Chiu TT, Patel N, Shaw AE, Bamburg JR, Klip A.

MOT 7307 Canadian Institutes of Health Research , NS40371 NINDS NIH HHS

	Figure 1. Down-regulation of Arp3 prevents insulin-induced actin remodeling and reduces GLUT4 translocation. (A) L6GLUT4myc myoblasts were transiently transfected with Arp3-GFP. After 3-h serum starvation, cells were stimulated with 100 nM insulin for 10 min. Subsequent staining of F-actin by rhodamine-phalloidin was performed to observe the changes in the localization of Arp3-GFP with respect to the remodeled actin. Representative images of four independent experiments are shown. Bars, 20 μm. (B) Myoblasts were transfected with 200 nM of nonrelated (NR) siRNA control or Arp3 siRNA for 72 h. Total cell lysates were prepared, and 10 μg protein was loaded and immunoblotted for Arp3 and actinin-1 (as loading control). Representative blots of five independent experiments are shown. (C) Myoblasts transfected with NR or Arp3 siRNA were treated with/without insulin for 10 min followed by staining surface GLUT4myc in nonpermeabilized cells and then permeabilized to label actin filaments with rhodamine-phalloidin. Dorsal actin remodeling was calculated from the pixel quantification in fluorescence optical cuts of the dorsal surface of adhered myoblasts (see Materials and Methods). Representative images of three independent experiments are shown. Bars, 20 μm. (D) Quantification of changes in insulin-stimulated dorsal actin remodeling relative to NR control (mean ± SE). (E) Quantification of fold increases in surface GLUT4myc relative to NR basal in NR and Arp3 knockdown conditions (mean ± SE, #p < 0.05).
	Figure 2. Down-regulation of p34 inhibits the formation of remodeled actin and decreases GLUT4 translocation. (A) L6GLUT4myc myoblasts were transfected with 200 nM of nonrelated (NR) siRNA control or p34 siRNA for 72 h. Total cell lysates were prepared, and 10 μg protein was loaded and immunoblotted for p34 and actinin-1 (as loading control). Representative blots of five independent experiments are shown. (B) Myoblasts transfected with NR or p34 siRNA were treated with/without insulin for 10 min followed by costaining of surface GLUT4myc in the nonpermeabilized state and then permeabilized to label actin filaments with rhodamine-phalloidin. Representative images of five independent experiments are shown. Bars, 20 μm. (C) Quantification of changes in insulin-stimulated dorsal actin remodeling relative to NR control (mean ± SE). (D) Quantification of fold increases in surface GLUT4myc relative to NR basal in NR and p34 knockdown conditions (mean ± SE, #p < 0.05). (E) p34 knockdown abolishes cortical actin remodeling in rounded-up myoblasts and prevents gain in surface GLUT4myc in response to insulin. Bars, 10 μm.
	Figure 3. 2D Western blots of ADF/cofilin from which the relative percent of cofilin, ADF, phosphorylated-cofilin, and phosphorylated ADF can be calculated. Basal and insulin-stimulated L6GLUT4myc myoblasts were lysed and subjected to 2D gel electrophoresis and assessment of ratios of ADF, cofilin, and their phosphorylated versions, essentially as described (Shaw et al., 2004). (A) Representative gel. Faint spot midway between cofilin and phospho-cofilin probably represents the cofilin-2 isoform. (B) Quantification of three experiments.
	Figure 4. Insulin stimulation in L6GLUT4myc muscle cells causes dephosphorylation of cofilin that is dependent on SSH1. L6GLUT4myc myoblasts were serum-starved for 3 h before insulin stimulation (100 nM) for the indicated time periods. (A) Total lysates immunoblotted for P-cofilin, cofilin, P-Akt, and actin (as loading control). Representative blots of four experiments are shown. (B) Quantification of insulin-dependent cofilin dephosphorylation expressed as P-Cofilin/Cofilin ratio relative to time 0 (n = 4, mean ± SE, #p < 0.05). (C) Lysates from myoblasts treated with SSH1 siRNA (siSSH1) were prepared to determine the knockdown effect on SSH1 and its contribution toward insulin-induced cofilin dephosphorylation and Akt phosphorylation by immunoblotting for P-cofilin and p-Akt (Ser-473). Myoblasts were (D) transfected with p34 siRNA or (E) subjected to 250 nM LB treatment for 30 min, and the effect of insulin on P-Cofilin was assessed. Representative blots of more than three independent experiments are shown.
	Figure 5. Cofilin localizes with insulin-induced remodeled actin and down-regulation of cofilin increases F-actin aggregates and reduces insulin-induced GLUT4 translocation. (A) L6GLUT4myc myoblasts were serum-starved for 3 h and stimulated with 100 nM insulin for 10 min, followed by labeling with rhodamine-phalloidin for F-actin and cofilin-specific antibody for endogenous cofilin. Representative images of three independent experiments are shown. Bars, 10 μm. (B) Total lysates from myoblasts transfected with NR or cofilin siRNA were prepared and immunoblotted for cofilin and actinin-1 (loading control). Representative blots of five independent experiments are shown. (C) Myoblasts with siNR or siCofilin were treated with/without insulin followed by costaining of surface GLUT4myc and F-actin. Representative images of four independent experiments are shown. Bars, 20 μm. (D) Quantification of dorsal polymerized actin relative to NR basal after cofilin knockdown (mean ± SE). (E) Quantification of fold increases in surface GLUT4myc relative to NR basal in NR and siCofilin conditions (mean ± SE, #p < 0.05).
	Figure 6. Knockdown of either p34 or cofilin does not alter Tf recycling. L6GLUT4myc myoblasts were treated with NR, p34, or cofilin siRNA. After 3-h serum starvation, to 125I-Tf recycling was determined as described in Materials and Methods, in basal (left)and insulin-stimulated (right) conditions. Tf recycling is displayed as the ratio of externalized Tf to internalized Tf (mean ± SD).
	Figure 7. Expression of Xenopus cofilin-WT-GFP, but not cofilin-S3E-GFP mutant, restores normal F-actin morphology and GLUT4 translocation. (A) L6GLUT4myc myoblasts transfected with NR or cofilin siRNA were further transfected with cDNA to either GFP as control, cofilin-WT-GFP, or cofilin-S3E-GFP. After serum starvation, F-actin was labeled with rhodamine-phalloidin in the basal state to detect changes in actin morphology. Representative images of six independent experiments are shown. Bars, 20 μm. (B) Quantification of F-actin aggregates relative to NR+GFP basal (mean ± SE). (C) Surface GLUT4myc levels in siCofilin-treated myoblasts cotransfected with cofilin-WT or S3E mutant expression in siCofilin myoblasts were measured by fluorescent detection of single cells and are presented as fold increases in surface GLUT4myc relative to NR basal are also shown (mean ± SE, n = 6, #p < 0.05).
	Figure 8. Proposed mechanism of insulin-regulated actin dynamics in muscle cells. Insulin stimulation in muscle cells promotes dynamic actin remodeling. The formation of the remodeled actin is achieved by the polymerization activity of Arp2/3 acting downstream of active Rac. The accumulation of polymerized F-actin poses a stimulatory factor in the phosphatase activity of SSH, which leads to net dephosphorylation and activation of cofilin. Hereon, the actin-severing function of cofilin maintains the flexibility of remodeled actin and enables regeneration of free monomeric actin for further polymerization. This active cycling of actin mediated by insulin keeps proper actin dynamics at the cortical zone in order to facilitate GLUT4 insertion onto the cellular surface.

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