Ma L, Cantley L C, Janmey P A, Kirschner M W
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Cell Biol. 1998 Mar 9;140(5):1125-36. doi: 10.1083/jcb.140.5.1125.
Both phosphoinositides and small GTP-binding proteins of the Rho family have been postulated to regulate actin assembly in cells. We have reconstituted actin assembly in response to these signals in Xenopus extracts and examined the relationship of these pathways. We have found that GTPgammaS stimulates actin assembly in the presence of endogenous membrane vesicles in low speed extracts. These membrane vesicles are required, but can be replaced by lipid vesicles prepared from purified phospholipids containing phosphoinositides. Vesicles containing phosphatidylinositol (4,5) bisphosphate or phosphatidylinositol (3,4,5) trisphosphate can induce actin assembly even in the absence of GTPgammaS. RhoGDI, a guanine-nucleotide dissociation inhibitor for the Rho family, inhibits phosphoinositide-induced actin assembly, suggesting the involvement of the Rho family small G proteins. Using various dominant mutants of these G proteins, we demonstrate the requirement of Cdc42 for phosphoinositide-induced actin assembly. Our results suggest that phosphoinositides may act to facilitate GTP exchange on Cdc42, as well as to anchor Cdc42 and actin nucleation activities. Hence, both phosphoinositides and Cdc42 are required to induce actin assembly in this cell-free system.
磷酸肌醇和Rho家族的小GTP结合蛋白都被认为可调节细胞中的肌动蛋白组装。我们在非洲爪蟾提取物中重建了对这些信号作出反应的肌动蛋白组装，并研究了这些途径之间的关系。我们发现，在低速提取物中存在内源性膜泡的情况下，GTPγS会刺激肌动蛋白组装。这些膜泡是必需的，但可以被由含有磷酸肌醇的纯化磷脂制备的脂质体所替代。含有磷脂酰肌醇（4,5）二磷酸或磷脂酰肌醇（3,4,5）三磷酸的脂质体即使在没有GTPγS的情况下也能诱导肌动蛋白组装。RhoGDI是Rho家族的鸟嘌呤核苷酸解离抑制剂，它能抑制磷酸肌醇诱导的肌动蛋白组装，这表明Rho家族的小G蛋白参与其中。使用这些G蛋白的各种显性突变体，我们证明了Cdc42对于磷酸肌醇诱导的肌动蛋白组装是必需的。我们的结果表明，磷酸肌醇可能起到促进Cdc42上的GTP交换以及锚定Cdc42和肌动蛋白成核活性的作用。因此，在这个无细胞系统中，磷酸肌醇和Cdc42都是诱导肌动蛋白组装所必需的。
