Optogenetic Control Of PI?4,5?P₂ And Its Function In GLUT4 Vesicle Trafficking In Adipocytes

Phosphoinositides are important signaling molecules for vesicular trafficking,its metabolism is closely linked with the the dysfunction of insulin-regulated GLUT4 translocation and is a major cause of type 2 diabetes in humans.Phosphoinositide-4,5-diphosphate[PI(4,5)P2]has been proved to be involved in insulin regulated GLUT4 transloction in adipocytes.However,based on the common biochemical and protein knockout methods used in previous studies,the action time is long,it remains unclear where and how PI(4,5)P2 functions in insulin-stimulated GLUT4 trafficking in adipocytes.Here,we combined optogenetics with TIRFM imaging,to monitor the temporal-spatial-dependent PI(4,5)P2 regulation on discrete steps of GLUT4 translocation in 3T3-L1 adipocytes.The detailed research contents and results are as follows:1.Rapid control of phosphoinositide metabolism in single cellThe 3T3-L1 preadipocytes were differentiated into mature adipocytes and the transfection method was optimized.Combined wih optogenetic modules of CIBN/CRY2,we were able to rapidly control the PI(4,5)P2 metabolism at different intracellular organelles,such as cell membrane,mitochondria,Golgi and GLUT4 vesicle.2.Effect of rapid depletion of intracellular PI(4,5)P2 on GLUT4 vesicletransloctionUtilizing 4' phosphatase Sacl and 5' phosphatase OCRL,we were able to decrease the intracellular PI(4,5)P2 upon blue light stimulation.Combined with IF and TIRFM imaging of living cells,the effects on GLUT4 vesicle transloction were systematically analyzed:the cell membrane is an important site for the function of PI(4,5)P2,for only the decrease of PI(4,5)P2 on the cell membrane can significantly inhibit the transport of GLUT4 and at the same time will significantly inhibit the GSVs fusion process.3.Effect of subcellular control of PI(4,5)P2 on GLUT4 translocationWe used light-controlled optogenetic module to selectively regulate PI(4,5)P2 metabolism at single GLUT4 vesicle level,which showed to promote GLUT4 vesicle undocking.In addition,it was found that undocking had no effect on the insulin-stimulated phosphorylation of Akt,but significantly inhibited the docking and fusion of GLUT4 vesicles under insulin stimulation.These results illustrate the important role of PI(4,5)P2 in GLUT4 vesicle exocytosis.4.Effect of subcellular control of PI(3,4,5)P3 on GLUT4 translocationUtilizing iSH2,a combination of p 11 O? in PI(3,4,5)P3,we can enhance the PI(4,5)P2 converted into PIP3 at the GLUT4 vesicle docking site.Combined with TIRFM imaging of living cells,we found that the regulation of this site in the basal state did not affect the total PIP3 content on the cell membrane,nor did it affect the docking process or the phosphorylation level of Akt,but significantly up-regulated the transport and fusion of GLUT4 in the basal state.These results suggest that during vesicle docking,PI(4,5)P2 may be phosphorylated to PI(3,4,5)P3,to facilitate subsequent GLUT4 vesicle fusion and translocation.In this work,the regulation mechanism of PI(4,5)P2 on the transport of GLUT4 vesicles in 3T3-L1 adipocytes was thoroughly investigated,and it is found that PI(4,5)P2 on the cell membrane was crucial to the insulin signal transduction and the transport process of insulin-stimulated GLUT4 vesicles.Our results suggest that PI(4,5)P2 plays dual functions at the cell surface,in which it not only regulates the proper activation of insulin signaling in general as a precursor of PIP3 synthesis,but also controls GLUT4 vesicle docking at the vesicle-membrane contact sites.These results have deepened understanding of the mechanism of GLUT4 transport regulation by phosphoinositide and provided new ideas for similar studies by optogenetic tools in the future.