Cano M L, Cassimeris L, Fechheimer M, Zigmond S H
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
J Cell Biol. 1992 Mar;116(5):1123-34. doi: 10.1083/jcb.116.5.1123.
While actin polymerization and depolymerization are both essential for cell movement, few studies have focused on actin depolymerization. In vivo, depolymerization can occur exceedingly rapidly and in a spatially defined manner: the F-actin in the lamellipodia depolymerizes in 30 s after chemoattractant removal (Cassimeris, L., H. McNeill, and S. H. Zigmond. 1990. J. Cell Biol. 110:1067-1075). To begin to understand the regulation of F-actin depolymerization, we have examined F-actin depolymerization in lysates of polymorphonuclear leukocytes (PMNs). Surprisingly, much of the cell F-actin, measured with a TRITC-phalloidin-binding assay, was stable after lysis in a physiological salt buffer (0.15 M KCl): approximately 50% of the F-actin did not depolymerize even after 18 h. This stable F-actin included lamellar F-actin which could still be visualized one hour after lysis by staining with TRITC-phalloidin and by EM. We investigated the basis for this stability. In lysates with cell concentrations greater than 10(7) cells/ml, sufficient globular actin (G-actin) was present to result in a net increase in F-actin. However, the F-actin stability was not solely because of the presence of free G-actin since addition of DNase I to the lysate did not increase the F-actin loss. Nor did it appear to be because of barbed end capping factors since cell lysates provided sites for barbed end polymerization of exogenous added actin. The stable F-actin existed in a macromolecular complex that pelleted at low gravitational forces. Increasing the salt concentration of the lysis buffer decreased the amount of F-actin that pelleted at low gravitational forces and increased the amount of F-actin that depolymerized. Various actin-binding and cross-linking proteins such as tropomyosin, alpha-actinin, and actin-binding protein pelleted with the stable F-actin. In addition, we found that alpha-actinin, a filament cross-linking protein, inhibited the rate of pyrenyl F-actin depolymerization. These results suggested that actin cross-linking proteins may contribute to the stability of cellular actin after lysis. The activity of crosslinkers may be regulated in vivo to allow rapid turnover of lamellipodia F-actin.
虽然肌动蛋白的聚合和解聚对细胞运动都至关重要，但很少有研究关注肌动蛋白的解聚。在体内，解聚可以极其迅速地以空间限定的方式发生：趋化因子去除后30秒内，片状伪足中的F-肌动蛋白就会解聚（卡西梅里斯，L.，H. 麦克尼尔，和S. H. 齐格蒙德。1990。《细胞生物学杂志》110：1067 - 1075）。为了开始理解F-肌动蛋白解聚的调控机制，我们检测了多形核白细胞（PMN）裂解物中的F-肌动蛋白解聚情况。令人惊讶的是，用TRITC-鬼笔环肽结合试验测量的细胞中大部分F-肌动蛋白，在生理盐缓冲液（0.15 M KCl）中裂解后是稳定的：即使在18小时后，仍有约50%的F-肌动蛋白没有解聚。这种稳定的F-肌动蛋白包括片状F-肌动蛋白，在裂解1小时后，通过用TRITC-鬼笔环肽染色和电子显微镜仍可观察到。我们研究了这种稳定性的基础。在细胞浓度大于10⁷个细胞/ml 的裂解物中，存在足够量的球状肌动蛋白（G-肌动蛋白），导致F-肌动蛋白净增加。然而，F-肌动蛋白的稳定性并非仅仅因为游离G-肌动蛋白的存在，因为向裂解物中添加DNase I并没有增加F-肌动蛋白的损失。这似乎也不是由于带刺末端封端因子，因为细胞裂解物为外源添加肌动蛋白的带刺末端聚合提供了位点。稳定的F-肌动蛋白存在于一个在低重力下会沉淀的大分子复合物中。增加裂解缓冲液的盐浓度会减少在低重力下沉淀的F-肌动蛋白量，并增加解聚的F-肌动蛋白量。各种肌动蛋白结合和交联蛋白，如原肌球蛋白、α-辅肌动蛋白和肌动蛋白结合蛋白，与稳定的F-肌动蛋白一起沉淀。此外，我们发现α-辅肌动蛋白，一种细丝交联蛋白，抑制了芘基F-肌动蛋白的解聚速率。这些结果表明，肌动蛋白交联蛋白可能有助于裂解后细胞肌动蛋白的稳定性。交联剂的活性可能在体内受到调控，以使片状伪足F-肌动蛋白能够快速周转。
