Rozelle A L, Machesky L M, Yamamoto M, Driessens M H, Insall R H, Roth M G, Luby-Phelps K, Marriott G, Hall A, Yin H L
Departments of Physiology and Biochemistry, University of Texas Southwestern Medical Center, Dallas 75390, USA.
Curr Biol. 2000 Mar 23;10(6):311-20. doi: 10.1016/s0960-9822(00)00384-5.
Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements.
Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation.
Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.
磷脂酰肌醇4,5 - 二磷酸（PIP₂）与肌动蛋白细胞骨架的调节和囊泡运输有关。它通过激活涉及威斯科特 - 奥尔德里奇综合征蛋白（WASP）和肌动蛋白相关蛋白复合物Arp2/3的途径来刺激肌动蛋白的从头聚合。其他研究表明，肌动蛋白从称为“脂筏”的富含胆固醇 - 鞘脂的膜微区聚合，其方式依赖于酪氨酸磷酸化。尽管肌动蛋白与囊泡运输有关，且脂筏是介导顶端定向囊泡运输的活性磷酸肌醇和酪氨酸激酶信号传导的位点，但尚不清楚肌动蛋白动力学的磷酸肌醇调节是否发生在脂筏中，或者它是否与囊泡运动相关。
合成PIP₂的I型磷脂酰肌醇磷酸5 - 激酶（PIP5KI）的过表达促进了膜结合囊泡的肌动蛋白聚合，形成可移动的肌动蛋白彗星。钒酸钠（PV），一种酪氨酸磷酸酶抑制剂，即使在没有PIP5KI过表达的情况下也能诱导彗星形成。PV增加了PIP₂水平，表明它通过改变PIP₂稳态和增加酪氨酸磷酸化来诱导彗星形成。血小板衍生生长因子（PDGF）增强了PV诱导的彗星形成，并且这些刺激共同增强了PIP5KI的作用。彗星头部的囊泡富含PIP5KIs和酪氨酸磷酸化蛋白。使用显性负性WASP构建体确定了WASP - Arp2/3的参与。内吞和外排标记物将富含脂筏的囊泡鉴定为彗星形成的优先位点。用甲基 - β - 环糊精提取胆固醇减少了彗星形成，证实脂筏促进彗星形成。
鞘脂 - 胆固醇脂筏是膜连接肌动蛋白聚合的优选平台。这是由原位PIP₂合成和通过WASP - Arp2/3途径的酪氨酸激酶信号传导介导的。肌动蛋白彗星可能为脂筏依赖性囊泡运输和顶端膜运输提供一种新机制。
