title: Arp2/3- and cofilin-coordinated actin dynamics is required for insulin-mediated GLUT4 translocation to the surface of muscle cells.
abstract: GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.
authors.affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: Tim Ting
authors.initials: TT
authors.lastname: Chiu
authors.forename: Nish
authors.initials: N
authors.lastname: Patel
authors.forename: Alisa E
authors.initials: AE
authors.lastname: Shaw
authors.forename: James R
authors.initials: JR
authors.lastname: Bamburg
authors.forename: Amira
authors.initials: A
authors.lastname: Klip
mesh_terms: D051339:Actin Depolymerizing Factors / Q000378:metabolism*; D051376:Actin-Related Protein 2-3 Complex / Q000172:deficiency / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D015180:Electrophoresis, Gel, Two-Dimensional; D055785:Gene Knockdown Techniques; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008840:Microfilament Proteins / Q000378:metabolism; D032342:Muscle Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D014168:Transferrin / Q000378:metabolism; D014981:Xenopus
chemical_list: D051339:Actin Depolymerizing Factors; D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D051275:Glucose Transporter Type 4; D007328:Insulin; D008840:Microfilament Proteins; C495694:Slc2a4 protein, rat; D014168:Transferrin; D049452:Green Fluorescent Proteins; C529152:SSH1 protein, rat; D010744:Phosphoric Monoester Hydrolases