Tidball J G, Spencer M J
Department of Physiological Science, University of California, Los Angeles 90024-1527.
J Cell Biol. 1993 Nov;123(3):627-35. doi: 10.1083/jcb.123.3.627.
Modifications in the interactions of the muscle cytoskeleton with the cell membrane occur during cell growth and adaptation, although the mechanisms regulating these interactions are unknown. We have observed that myotendinous junctions (MTJs), which are the primary sites of turnover of the thin filament-membrane associations in skeletal muscle, are greatly enriched in receptors for PDGF. The high concentration of PDGF receptors at MTJs suggested to us that receptor binding may initiate cytoskeletal remodeling in skeletal muscle. We tested this possibility by examining the organization and phosphorylation of cytoskeletal components of L6 myocytes after PDGF stimulation. We have found that 10 min after PDGF stimulation, L6 myoblasts exhibit no stress fibers discernible by phalloidin binding, and that vinculin relocates from focal contacts into a diffuse cytoplasmic distribution. After 60 min of incubation, these changes are largely reversed. Indirect immunofluorescence shows that at 10-min PDGF stimulation, there are no changes in the distribution of talin, the beta 1 subunit of integrin, pp125FAK or desmin. Phosphotyrosine distribution changes upon stimulation from focal contacts to being located both in focal contacts and granules concentrated in perinuclear regions. These granules also immunolabel with anti-PDGF receptor Immunoprecipitations with anti-phosphotyrosine show that polypeptides at 180 and 230 kD show the greatest increase in tyrosine phosphorylation after PDGF stimulation. Immunoblots of anti-phosphotyrosine precipitates show that these polypeptides are the PDGF receptor and talin. We also examined the possibility that the cytoskeletal reorganization observed may result from calpain activation caused by elevated intracellular calcium induced by PDGF stimulation. However, immunoblots of control and stimulated cells show no decrease in the inactive calpain proenzyme or increase in the proteolytic, autolyzed forms of calpain pursuant to stimulation. Furthermore, stimulation produces no increase in the proportion of the 190-kD talin fragment characteristic of calpain-mediated cleavage. The retention of talin and integrin at focal contacts after talin phosphorylation, while vinculin is redistributed, indicate that phosphorylation of talin in PDGF-stimulated cells leads to separation of talin-vinculin associations but not talin-integrin associations. We propose that PDGF binding to PDGF receptors at MTJs may provide one means of regulating myofibril associations with the muscle cell membrane.
在细胞生长和适应过程中，肌肉细胞骨架与细胞膜之间的相互作用会发生改变，尽管调节这些相互作用的机制尚不清楚。我们观察到，肌腱连接点（MTJ）是骨骼肌中细肌丝 - 膜结合更新的主要部位，富含血小板衍生生长因子（PDGF）受体。MTJ处高浓度的PDGF受体让我们推测受体结合可能引发骨骼肌中的细胞骨架重塑。我们通过检测PDGF刺激后L6肌细胞细胞骨架成分的组织和磷酸化情况来验证这一可能性。我们发现，PDGF刺激10分钟后，L6成肌细胞中通过鬼笔环肽结合无法辨别出应力纤维，纽蛋白从粘着斑重新分布到弥漫性的细胞质中。孵育60分钟后，这些变化在很大程度上逆转。间接免疫荧光显示，在PDGF刺激10分钟时，踝蛋白、整合素β1亚基、pp125FAK或结蛋白的分布没有变化。磷酸酪氨酸的分布在刺激后从粘着斑改变为同时位于粘着斑和集中在核周区域的颗粒中。这些颗粒也能用抗PDGF受体进行免疫标记。用抗磷酸酪氨酸进行免疫沉淀显示，180和230 kD的多肽在PDGF刺激后酪氨酸磷酸化增加最为显著。抗磷酸酪氨酸沉淀物的免疫印迹显示这些多肽是PDGF受体和踝蛋白。我们还研究了观察到的细胞骨架重组可能是由PDGF刺激诱导的细胞内钙升高导致的钙蛋白酶激活所引起的这一可能性。然而，对照细胞和受刺激细胞的免疫印迹显示，在刺激后非活性钙蛋白酶原酶没有减少，钙蛋白酶的蛋白水解、自溶形式也没有增加。此外，刺激后钙蛋白酶介导的裂解所特有的190 - kD踝蛋白片段的比例没有增加。在踝蛋白磷酸化后，踝蛋白和整合素在粘着斑处保留，而纽蛋白重新分布，这表明在PDGF刺激的细胞中踝蛋白的磷酸化导致踝蛋白 - 纽蛋白结合的分离，但不导致踝蛋白 - 整合素结合的分离。我们提出，PDGF与MTJ处的PDGF受体结合可能是调节肌原纤维与肌肉细胞膜结合的一种方式。
