Paxillin-beta-catenin interactions are involved in Rac/Cdc42-mediated endothelial barrier-protective response to oxidized phospholipids

Oxidized phospholipids may appear in the pulmonary circulation as a result of acute lung injury or inflammation. We have previously described barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on human pulmonary endothelial cells (EC) mediated by small GTPases Rac and Cdc42. This work examined OxPAPC-induced focal adhesion (FA) and adherens junction (AJ) remodeling and potential interactions between FA and AJ protein complexes involved in OxPAPC-induced EC barrier enhancement. Immunofluorescence analysis, subcellular fractionation, and coimmunoprecipitation assays have shown that OxPAPC induced translocation and peripheral accumulation of FA complexes containing paxillin, focal adhesion kinase, vinculin, GIT1, and GIT2, increased association of AJ proteins vascular endothelial-cadherin, p120-catenin, alpha-, beta-, and gamma-catenins, and dramatically enhanced cell junction areas covered by AJ. Coimmunoprecipitation, pulldown assays, and confocal microscopy studies have demonstrated that OxPAPC promoted novel interactions between FA and AJ complexes via paxillin and beta-catenin association, which was critically dependent on Rac and Cdc42 activities and was abolished by pharmacological or small interfering RNA (siRNA)-mediated inhibition of Rac and Cdc42. Depletion of beta-catenin using the siRNA approach attenuated OxPAPC-induced paxillin translocation to the cell periphery, but also significantly decreased interaction of paxillin with AJ protein complex. In turn, paxillin knockdown by specific siRNA attenuated AJ enhancement in response to OxPAPC. These results show for the first time the novel interactions between FA and AJ protein complexes critical for EC barrier regulation by OxPAPC.     

Investigating lasp-2 in cell adhesion: new binding partners and roles in motility

Focal adhesions are intricate protein complexes that facilitate cell attachment, migration, and cellular communication. Lasp-2 (LIM-nebulette), a member of the nebulin family of actin-binding proteins, is a newly identified component of these complexes. To gain further insights into the functional role of lasp-2, we identified two additional binding partners of lasp-2: the integral focal adhesion proteins vinculin and paxillin. Of interest, the interaction of lasp-2 with its binding partners vinculin and paxillin is significantly reduced in the presence of lasp-1, another nebulin family member. The presence of lasp-2 appears to enhance the interaction of vinculin and paxillin with each other; however, as with the interaction of lasp-2 with vinculin or paxillin, this effect is greatly diminished in the presence of excess lasp-1. This suggests that the interplay between lasp-2 and lasp-1 could be an adhesion regulatory mechanism. Lasp-2’s potential role in metastasis is revealed, as overexpression of lasp-2 in either SW620 or PC-3B1 cells—metastatic cancer cell lines—increases cell migration but impedes cell invasion, suggesting that the enhanced interaction of vinculin and paxillin may functionally destabilize focal adhesion composition. Taken together, these data suggest that lasp-2 has an important role in coordinating and regulating the composition and dynamics of focal adhesions.     

Changes in the phosphorylation and distribution of vinculin during nerve growth factor induced neurite outgrowth

The mechanism of neurite initiation and elongation was studied using nerve growth factor (NGF) treatment of PC12 cells. The distribution of focal adhesion sites and of the cytoskeletal protein vinculin was determined in large, fused, multinucleated PC12 cells. In the absence of NGF, focal adhesion sites as seen by interference reflection microscopy were restricted to the cell periphery in a regular distribution. Vinculin assemblies (foci), observed by indirect immunofluorescence microscopy using affinity purified anti-vinculin antibodies, were restricted to the cell periphery at focal adhesion sites. Within 4 hr after NGF treatment of the cells, the distribution of both vinculin and focal adhesion sites began to change. Focal adhesion sites became restricted to discrete protruding portions of the cell periphery. Larger, brighter vinculin foci appeared at the tips of the cell margin extensions, concomitant with the loss of foci at locations between the protrusions. As neurites elongated focal adhesion sites and vinculin foci remained with the tips of the growth cone extensions. Both focal adhesion sites and vinculin foci were rarely seen in the perikarya of cells with elongating neurites, and these were always confined to extended portions of the cell body margin. Occasionally, vinculin foci could be seen at the proximal portion of the neurite, at bending elbows, and at discrete expansions along the length. By immunoprecipitation of vinculin from 32P-labeled cells, vinculin phosphorylation was found to be increased within 1 hr of NGF treatment. The role of vinculin phosphorylation and assembly in the formation and directional elongation of neuritic processes in response to NGF is discussed.     

Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells

Microinjection of fluorophore-tagged cytoskeletal proteins has been a useful tool in studies of formation of focal adhesions (FA). We used this method to study the maintenance of adherens junctions (AJ) and tight junctions (TJ) of epithelial Madin-Darby bovine kidney cells. We chose alpha-actinin and vinculin as markers, because they are present both at adherens junctions and focal adhesions and their binding partners have been well characterized. Isolated FITC-labelled chicken alpha-actinin and vinculin were injected into confluent cells where they were rapidly incorporated both in FAs and AJs. The FAs remained unchanged, whereas cell-cell contacts began to fade within an hour after injection and the cells were joined to polykaryons having 5 to 13 nuclei. Short fragments of cell membranes containing injected proteins, actin, beta-catenin, cadherin, claudin, occludin and ZO-1 were visible inside the polykaryons indicating that both AJs and TJs were disintegrated as a single complex. Microinjected FITC-labelled vinculin head domain was also incorporated to both AJs and FAs, but instead of fusions it rapidly induced the detachment of the cells from the substratum probably due to high affinity of vinculin head to talin. Vinculin tail domain had no apparent effect on the cell morphology. Since small GTPases are involved in the building up of AJs, we injected active and inactive forms of cdc42 and rac proteins together with vinculin to see their effect. Active forms reduced the formation of polykaryons presumably by strengthening AJs, whereas inactive forms had no apparent effect. We suggest that excess alpha-actinin and vinculin uncouple the cell-cell adhesion junctions from the intracellular cytoskeleton which leads to fragmentation of junctional complexes and subsequent cell fusion. The results show that cell-cell adhesion sites are more dynamic and more sensitive than FAs to an imbalance in the amount of free alpha-actinin and intact vinculin.     

Rac1 inactivation by lethal toxin from Clostridium sordellii modifies focal adhesions upstream of actin depolymerization

Inactivation of different small GTPases upon their glucosylation by lethal toxin from Clostridium sordellii strain IP82 (LT‐82) is already known to lead to cell rounding, adherens junction (AJ) disorganization and actin depolymerization. In the present work, we observed that LT‐82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Among the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing Rac1^V12. Rac1 inactivation by LT‐82 modifies interactions between proteins from AJ and FA complexes as shown by pull‐down assays. We showed that in Triton X‐100‐insoluble membrane proteins from these complexes, namely E‐cadherin, β‐catenin, p120‐catenin and talin, are decreased upon LT‐82 intoxication, a treatment that also induces a rapid decrease in cell phosphoinositide content. Therefore, we proposed that Rac inactivation by LT‐82 alters phosphoinositide metabolism leading to FA and AJ complex disorganization and actin depolymerization.     

Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells

In this study, we examined the effects of shark cartilage extract on the attachment and spreading properties and the focal adhesion structure of cultured bovine pulmonary artery endothelial cells. Treatment with cartilage extract resulted in cell detachment from the substratum. Immunofluorescence staining of those treated cells that remained attached showed that, instead of being present in both central and peripheral focal adhesions as in control cells, both integrin alpha(v)beta(3) and vinculin were found only in peripheral focal adhesion and thinner actin filament bundles were seen. In addition to causing cell detachment, cartilage extract partially inhibited the initial adherence of the cells to the substratum in a dose-dependent manner. Integrin alpha(v)beta(3) and vinculin staining of these cells also showed a peripheral focal adhesion distribution pattern. Vitronectin induced cell spreading in the absence of serum, but was blocked by simultaneous incubation with cartilage extract, which was shown to inhibit both integrin alpha(v)beta(3) and vinculin recruitment to focal adhesion and the formation of stress fibers. Dot binding assays showed that these inhibitory effects on cell attachment and spreading were not due to direct binding of cartilage extract components to integrin alpha(v)beta(3) or vitronectin. Shark cartilage chondroitin sulfate had no inhibitory effect on either cell attachment or spreading of endothelial cells. These results show that the inhibitory effects of cartilage extract on cell attachment and spreading are mediated by modification of the organization of focal adhesion proteins.     

Actomyosin tension is required for correct recruitment of adherens junction components and zonula occludens formation

The adherens junction (AJ) densely associated with actin filaments is a major cell-cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly.     

Protein kinase G signaling disrupts Rac1-dependent focal adhesion assembly in liver specific pericytes

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMP-dependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1.     

Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II–mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II–dependent manner and to examine the mechanism for their myosin II–sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II–mediated FAK phosphorylation of paxillin that promotes vinculin FA recruitment to reinforce the cytoskeletal ECM linkage and drive FA maturation.     

A lipid-regulated docking site on vinculin for protein kinase C.

During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase Calpha and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase Calpha to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase Calpha reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to < or =50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase Calpha and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes.     

Changes in membrane lipid composition cause alterations in epithelial cell–cell adhesion structures in renal papillary collecting duct cells

In epithelial tissues, adherens junctions (AJ) mediate cell–cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell–cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell–cell adhesion structures in renal papillary collecting duct cells.     

N-glycosylation affects the molecular organization and stability of E-cadherin junctions

Epithelial cell-cell adhesion is mediated by E-cadherin, an intercellular N-glycoprotein adhesion receptor that functions in the assembly of multiprotein complexes anchored to the actin cytoskeleton named adherens junctions (AJs). E-cadherin ectodomains 4 and 5 contain three potential N-glycan addition sites, although their significance in AJ stability is unclear. Here we show that sparse cells lacking stable AJs produced E-cadherin that was extensively modified with complex N-glycans. In contrast, dense cultures with more stable AJs had scarcely N-glycosylated E-cadherin modified with high mannose/hybrid and limited complex N-glycans. This suggested that variations in AJ stability were accompanied by quantitative and qualitative changes in E-cadherin N-glycosylation. To further examine the role of N-glycans in AJ function, we generated E-cadherin N-glycosylation variants lacking selected N-glycan addition sites. Characterization of these variants in CHO cells, lacking endogenous E-cadherin, revealed that site 1 on ectodomain 4 was modified with a prominent complex N-glycan, site 2 on ectodomain 5 did not have a substantial oligosaccharide, and site 3 on ectodomain 5 was decorated with a high mannose/hybrid N-glycan. Removal of complex N-glycan from ectodomain 4 led to a dramatically increased interaction of E-cadherin-catenin complexes with vinculin and the actin cytoskeleton. The latter effect was further enhanced by the deletion of the high mannose/hybrid N-glycan from site 3. In MDCK cells, which produce E-cadherin, a variant lacking both complex and high mannose/hybrid N-glycans functioned like a dominant positive displaying increased interaction with gamma-catenin and vinculin compared with the endogenous E-cadherin. Collectively, our studies show that N-glycans, and complex oligosaccharides in particular, destabilize AJs by affecting their molecular organization.     

Microtubule-Dependent Modulation of Adhesion Complex Composition

The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding α₅β₁ integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, α₅-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, α₅-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183.     

Changes in membrane lipid composition cause alterations in epithelial cell-cell adhesion structures in renal papillary collecting duct cells

In epithelial tissues, adherens junctions (AJ) mediate cell-cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell-cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell-cell adhesion structures in renal papillary collecting duct cells.     

Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells

We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.     

Focal adhesion molecules as potential target of lead toxicity in NRK-52E cell line

In this study, we investigated the influence of inorganic lead (Pb(II)), an environmental pollutant having nephrotoxic action, on the focal adhesion (FA) organization of a rat kidney epithelial cell line (NRK-52E). In particular, we evaluated the effects of the metal on the recruitment of paxillin, focal adhesion kinase, vinculin and cytoskeleton proteins at the FAs complexes. We provided evidences that, in proliferating NRK-52E cell cultures, low concentrations of Pb(II) affect the cell adhesive ability and stimulate the disassembly of FAs, thus inhibiting the integrin-activated signalling. These effects appeared to be strictly associated to the Pb-induced arrest of cell cycle at G0/G1 phase also proved in this cell line.     

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking- focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.     

Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.     

Roles for the tubulin- and PTP-PEST-binding paxillin LIM domains in cell adhesion and motility

Cell dynamics mediated through cell-extracellular matrix contacts, such as adhesion and motility involve the precise regulation of large complexes of structural and signaling molecules called focal adhesions (FAs). Paxillin is a multi-domain FA adaptor protein containing five amino-terminal paxillin leucine-aspartate repeat (LD) motifs and four carboxyl-terminal Lin-11 Isl-1 and Mec-3 (LIM) domains. The LD motifs support paxillin binding to actopaxin, integrin linked kinase (ILK), FA kinase (FAK), paxillin kinase linker (PKL) and vinculin. Of the LIM domains, LIM2 and 3 comprise the paxillin FA-targeting motif, with phosphorylation of these domains modulating paxillin targeting and cell adhesion to fibronectin (Fn). The identity of the paxillin FA targeting partner remains to be determined; however, the LIM domains mediate interactions with tubulin and the protein-tyrosine phosphatase (PTP)-PEST. PTP-PEST binding requires both LIM3 and 4, whereas, the precise LIM target of tubulin binding is not known. In this report, we demonstrate that the individual paxillin LIM2 and 3 domains support specific binding to tubulin and suggest a potential role for this interaction in the regulation of paxillin sub-cellular compartmentalization. In addition, expression of paxillin molecules with mutations in the tubulin- and PTP-PEST-binding LIM domains differentially impaired Chinese hamster ovary K1 (CHO.K1) cell adhesion and migration to Fn. Perturbation of LIM3 or 4 inhibited adhesion while mutation of LIM2 or 4 decreased cell motility. Interestingly, expression of tandem LIM2-3 inhibited cell adhesion and spreading while LIM3-4 stimulated a well-spread polarized phenotype. These data offer further support for a critical role for paxillin in cell adhesion and motility.