Phorbol myristate acetate induces changes on F-actin and vinculin content in immature rat Sertoli cells

Actin and vinculin are two of the most abundant cytoskeletal proteins, widely expressed in nearly all types of eukaryotic cells. It has been well established that long-term exposure to the tumor promoter phorbol myristate acetate (PMA) affects Sertoli cell morphology, as well as F-actin and vinculin organization in vitro. To analyze in a quantitative manner the F-actin and vinculin content of rat immature Sertoli cells in vitro in response to PMA exposure, cytoskeletal fractions were prepared following extraction with Triton X-100. Analysis of the isolated cytoskeletal fractions by immunoblotting showed that exposure of immature rat Sertoli cells to PMA for 8h has an appreciable effect on the cellular level of both the actin and vinculin. Interestingly, as revealed by using calphostin C, a specific protein kinase C inhibitor, the observed F-actin and vinculin changes are most probably mediated by a mechanism that depends on protein kinase activity. A discussion is made concerning PKC modulation by PMA and the subsequent actin and vinculin quantitative changes and reorganization, phenomena that have been closely related to cell transformation.     

纽带蛋白及其与微丝和膜结合的研究

 用落球粘度计测量低剪切粘度的方法研究了纽带蛋白-肌动蛋白的相互作用。与以前关于纽带蛋白可使肌动蛋白粘度降低并推测它抑制微丝间相互作用的报道不同,10—50μg/ml的纽带蛋白引起肌动蛋白细丝溶液粘度略有增加。电镜观察表明,由于纽带蛋白的作用,肌动蛋白丝聚集成束。 远紫外圆二色谱显示,在pH7.0下,220和207nm处呈双负峰,190nm处有一正峰。按三波长法计算,其α螺旋和β折叠含量分别占41％和22％。研究了pH,磷脂和去垢剂对其构象的影响。疏水性结合的和水溶性的纽带蛋白相比,存在少量构象差别。联系这一蛋白处在细胞骨架与质膜相联系的特殊位置,推测在体内,纽带蛋白与脂结合后也可能发生类似的构象变化。     

原代培养脊髓神经元线状溶酶体与细胞骨架蛋白-纽蛋白关系的研究

目的研究原代培养脊髓神经元线状溶酶体(nematolysosome)的形成与分布及其与细胞骨架蛋白-纽蛋白(vinculin)的关系.方法用细胞松弛素D(cytochalasin D,CD)及佛波醇酯(phorbol myristate acetate,PMA)处理原代培养脊髓神经元,用免疫荧光双标记纽蛋白及组织蛋白酶D(cathepsin D)、酸性磷酸酶(ACPase)、电镜细胞化学及共焦激光扫描显微镜方法研究线状溶酶体与纽蛋白的关系.结果在正常对照组神经元,组织蛋白酶D(标记溶酶体)与纽蛋白分布于胞质及突起内;在CD及PMA处理神经元,纽蛋白及组织蛋白酶D的分布呈向心性移动,但集聚的部位不同;电镜酶细胞化学方法显示CD组及PMA组神经元内线状溶酶体均增多.结论组织蛋白酶D及纽蛋白在培养脊髓神经元内协同分布,CD及PMA均可引起二者分布的变化,提示纽蛋白可通过增强细胞内吞体/溶酶体系统活动而使线状溶酶体增加,也可通过促进丝状肌动蛋白聚合而影响线状溶酶体的形成及运动.     

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.     

An Autoinhibited Structure of α-Catenin and Its Implications for Vinculin Recruitment to Adherens Junctions

α-Catenin is an actin- and vinculin-binding protein that regulates cell-cell adhesion by interacting with cadherin adhesion receptors through β-catenin, but the mechanisms by which it anchors the cadherin-catenin complex to the actin cytoskeleton at adherens junctions remain unclear. Here we determined crystal structures of αE-catenin in the autoinhibited state and the actin-binding domain of αN-catenin. Together with the small-angle x-ray scattering analysis of full-length αN-catenin, we deduced an elongated multidomain assembly of monomeric α-catenin that structurally and functionally couples the vinculin- and actin-binding mechanisms. Cellular and biochemical studies of αE- and αN-catenins show that αE-catenin recruits vinculin to adherens junctions more effectively than αN-catenin, partly because of its higher affinity for actin filaments. We propose a molecular switch mechanism involving multistate conformational changes of α-catenin. This would be driven by actomyosin-generated tension to dynamically regulate the vinculin-assisted linkage between adherens junctions and the actin cytoskeleton.     

Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein

The focal adhesion protein vinculin is an actin-binding protein involved in the mechanical coupling between the actin cytoskeleton and the extracellular matrix. An autoinhibitory interaction between the N-terminal head (Vh) and the C-terminal tail (Vt) of vinculin masks an actin filament side-binding domain in Vt. The binding of several proteins to Vh disrupts this intramolecular interaction and exposes the actin filament side-binding domain. Here, by combining kinetic assays and microscopy observations, we show that Vt inhibits actin polymerization by blocking the barbed ends of actin filaments. In low salt conditions, Vt nucleates actin filaments capped at their barbed ends. We determined that the interaction between vinculin and the barbed end is characterized by slow association and dissociation rate constants. This barbed end capping activity requires C-terminal amino acids of Vt that are dispensable for actin filament side binding. Like the side-binding domain, the capping domain of vinculin is masked by an autoinhibitory interaction between Vh and Vt. In contrast to the side-binding domain, the capping domain is not unmasked by the binding of a talin domain to Vh and requires the dissociation of an additional autoinhibitory interaction. Finally, we show that vinculin and the formin mDia1, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends.     

Vinculin Regulates Osteoclast Function

Osteoclastic bone resorption depends upon the cell''s ability to organize its cytoskeleton. Because vinculin (VCL) is an actin-binding protein, we asked whether it participates in skeletal degradation. Thus, we mated VCL^fl/fl mice with those expressing cathepsin K-Cre (CtsK-VCL) to delete the gene in mature osteoclasts or lysozyme M-Cre (LysM-VCL) to target all osteoclast lineage cells. VCL-deficient osteoclasts differentiate normally but, reflecting cytoskeletal disorganization, form small actin rings and fail to effectively resorb bone. In keeping with inhibited resorptive function, CtsK-VCL and LysM-VCL mice exhibit a doubling of bone mass. Despite cytoskeletal disorganization, the capacity of VCL^−/− osteoclastic cells to normally phosphorylate c-Src in response to αvβ3 integrin ligand is intact. Thus, integrin-activated signals are unrelated to the means by which VCL organizes the osteoclast cytoskeleton. WT VCL completely rescues actin ring formation and bone resorption, as does VCL^P878A, which is incapable of interacting with Arp2/3. As expected, deletion of the VCL tail domain (VCL^1–880), which binds actin, does not normalize VCL^−/− osteoclasts. The same is true regarding VCL^I997A, which also prevents VCL/actin binding, and VCL^A50I and VCL^811–1066, both of which arrest talin association. Thus, VCL binding talin, but not Arp2/3, is critical for osteoclast function, and its selective inhibition retards physiological bone loss.     

Vinculin E29R mutation changes cellular mechanics

We investigated the effect of the point mutation E29R on vinculin under cell mechanical aspects. MEFvcl KO cells were transfected with intact eGFP-vinculin (rescue) or mutant E29R vinculin. Cellular stiffness and adhesion strength of mutant E29R vinculin were considerably higher compared to rescue and MEFvcl KO cells. 2D traction microscopy also indicated markedly higher strain energy in E29R mutant cells compared to rescue and MEFvcl KO cells. Fluorescence recovery after photobleaching showed that the recovery time for mutant E29R cells was drastically slower than for MEFvcl rescue cells and that the mobile fraction was larger for rescue compared to E29R mutant cells. These results indicate that E29R mutation might prime the vinculin head for F-actin binding, which results in higher cell stiffness, contractile force, and strengthening of focal adhesions.     

Identification and isolation of vinculin from platelets

A vinculin-like protein was identified in chicken as well as in bovine platelets by ELISA competitive binding assay using antibodies against vinculin from chicken gizzard. By a modified procedure (J. Biol. Chem. (1980) 255, 1194–1199) we succeeded in isolating bovine platelet vinculin to apparent homogeneity. The structural identity of platelet and chicken gizzard vinculin was demonstrated by circular dichroism analysis. It was also shown that platelet vinculin induces a significant decrease in the low shear viscosity of F-actin. Vinculin, in all probability, plays an important role in the organization of actin filaments in platelets, especially in the linkages of microfilaments to the membrane.