Phosphoinositides are important signaling molecules involved in the regulation of vesicular trafficking. It has been implicated that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is involved in insulin-regulated GLUT4 translocation in adipocytes. However, it remains unclear where and how PI(4,5)P₂ regulates discrete steps of GLUT4 vesicle translocation in adipocytes, especially on the exocytic arm of regulation. Here, we employed optogenetic tools to acutely control the PI(4,5)P₂ metabolism in living cells. By combination of TIRFM imaging, we were able to monitor the temporal-spatial-dependent PI(4,5)P₂ regulation on discrete steps of GLUT4 translocation in adipocytes. We found that the plasma membrane localized PI(4,5)P₂ is crucial for proper insulin signaling propagation and for insulin-stimulated GLUT4 vesicle translocation in 3T3-L1 adipocytes. Global depletion of PI(4,5)P₂ on the cell surface blunted insulin-stimulated Akt phosphorylation and abolished insulin effects in promotion of the docking and fusion of GLUT4 vesicle with the plasma membrane. Furthermore, by development of a novel optogenetic module to selectively modulate PI(4,5)P₂ levels on the GLUT4 vesicle docking site, we identified an important regulatory role of PI(4,5)P₂ in controlling of vesicle docking process. Local depletion of PI(4,5)P₂ at the vesicle docking site promoted GLUT4 vesicle undocking, diminished insulin-stimulated GLUT4 vesicle docking and fusion, but without perturbation of insulin signaling propagation in adipocytes. Our results provide strong evidence that cell surface PI(4,5)P₂ plays two distinct functions on regulation of the exocytic trafficking of GLUT4 in adipocytes. PI(4,5)P₂ not only regulates the proper activation of insulin signaling in general but also controls GLUT4 vesicle docking process at the vesicle-membrane contact sites.

磷脂酰肌醇是参与调节水泡运输的重要信号分子。已经暗示磷脂酰肌醇4，5-二磷酸[PI（4，5）P ₂ ]参与脂肪细胞中胰岛素调节的GLUT4易位。但是，尚不清楚PI（4,5）P ₂在哪里以及如何调节脂肪细胞中GLUT4囊泡易位的离散步骤，尤其是在调节的胞外臂上。在这里，我们采用了光遗传学工具来急性控制活细胞中的PI（4,5）P ₂代谢。通过组合的TIRFM成像，我们能够监测时空依赖的PI（4,5）P ₂调节脂肪细胞中GLUT4易位的离散步骤。我们发现质膜定位PI（4,5）P₂对于3T3-L1脂肪细胞中正确的胰岛素信号传导传播和胰岛素刺激的GLUT4囊泡转运至关重要。细胞表面PI（4,5）P _2的整体耗竭减弱了胰岛素刺激的Akt磷酸化，并废除了胰岛素促进GLUT4囊泡与质膜融合和融合的作用。此外，通过开发新型的光遗传模块来选择性地调节GLUT4囊泡停靠位点上的PI（4,5）P ₂水平，我们确定了PI（4,5）P ₂在控制囊泡停靠过程中的重要调节作用。PI（4,5）P _2的局部耗竭在囊泡对接位点促进了GLUT4囊泡的对接，减少了胰岛素刺激的GLUT4囊泡的对接和融合，但没有干扰胰岛素信号在脂肪细胞中的传播。我们的结果提供了有力的证据，表明细胞表面PI（4,5）P ₂在调节脂肪细胞中GLUT4的胞外运输中起着两个不同的功能。PI（4,5）P ₂不仅通常调节胰岛素信号的适当激活，而且还控制GLUT4囊泡膜接触部位的对接过程。