Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes.
Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes.
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发表时间：
2000-01
影响因子：
4
通讯作者：
Z. Khayat;P. Tong;Karen Yaworsky;Robert J. Bloch;Amira Klip
中科院分区：
文献类型：
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作者：
Z. Khayat;P. Tong;Karen Yaworsky;Robert J. Bloch;Amira Klip
我们检查了胰岛素对肌动蛋白微丝的时间重组及其参与表达 myc 标记的葡萄糖转运蛋白 4 (GLUT4myc) 的 L6 肌管中信号分子和葡萄糖转运蛋白的定位。扫描电子显微镜显示胰岛素治疗后背细胞表面发生动态变形（膜皱褶）。在未刺激的细胞中，鬼笔环肽标记的肌动蛋白丝与细胞的纵轴平行。磷脂酰肌醇 3-激酶 p85 调节亚基的免疫染色呈弥漫性点状，GLUT4myc 呈核周。胰岛素治疗3分钟后，肌动蛋白重组形成结构；这些结构在 10 分钟时从肌管的背表面突出，并在 30 分钟时在肌质中凝结成不太突出的病灶。 p85 多肽在所有时间点都与这些结构共定位。细胞松弛素 D 或 latrunculin B 可以阻止肌动蛋白重塑和 p85 重新定位到肌动蛋白结构。还观察到 GLUT4myc 募集到富含肌动蛋白的投射中，但仅在胰岛素治疗 10 分钟后才观察到。无论胰岛素刺激如何，大部分 p85 和一部分 (45%) GLUT4 在 Triton X-100 不溶性材料中回收，该材料也富含肌动蛋白。相比之下，vp165（一种在形态上与 GLUT4 囊泡共定位的跨膜氨肽酶）完全可溶于胰岛素处理和对照肌管的 Triton X-100 提取物中。将显性抑制性 Rac1 (N17) 瞬时转染至 L6 肌管可阻止背侧肌动蛋白结构的形成，并阻止胰岛素诱导的 GLUT4myc 易位至细胞表面。我们认为，胰岛素依赖性肌动蛋白结构的形成促进了 PI3-K (p85) 与 GLUT4 囊泡的结合，并可能促进 GLUT4 到达细胞表面。
We examined the temporal reorganization of actin microfilaments by insulin and its participation in the localization of signaling molecules and glucose transporters in L6 myotubes expressing myc-tagged glucose transporter 4 (GLUT4myc). Scanning electron microscopy revealed a dynamic distortion of the dorsal cell surface (membrane ruffles) upon insulin treatment. In unstimulated cells, phalloidin-labeled actin filaments ran parallel to the longitudinal axis of the cell. Immunostaining of the p85 regulatory subunit of phosphatidylinositol 3-kinase was diffusely punctate, and GLUT4myc was perinuclear. After 3 minutes of insulin treatment, actin reorganized to form structures; these structures protruded from the dorsal surface of the myotubes by 10 minutes and condensed in the myoplasm into less prominent foci at 30 minutes. The p85 polypeptide colocalized with these structures at all time points. Actin remodeling and p85 relocalization to actin structures were prevented by cytochalasin D or latrunculin B. GLUT4myc recruitment into the actin-rich projections was also observed, but only after 10 minutes of insulin treatment. Irrespective of insulin stimulation, the majority of p85 and a portion (45%) of GLUT4 were recovered in the Triton X-100-insoluble material that was also enriched with actin. In contrast, vp165, a transmembrane aminopeptidase that morphologically colocalized with GLUT4 vesicles, was fully soluble in Triton X-100 extracts of both insulin-treated and control myotubes. Transient transfection of dominant inhibitory Rac1 (N17) into L6 myotubes prevented formation of dorsal actin structures and blocked insulin-induced GLUT4myc translocation to the cell surface. We propose that insulin-dependent formation of actin structures facilitates the association of PI3-K (p85) with GLUT4 vesicles and, potentially, the arrival of GLUT4 at the cell surface.