Zigmond S H, Joyce M, Yang C, Brown K, Huang M, Pring M
Department of Biology, University of Pennsylvania. Philadelphia, Pennsylvania 19104-6018, USA.
J Cell Biol. 1998 Aug 24;142(4):1001-12. doi: 10.1083/jcb.142.4.1001.
Cdc42, activated with GTPgammaS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool. Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 micron in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 micron. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.
用GTPγS激活的Cdc42可诱导裂解的中性粒细胞上清液中的肌动蛋白聚合。这种聚合作用，与激动剂诱导的聚合作用一样，需要在丝的带刺末端进行延伸。为了确定游离带刺末端的产生是否足以诱导肌动蛋白聚合，将血影蛋白-肌动蛋白种子或剪切的F-肌动蛋白丝形式的游离带刺末端添加到细胞上清液中。两者均未诱导聚合。此外，血影蛋白-肌动蛋白种子的存在并未增加Cdc42诱导的聚合速率，这表明Cdc42的存在并未促进血影蛋白-肌动蛋白种子的聚合，而如果Cdc42抑制加帽或从隔离池中释放G-肌动蛋白，情况可能就是如此。电子显微镜显示，Cdc42诱导的丝迅速伸长，在15秒内平均长度超过1微米。由血影蛋白-肌动蛋白种子形成的丝的平均长度<0.4微米。如果血影蛋白-肌动蛋白种子在被加帽之前以可比的速率伸长，它们会诱导出更长的丝。在15秒至5分钟之间，Cdc42诱导的丝的平均长度几乎没有变化，这表明这段时间内F-肌动蛋白的增加是由于丝数量的增加。这些数据表明，Cdc42诱导的肌动蛋白聚合既需要产生游离带刺末端，也需要在这些末端促进延伸。
