suggesting the involvement of myosin-II mediated contractile force in the focal adhesion dynamics. These findings suggest that α-actinin not only serves as a physical linker between cytoskeleton and integrin, but also participates in force transmission at adhesion sites to facilitate adhesion?s growth.     

Tropomyosin Isoform Expression Regulates the Transition of Adhesions To Determine Cell Speed and Direction

The balance of transition between distinct adhesion types contributes to the regulation of mesenchymal cell migration, and the characteristic association of adhesions with actin filaments led us to question the role of actin filament-associating proteins in the transition between adhesive states. Tropomyosin isoform association with actin filaments imparts distinct filament structures, and we have thus investigated the role for tropomyosins in determining the formation of distinct adhesion structures. Using combinations of overexpression, knockdown, and knockout approaches, we establish that Tm5NM1 preferentially stabilizes focal adhesions and drives the transition to fibrillar adhesions via stabilization of actin filaments. Moreover, our data suggest that the expression of Tm5NM1 is a critical determinant of paxillin phosphorylation, a signaling event that is necessary for focal adhesion disassembly. Thus, we propose that Tm5NM1 can regulate the feedback loop between focal adhesion disassembly and focal complex formation at the leading edge that is required for productive and directed cell movement.Among the different modes of migration that cells adopt, mesenchymal cell migration is dependent on integrin-based adhesion to the extracellular matrix (14), and the cellular mechanisms regulating integrin adhesion formation and turnover (adhesion dynamics) are integral to this process. The fate of integrin adhesions is intimately linked with filaments of polymerized actin (4). At the molecular level, actin filaments are highly dynamic, and this aspect of actin polymer biology provides an important control mechanism by which cells can organize filaments into structures with distinct properties. Tropomyosins are a multi-isoform family of actin-associating proteins that confer isoform-specific regulation of diverse actin filaments (3, 16, 34, 35). The interdependence of integrin adhesions and actin filaments suggests that expression of actin-associated proteins such as the tropomyosins may represent a mechanism for the regulation of adhesion dynamics that determine cell migration.In migrating cells small integrin-based focal complexes form at the periphery of lamellipodial extensions (32). These complexes are characterized by their subcellular distribution, dot-like shape, dependence on Rac activity, phosphorylated paxillin, and association with the network of short, branched actin filaments at the leading edge. The focal complexes are short lived (43) but provide strong traction forces at the leading edge (2) and most likely regulate directional migration (19). Subsets of focal complexes mature into focal adhesions, structures characterized by: Rho GTPase and Rho kinase dependence, dash-like shape, high levels of paxillin and phosphorylated paxillin, and low levels of the actin-binding molecule tensin (43, 44). The focal adhesions play an important role in anchoring bundles of polymerized actin stress fibers, providing the contractile force necessary for the translocation of the cell body during migration. There are at least three distinct classes of stress fibers observed in migrating cells (20, 27). Dorsal stress fibers are inserted into focal adhesions at the ventral surface of the cell. The distal end of the dorsal fibers can associate with a second type of actin fiber, the transverse arcs that run parallel to the leading edge and are not directly connected to focal adhesions. Ventral stress fibers have focal adhesions at either end and can be established following the contraction of two dorsal stress fibers and the associated transverse arc to form one actin bundle (20).Increased ventral stress fibers and focal adhesions are characteristic of nonmotile cells, in contrast, cell migration depends on focal adhesion turnover at the leading edge, allowing the formation of newly protruding regions of membrane and focal complex formation (28, 39). While the precise mechanism of focal adhesion turnover is incompletely understood, activation and phosphorylation of Src kinase, p130Cas, and paxillin (13, 39, 45) have all been implicated in focal adhesion turnover. A biphasic relationship between cell adhesion and cell speed suggests that conditions that alter the turnover rate of focal adhesions (either too much or too little) can reduce cell speed (18, 22).In cells with a fibroblastic phenotype, increased levels of acto-myosin contractility promote focal adhesion transition to fibrillar adhesions (also known as ECM contacts) (6, 7): elongated, thin, central arrays of dots or elongated fibrils that characteristically contain tensin but low levels of phosphorylated paxillin (29, 44, 45) and bind fibrils of fibronectin parallel to actin bundles (23, 29). These adhesions are formed by ligand-occupied fibronectin integrin receptor translocation from focal adhesions along bundles of actin filaments toward the cell center, and the process is dependent on an intact actin cytoskeleton and myosin activity (29). Receptor translocation stimulates matrix reorganization by transmitting cytoskeleton-generated tension through the integrin receptors onto the surrounding matrix (25, 29). The rate of receptor translocation is apparently independent from the rate of cell migration (29). However, the cytoskeletal tension that causes the fibrillar adhesion formation is also reported to decrease paxillin phosphorylation (45). Since phosphorylated paxillin is required for the generation of new focal complexes (45), conditions which switch the balance of adhesion in favor of fibrillar adhesion should presumably result in significantly reduced paxillin phosphorylation, leading to reduced focal adhesion turnover and correspondingly decreased cell migration.The cytoskeletal tropomyosin Tm5NM1 is a broadly distributed isoform (37) that alters cell shape (34), localizes to and promotes stress fibers that are resistant to actin depolymerizing drugs (9), enhances myosin IIA activation and recruitment to stress fibers, and inhibits cell migration (3). Therefore, we hypothesized that Tm5NM1 expression might determine cell migration by coordinating actin-dependent transition toward a predominance of focal adhesions and fibrillar adhesions. Using overexpression, knockdown, and genetic knockout models, we demonstrate that Tm5NM1 inhibits cell migration by promoting selective stabilization of focal adhesions and transition to fibrillar adhesions via the regulation of paxillin phosphorylation.