Kolega J, Kumar S
Department of Anatomy and Cell Biology, State University of New York at Buffalo, School of Medicine and Biomedical Sciences 14214, USA.
Cell Motil Cytoskeleton. 1999;43(3):255-68. doi: 10.1002/(SICI)1097-0169(1999)43:3<255::AID-CM8>3.0.CO;2-T.
During the crawling movements of non-muscle cells, myosin II-containing structures assemble and disassemble with a high degree of spatial and temporal heterogeneity. In order to understand how this is controlled, we examined factors that influence the association of myosin II with detergent-resistant cytoskeletons of cultured endothelial cells. Treatment of cells with 0.05% Triton X-100 in an actin-stabilizing buffer released approximately 42% of the myosin II from the cytoplasm. Most remaining myosin II was dissociated from the cytoskeleton by treatment with ATP or AMPPNP, but not ADP, suggesting that myosin II is retained as ATP-sensitive filaments or via rigor-like binding to F-actin. Disruption of actin filaments with cytochalasin or latrunculin prior to detergent permeabilization sharply decreased the amount of myosin II retained, suggesting the latter type of association. Because phosphorylation of myosin II affects filament assembly and actin binding in vitro, phosphorylation levels in soluble and cytoskeletal myosin II were measured. Phosphorylation of myosin heavy chains was not significantly different between the two fractions, but regulatory light chains of cytoskeletal myosin II were 5 times more phosphorylated than in soluble myosin II. Tryptic-peptide mapping showed that cytoskeletal light chains were phosphorylated predominantly at serine 19/threonine 18, which regulates myosin II assembly in vitro, whereas soluble light chains were not phosphorylated or were phosphorylated at threonine 9. Treating cells with the kinase inhibitor, staurosporine, prior to permeabilization decreased light-chain phosphorylation with concomitant reduction in myosin retention. These observations suggest that assembly of myosin II into cytoskeletal structures, where it can generate and resist forces, is regulated in vivo by phosphorylation of myosin light chains at serine 19/threonine 18.
在非肌肉细胞的爬行运动过程中，含肌球蛋白II的结构以高度的空间和时间异质性进行组装和拆卸。为了了解这一过程是如何被控制的，我们研究了影响肌球蛋白II与培养的内皮细胞抗去污剂细胞骨架结合的因素。在肌动蛋白稳定缓冲液中用0.05%的 Triton X-100处理细胞，可使约42%的肌球蛋白II从细胞质中释放出来。大多数剩余的肌球蛋白II通过ATP或AMPPNP处理从细胞骨架上解离，但ADP不能使其解离，这表明肌球蛋白II以对ATP敏感的细丝形式保留，或通过类似僵直的方式与F-肌动蛋白结合。在用去污剂通透细胞之前，用细胞松弛素或拉春库林破坏肌动蛋白丝，会显著减少保留的肌球蛋白II的量，提示存在后一种结合方式。由于肌球蛋白II的磷酸化在体外会影响细丝组装和与肌动蛋白的结合，因此我们测量了可溶性和细胞骨架肌球蛋白II中的磷酸化水平。两个组分中肌球蛋白重链的磷酸化没有显著差异，但细胞骨架肌球蛋白II的调节轻链的磷酸化程度比可溶性肌球蛋白II高5倍。胰蛋白酶肽图谱显示，细胞骨架轻链主要在丝氨酸19/苏氨酸18处磷酸化，这在体外调节肌球蛋白II的组装，而可溶性轻链未磷酸化或在苏氨酸9处磷酸化。在通透细胞之前用激酶抑制剂星形孢菌素处理细胞，会降低轻链磷酸化，并伴随肌球蛋白保留量的减少。这些观察结果表明，肌球蛋白II组装到细胞骨架结构中（在其中它可以产生和抵抗力），在体内是由肌球蛋白轻链在丝氨酸19/苏氨酸18处的磷酸化调节的。
