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.

Agnew, Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site. 1995, Pubmed

Agnew, Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site. 1995, Pubmed
Ashworth, ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion. 2003, Pubmed , Xenbase
Bernards, GAP control: regulating the regulators of small GTPases. 2004, Pubmed
Brozinick, Disruption of cortical actin in skeletal muscle demonstrates an essential role of the cytoskeleton in glucose transporter 4 translocation in insulin-sensitive tissues. 2004, Pubmed
Bubb, Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. 1994, Pubmed
Edwards, Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. 1999, Pubmed
Eyster, Expression of constitutively active Akt/protein kinase B signals GLUT4 translocation in the absence of an intact actin cytoskeleton. 2005, Pubmed
Foster, Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC). 2006, Pubmed
Garvey, Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance. 1998, Pubmed
Goley, The ARP2/3 complex: an actin nucleator comes of age. 2006, Pubmed
Gonzalez, Insulin-modulated Akt subcellular localization determines Akt isoform-specific signaling. 2009, Pubmed
Gonzalez, Insulin signaling diverges into Akt-dependent and -independent signals to regulate the recruitment/docking and the fusion of GLUT4 vesicles to the plasma membrane. 2006, Pubmed
Huang, The GLUT4 glucose transporter. 2007, Pubmed
Insall, Actin dynamics at the leading edge: from simple machinery to complex networks. 2009, Pubmed
Ishikura, Muscle cells engage Rab8A and myosin Vb in insulin-dependent GLUT4 translocation. 2008, Pubmed
Ishikura, Rabs 8A and 14 are targets of the insulin-regulated Rab-GAP AS160 regulating GLUT4 traffic in muscle cells. 2007, Pubmed
Ishikura, Small G proteins in insulin action: Rab and Rho families at the crossroads of signal transduction and GLUT4 vesicle traffic. 2008, Pubmed
JeBailey, Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulin-induced actin remodeling. 2004, Pubmed
JeBailey, Ceramide- and oxidant-induced insulin resistance involve loss of insulin-dependent Rac-activation and actin remodeling in muscle cells. 2007, Pubmed
Jiang, A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling. 2002, Pubmed
Jovceva, Dynamic cofilin phosphorylation in the control of lamellipodial actin homeostasis. 2007, Pubmed
Kanzaki, Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling. 2001, Pubmed
Kanzaki, Small GTP-binding protein TC10 differentially regulates two distinct populations of filamentous actin in 3T3L1 adipocytes. 2002, Pubmed , Xenbase
Khayat, Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes. 2000, Pubmed
Klip, Recruitment of GLUT-4 glucose transporters by insulin in diabetic rat skeletal muscle. 1990, Pubmed
Korobova, Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells. 2008, Pubmed , Xenbase
Krook, Sending the signal: molecular mechanisms regulating glucose uptake. 2004, Pubmed
Kurita, Molecular dissection of the mechanisms of substrate recognition and F-actin-mediated activation of cofilin-phosphatase Slingshot-1. 2008, Pubmed
Larance, The GLUT4 code. 2008, Pubmed
Machesky, Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex. 1999, Pubmed
McCarthy, Loss of cortical actin filaments in insulin-resistant skeletal muscle cells impairs GLUT4 vesicle trafficking and glucose transport. 2006, Pubmed
Meberg, Actin depolymerizing factor and cofilin phosphorylation dynamics: response to signals that regulate neurite extension. 1998, Pubmed
Miki, IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. 2000, Pubmed
Mora, An adipocentric view of signaling and intracellular trafficking. 2002, Pubmed
Moriyama, Phosphorylation of Ser-3 of cofilin regulates its essential function on actin. 1996, Pubmed
Mîinea, AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain. 2005, Pubmed
Nagata-Ohashi, A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia. 2004, Pubmed
Ng, Rapid activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes. 2008, Pubmed
Nishita, Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion. 2004, Pubmed
Nishita, Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration. 2005, Pubmed
Niu, Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis. 2003, Pubmed
Niwa, Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. 2002, Pubmed
Omata, Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes. 2000, Pubmed
Ono, Characterization of a novel cofilin isoform that is predominantly expressed in mammalian skeletal muscle. 1994, Pubmed
Ono, Mechanism of depolymerization and severing of actin filaments and its significance in cytoskeletal dynamics. 2007, Pubmed
Patel, Intracellular segregation of phosphatidylinositol-3,4,5-trisphosphate by insulin-dependent actin remodeling in L6 skeletal muscle cells. 2003, Pubmed
Patel, Cellular location of insulin-triggered signals and implications for glucose uptake. 2006, Pubmed
Peyrollier, A role for the actin cytoskeleton in the hormonal and growth-factor-mediated activation of protein kinase B. 2000, Pubmed
Pollard, Regulation of actin filament assembly by Arp2/3 complex and formins. 2007, Pubmed
Randhawa, GLUT4 vesicle recruitment and fusion are differentially regulated by Rac, AS160, and Rab8A in muscle cells. 2008, Pubmed
Ridley, The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. 1992, Pubmed
Rossman, GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. 2005, Pubmed
Ruderman, Activation of phosphatidylinositol 3-kinase by insulin. 1990, Pubmed
Sano, Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation. 2003, Pubmed
Sano, Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane. 2007, Pubmed
Shaw, Cross-reactivity of antibodies to actin- depolymerizing factor/cofilin family proteins and identification of the major epitope recognized by a mammalian actin-depolymerizing factor/cofilin antibody. 2004, Pubmed
Sidani, Cofilin determines the migration behavior and turning frequency of metastatic cancer cells. 2007, Pubmed
Soosairajah, Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin. 2005, Pubmed
Spector, Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. 1983, Pubmed
Suetsugu, Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex. 1999, Pubmed
Talior-Volodarsky, Alpha-actinin-4 is selectively required for insulin-induced GLUT4 translocation. 2008, Pubmed
Thong, The Rab GTPase-activating protein AS160 integrates Akt, protein kinase C, and AMP-activated protein kinase signals regulating GLUT4 traffic. 2007, Pubmed
Tong, Insulin-induced cortical actin remodeling promotes GLUT4 insertion at muscle cell membrane ruffles. 2001, Pubmed
Trifaró, Cytoskeletal control of vesicle transport and exocytosis in chromaffin cells. 2008, Pubmed
Tsakiridis, Disassembly of the actin network inhibits insulin-dependent stimulation of glucose transport and prevents recruitment of glucose transporters to the plasma membrane. 1994, Pubmed
Tsakiridis, Multiple roles of phosphatidylinositol 3-kinase in regulation of glucose transport, amino acid transport, and glucose transporters in L6 skeletal muscle cells. 1995, Pubmed
Török, Insulin but not PDGF relies on actin remodeling and on VAMP2 for GLUT4 translocation in myoblasts. 2004, Pubmed
Ueda, Crucial role of the small GTPase Rac1 in insulin-stimulated translocation of glucose transporter 4 to the mouse skeletal muscle sarcolemma. 2010, Pubmed
Ueyama, GLUT-4myc ectopic expression in L6 myoblasts generates a GLUT-4-specific pool conferring insulin sensitivity. 1999, Pubmed
Van Troys, Ins and outs of ADF/cofilin activity and regulation. 2008, Pubmed
Wang, Actin filaments participate in the relocalization of phosphatidylinositol3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes. 1998, Pubmed
Wang, Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. 1999, Pubmed
Weed, Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex. 2000, Pubmed
Yan, CART: an Hrs/actinin-4/BERP/myosin V protein complex required for efficient receptor recycling. 2005, Pubmed
Yang, Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. 1998, Pubmed
Zaid, Insulin action on glucose transporters through molecular switches, tracks and tethers. 2008, Pubmed
Zierath, Insulin action on glucose transport and plasma membrane GLUT4 content in skeletal muscle from patients with NIDDM. 1996, Pubmed