Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes

We examined the temporal reorganization of actin microfilaments by insulin and its participation in the localization of signaling molecules and glucos e transporters in L6 myotubes expressing myc-tagged glucose transporter 4 ( GLUT4myc), Scanning electron microscopy revealed a dynamic distortion of th e dorsal cell surface (membrane ruffles) upon insulin treatment. In unstimu lated cells, phalloidin-labeled actin filaments ran parallel to the longitu dinal asis of the cell. Immunostaining of the p85 regulatory subunit of pho sphatidylinositol 3-kinase was diffusely punctate, and GLUT4myc was perinuc lear, After 3 minutes of insulin treatment, actin reorganized to form struc tures; these structures protruded from the dorsal surface of the myotubes b y 10 minutes and condensed in the myoplasm into less prominent foci at 30 m inutes. The p85 polypeptide colocalized with these structures at all time p oints. Actin remodeling and p85 relocalization to actin structures were pre vented by cytochalasin D or latrunculin B. GLUT4myc recruitment into the ac tin-rich projections was also observed, but only after 10 minutes of insuli n treatment. Irrespective of insulin stimulation, the majority of p85 and a portion (45%) of GLUT4 were recovered in the Triton X-100-insoluble materi al that was also enriched with actin, In contrast, vp165, a transmembrane a minopeptidase that morphologically colocalized with GLUT4 resides, was full y soluble in Triton X-100 extracts of both insulin-treated and control myot ubes. Transient transfection of dominant inhibitory Rad (N17) into L6 myotu bes prevented formation of dorsal actin structures and blocked insulin-indu ced GLUT4myc translocation to the cell surface. We propose that insulin-dep endent formation of actin structures facilitates the association of PI3-K ( p85) with GLUT4 vesicles and, potentially, the arrival of GLUT4 at the cell surface.