Smooth muscle adherens junctions associated proteins are stable at the cell periphery during relaxation and activation

This study was performed to determine the stability of the adherens junction (AJ)-associated proteins at the smooth muscle cell (SMC) plasma membrane during relaxing and activating conditions. Dog stomach, ileum, colon, and trachea tissues were stored in Ca²⁺-free PSS or regular PSS or were activated in 10 µM carbachol in PSS before rapid freezing. The tissues were subsequently sectioned and immunoreacted using antibodies for vinculin, talin, fibronectin, and caveolin to determine their cellular distribution in these tissues under these conditions. In all four tissues and under all three conditions, the distribution of these four proteins remained localized to the periphery of the cell. In transverse tissue sections, the AJ-associated proteins formed a distinct punctate pattern around the periphery of the SMCs at the plasma membrane. These domains alternated with the caveolae (as identified by the presence of caveolin). In longitudinal tissue sections, the AJ-associated proteins formed continuous tracks or staves, while the caveolae remained punctate in this dimension as well. Caveolin is not present in the tapered ends of the SMCs, where the AJ-associated proteins appear continuous around the periphery. Densitometry of the fluorophore distribution of these proteins showed no shift in their localization from the SMC periphery when the tissues were relaxed or when they were activated before freezing. These results suggest that under physiologically relaxing and activating conditions, AJ-associated proteins remain stably localized at the plasma membrane. vinculin; talin; fibronectin; caveolin; stomach; ileum; colon; trachea     

Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane

Cytoskeletal reorganization of the smooth muscle cell in response to contractile stimulation may be an important fundamental process in regulation of tension development. We used confocal microscopy to analyze the effects of cholinergic stimulation on localization of the cytoskeletal proteins vinculin, paxillin, talin and focal adhesion kinase (FAK) in freshly dissociated tracheal smooth muscle cells. All four proteins were localized at the membrane and throughout the cytoplasm of unstimulated cells, but their concentration at the membrane was greater in acetylcholine (ACh)-stimulated cells. Antisense oligonucleotides were introduced into tracheal smooth muscle tissues to deplete paxillin protein, which also inhibited contraction in response to ACh. In cells dissociated from paxillin-depleted muscle tissues, redistribution of vinculin to the membrane in response to ACh was prevented, but redistribution of FAK and talin was not inhibited. Muscle tissues were transfected with plasmids encoding a paxillin mutant containing a deletion of the LIM3 domain (paxillin LIM3 dl 444–494), the primary determinant for targeting paxillin to focal adhesions. Expression of paxillin LIM3 dl in muscle tissues also inhibited contractile force and prevented cellular redistribution of paxillin and vinculin to the membrane in response to ACh, but paxillin LIM3 dl did not inhibit increases in intracellular Ca²⁺ or myosin light chain phosphorylation. Our results demonstrate that recruitment of paxillin and vinculin to smooth muscle membrane is necessary for tension development and that recruitment of vinculin to the membrane is regulated by paxillin. Vinculin and paxillin may participate in regulating the formation of linkages between the cytoskeleton and integrin proteins that mediate tension transmission between the contractile apparatus and the extracellular matrix during smooth muscle contraction. tissue transfection; plasmids; cytoskeleton; talin; immunofluorescence     

Identification and subcellular location of talin in various cell types and tissues by means of [125I]vinculin overlay, immunoblotting and immunocytochemistry

In the present study, we have examined the cellular and subcellular distribution of talin in several tissues of the chicken. By immunocytochemistry, Western Blot analysis and [125I]vinculin overlay, talin was demonstrated in most of the main tissues and cell types of the body. Corresponding to the property of talin to bind to the fibronectin receptor, talin was found to be confined to the site of the plasma membrane that abuts the extracellular matrix in various types of mesenchymal and epithelial cells. In the central nervous system talin was almost exclusively confined to cells of the connective tissue, i.e., blood vessels and the connective tissue sheaths. No evidence was obtained for the association of talin with any type of intercellular junction. In nonadhering cells such as circulating platelets and leukocytes, talin displayed a diffuse distribution throughout the cytoplasm. These findings suggest a general role for talin in certain aspects of cellular adhesion to the extracellular matrix.     

Integrin on developing and adult skeletal muscle

Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu     

Preliminary characterization of cell surface-extracellular matrix linkage complexes in cultured retinal pigmented epithelial cells

In this report, we describe the relative distribution of vinculin, talin, and fibronectin in cultured retinal pigmented epithelial cells from chick embryo eyes. We show that in these cells vinculin is present in both focal cell-substratum and cell-cell contacts, whereas talin is present only in the cell-substratum contacts. When cells are double-labeled for talin and fibronectin and viewed at the substratum level, fibronectin is not detectable and talin is concentrated in plaques corresponding to focal contacts. However, when the same cells are viewed at the apical level, both talin and fibronectin are present in a fibrillar pattern. In addition to fibrils which are both talin- and fibronectin-positive, there are areas which are either talin-positive and fibronectin-negative or, vice versa, talin-negative and fibronectin-positive. These observations indicate an interesting variability in the composition of transmembrane linkages in retinal pigmented epithelial cells in vitro.     

Kinetic determination of focal adhesion protein formation

I examined the binding kinetics between integrin (alpha(IIb)beta(3)) and purified focal adhesion proteins, including alpha-actinin, filamin, vinculin, talin, and F-actin. Using static light-scatter technique, I observed affinities of the order talin > filamin > F-actin > alpha-actinin > (talin when bound to vinculin) which were lower when integrin was complexed with fibronectin. No binding between integrin and vinculin was detected. The calculated dissociation constants (K(d)) ranged between 0.4 microM and 5 microM. These results in part confirm previously published data using different methods. The modest affinity with which the focal adhesion proteins interact in vitro might be indicative of how cells, e.g., thrombocytes, gain a high degree of versatility and velocity.     

Talin and vinculin in the oocytes, eggs, and early embryos of Xenopus laevis: a developmentally regulated change in distribution

We have investigated the expression and distribution of talin and vinculin in the oocytes, eggs, and embryos of Xenopus laevis. Antibodies to the previously characterized avian proteins stain several different Xenopus cell types identically by immunofluorescence: adhesion plaques of cultured kidney (A6) cells, the cell peripheries of oviduct cells, and the postsynaptic neuromuscular junctions of tadpole tail muscle fibers. These antibodies also identify cognate proteins of the appropriate sizes on immunoblots of A6 cell and oviduct lysates. Using these antibodies on ovarian tissue, we find talin to be highly localized at the cortices of oocytes and vinculin to be in the oocyte cytoplasm and absent from the oocyte cortex. In the cells of the ovarian layers that surround the oocytes, talin and vinculin can be detected as soluble and cytoskeletal components. Vinculin is first detectable as a cytoskeletal component in eggs, appearing some time during or between oocyte maturation and oviposition. During early embryo development, talin and vinculin are colocalized in the cortex of cleavage furrows and blastomeres. Thus, Xenopus oocytes and eggs display different distributions of talin and vinculin. The change from unlinked localization to colocalization appears to be developmentally regulated, occurring during the transition from oocyte to egg.     

Focal contacts: transmembrane links between the extracellular matrix and the cytoskeleton

The sites of tightest adhesion that form between cells and substrate surfaces in tissue culture are termed focal contacts. The external faces of focal contacts include specific receptors, belonging to the integrin family of proteins, for fibronectin and vitronectin, two common components of extracellular matrices. On the internal (cytoplasmic) side of focal contacts, several proteins, including talin and vinculin, mediate interactions with the actin filament bundles of the cytoskeleton. The changes that occur in focal contacts as a result of viral transformation are discussed.     

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.     

Keap1 in adhesion complexes

Cell adhesion complexes are sensors that interact with the extracellular environment and allow for the transmission of signals found outside the cell across the plasma membrane to the cell interior. Keap1 is a newly identified component of cell adhesion complexes. We investigated Keap1's association with these complexes in diverse tissues and cell types. Keap1 is present in focal adhesion (FA)-like assemblies in kidney proximal tubule cells where it colocates with actin. In liver, Keap1 is found in the adherens junctions (AJ) and at the base of the bile canaliculi. To study Keap1's involvement in both the integrin-based FA and the cadherin-based AJ, we induced formation of these complexes in fibroblasts, using a serum starvation followed by a serum supplementation method. When compared with vinculin, a component of all FA, we found that Keap1 assembles only in the peripheral FA. Within the peripheral FA, Keap1 was present in distinct foci along the length of the FA and these foci were different from vinculin, talin, paxillin, and phospho-tyrosine rich regions of the FA. Unlike most FA components, Keap1 was also recruited to the newly formed AJ. As Keap1 homologues are actin-bundling proteins, we hypothesize that Keap1's function is to bundle F-actin within these diverse types of cell adhesion components.     

Scaffolds for engineering smooth muscle under cyclic mechanical strain conditions

Cyclic mechanical strain has been demonstrated to enhance the development and function of engineered smooth muscle (SM) tissues, but appropriate scaffolds for engineering tissues under conditions of cyclic strain are currently lacking. These scaffolds must display elastic behavior, and be capable of inducing an appropriate smooth muscle cell (SMC) phenotype in response to mechanical signals. In this study, we have characterized several scaffold types commonly utilized in tissue engineering applications in order to select scaffolds that exhibit elastic properties under appropriate cyclic strain conditions. The ability of the scaffolds to promote an appropriate SMC phenotype in engineered SM tissues under cyclic strain conditions was subsequently analyzed. Poly(L-lactic acid)-bonded polyglycolide fiber-based scaffolds and type I collagen sponges exhibited partially elastic mechanical properties under cyclic strain conditions, although the synthetic polymer scaffolds demonstrated significant permanent deformation after extended times of cyclic strain application. SM tissues engineered with type I collagen sponges subjected to cyclic strain were found to contain more elastin than control tissues, and the SMCs in these tissues exhibited a contractile phenotype. In contrast, SMCs in control tissues exhibited a structure more consistent with the nondifferentiated, synthetic phenotype. These studies indicate the appropriate choice of a scaffold for engineering tissues in a mechanically dynamic environment is dependent on the time frame of the mechanical stimulation, and elastic scaffolds allow for mechanically directed control of cell phenotype in engineered tissues.     

Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices

The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM component with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture conditions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 x 5 mm, 2-mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer matrices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matrices was a function of cell concentration in the seeding solution, but under all conditions a larger number (approximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were obtained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture conditions were next studied by culturing cell-polymer constructs in a stirred bioreactor versus static culture conditions. The stirred culture of SMC-seeded polymer matrices resulted in tissues with a cell density of 6.4 +/- 0.8 x 10(8) cells/cm3 after 5 weeks, compared to 2.0 +/- 1.1 x 10(8) cells/cm3 with static culture. The elastin and collagen synthesis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred bioreactor was 24 +/- 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were implanted in rats for various times. In summary, the system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM. This engineered tissue may find clinical applications and provide a tool to study molecular mechanisms in vascular development.     

Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin

The cytoskeletal proteins talin and vinculin are localized at cell‐matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F‐actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four‐ and five‐helix bundles that harbor amphipathic α‐helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four‐helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α‐helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four‐helix bundle.     

GDF11 prevents the formation of thoracic aortic dissection in mice: Promotion of contractile transition of aortic SMCs

Thoracic aortic dissection (TAD) is an aortic disease associated with dysregulated extracellular matrix composition and de-differentiation of vascular smooth muscle cells (SMCs). Growth Differentiation Factor 11 (GDF11) is a member of transforming growth factor β (TGF-β) superfamily associated with cardiovascular diseases. The present study attempted to investigate the expression of GDF11 in TAD and its effects on aortic SMC phenotype transition. GDF11 level was found lower in the ascending thoracic aortas of TAD patients than healthy aortas. The mouse model of TAD was established by β-aminopropionitrile monofumarate (BAPN) combined with angiotensin II (Ang II). The expression of GDF11 was also decreased in thoracic aortic tissues accompanied with increased inflammation, arteriectasis and elastin degradation in TAD mice. Administration of GDF11 mitigated these aortic lesions and improved the survival rate of mice. Exogenous GDF11 and adeno-associated virus type 2 (AAV-2)-mediated GDF11 overexpression increased the expression of contractile proteins including ACTA2, SM22α and myosin heavy chain 11 (MYH11) and decreased synthetic markers including osteopontin and fibronectin 1 (FN1), indicating that GDF11 might inhibit SMC phenotype transition and maintain its contractile state. Moreover, GDF11 inhibited the production of matrix metalloproteinase (MMP)-2, 3, 9 in aortic SMCs. The canonical TGF-β (Smad2/3) signalling was enhanced by GDF11, while its inhibition suppressed the inhibitory effects of GDF11 on SMC de-differentiation and MMP production in vitro. Therefore, we demonstrate that GDF11 may contribute to TAD alleviation via inhibiting inflammation and MMP activity, and promoting the transition of aortic SMCs towards a contractile phenotype, which provides a therapeutic target for TAD.     

Presence of Cx37 and lack of desmin in smooth muscle cells are early markers for arteriogenesis

In search of early structural markers of arteriogenesis, we studied the expression of gap junction proteins as well as of contractile and cytoskeletal proteins in smooth muscle cells (SMCs) during coronary collateral vessel growth induced by chronic occlusion of the left circumflex artery (LCx) in the dog heart. We used confocal microscopy with antibodies against connexin37 (Cx37), alpha-smooth muscle actin (alpha-SM actin), calponin, desmin and vinculin. The quantitative confocal analysis of immunofluorescence intensity showed that (1) in normal vessels (NV), Cx37 was present in endothelium only, not in SMC. Calponin, alpha-SM actin, desmin and vinculin were evenly expressed in SMC. (2) In early growing V (EV) with minimal intima formation, alpha-SM actin, calponin and vinculin showed little change in SMC, but desmin was 3.3 times lower than in NV, and Cx37 was induced (NV 0 arbitrary units/microm2, EV 50.3). (3) In actively growing V (AV), alpha-SM actin, calponin and vinculin were 3-, 3.3- and 2.9-fold lower, respectively, in the neointima as compared to the media. However, Cx37 was 48.2 AU/microm2 in the media and 15.8 AU/microm2 in the neointima. Desmin was almost absent in the neointima and 5-fold reduced in the media. SMC, strongly positive for alpha-SM actin and calponin, expressed Cx37. Our findings indicate that induction of Cx37 and reduction of desmin precede the phenotypic changes of SMCs, which are characterized by down-regulation of alpha-SM actin, calponin and vinculin, and the formation of a neointima. An altered expression of Cx37 and desmin, therefore, are early markers for arteriogenesis in dog heart.     

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.