Min. As shown in Figure 2B, Lat B pretreatment prevented insulinmediated

Min. As shown in Figure 2B, Lat B pretreatment prevented insulinmediated actin 78919-13-8 purchase 10236-47-2 remodeling and resulted in complete dispersal of nexilin. Moreover, disassembly of the actin cytoskeleton coincided with diminished Akt activation as determined by the intensity ofthe Ser 473 Akt phosphorylation signal (Fig. 2C). These results suggest that the spatial patterning of nexilin is linked to actin remodeling induced by insulin. We next tested the effect of Lat B treatment on IRS1-nexilin interactions. Interestingly, while exposure of L6 myotubes to Lat B was without effect on insulin-induced IRS1 tyrosine phosphory-Nexilin Binds and Regulates IRSFigure 3. Insulin-induced dissociation of IRS1/nexilin complex is dependent on F-actin remodeling. Left panel, IRS1 was immunoprecipitated from L6 myotubes that were either starved or insulin stimulated (100 nM) for the indicated times. Immune complexes were probed with anti-phosphotyrosine 4G10 or nexilin abs. WCL, whole cell lysates; Right Panel, Latrunculin B (20 mM, 20 min) or Jaspakinolide (2 mM, 30 min) treatment of L6 myotubes is without effect on the 18325633 phosphorylation status of IRS1 but inhibits insulin-induced IRS1/nexilin disassembly. doi:10.1371/journal.pone.0055634.glation, Lat B treatment blocked the disassembly of the IRS1/ nexilin complex in response to insulin, suggesting that efficient dissociation of this signaling complex is dependent on dynamic reorganization of the actin network (Fig. 3). This result prompted the assessment of actin filament stabilization on IRS1-nexilin interactions. To this end, jasplakinolide, which stabilizes F-actin filaments by inhibiting filament disassembly was used to treat L6 myotubes at the end of the starvation period. As with Lat B treatment, jasplakinolide pre-treatment had no effect on IRS1 tyrosine phosphorylation but was seen to mitigate insulin-induced disassociation of the IRS1/nexilin complex. Together, these results are consistent with the notion that insulin-elicited actin remodeling dynamically regulates IRS1?nexilin interactions. To study the functional requirement for nexilin in insulindependent signaling in skeletal muscle cells we began by assessing the effects of siRNA knockdown of nexilin (siNex) on the tyrosine phosphorylation status of IRS1 in response to insulin. As shown in Figure 4A disassembly of the IRS1/nexilin signaling complex correlated temporally with induction of IRS1 phosphorylation in response to insulin exposure, however, siRNA-mediated silencing of nexilin in L6 myotubes did not appear to have any discernible effects on the phosphotyrosine levels of IRS1 in cells incubated with maximal concentrations (100 nM) of insulin. We next sought to evaluate IRS1 tyrosine phosphorylation status under a submaximal dose (10 nM) of insulin. Interestingly, we found that silencing of nexilin under these conditions led to enhancedassociation of the p85/IRS1 signaling complex at earlier time points in the absence of any changes in IRS1 tyrosine phosphorylation (Fig. 4B). Thus our data would appear to indicate that the temporal release of IRS1 from nexilin/cytoskeletal scaffolds increases its coupling efficiency to downstream signalling intermediates. To test this idea, we assessed the effect of nexilin knockdown on PI3K activation and phosphatidylinositol-3,4,5triphosphate (PIP3) production using single cell assays. To this end we made use of a fluorescent indicator consisting of green fluorescent protein (GFP)-tag fused to the pleckstrin homol.Min. As shown in Figure 2B, Lat B pretreatment prevented insulinmediated actin remodeling and resulted in complete dispersal of nexilin. Moreover, disassembly of the actin cytoskeleton coincided with diminished Akt activation as determined by the intensity ofthe Ser 473 Akt phosphorylation signal (Fig. 2C). These results suggest that the spatial patterning of nexilin is linked to actin remodeling induced by insulin. We next tested the effect of Lat B treatment on IRS1-nexilin interactions. Interestingly, while exposure of L6 myotubes to Lat B was without effect on insulin-induced IRS1 tyrosine phosphory-Nexilin Binds and Regulates IRSFigure 3. Insulin-induced dissociation of IRS1/nexilin complex is dependent on F-actin remodeling. Left panel, IRS1 was immunoprecipitated from L6 myotubes that were either starved or insulin stimulated (100 nM) for the indicated times. Immune complexes were probed with anti-phosphotyrosine 4G10 or nexilin abs. WCL, whole cell lysates; Right Panel, Latrunculin B (20 mM, 20 min) or Jaspakinolide (2 mM, 30 min) treatment of L6 myotubes is without effect on the 18325633 phosphorylation status of IRS1 but inhibits insulin-induced IRS1/nexilin disassembly. doi:10.1371/journal.pone.0055634.glation, Lat B treatment blocked the disassembly of the IRS1/ nexilin complex in response to insulin, suggesting that efficient dissociation of this signaling complex is dependent on dynamic reorganization of the actin network (Fig. 3). This result prompted the assessment of actin filament stabilization on IRS1-nexilin interactions. To this end, jasplakinolide, which stabilizes F-actin filaments by inhibiting filament disassembly was used to treat L6 myotubes at the end of the starvation period. As with Lat B treatment, jasplakinolide pre-treatment had no effect on IRS1 tyrosine phosphorylation but was seen to mitigate insulin-induced disassociation of the IRS1/nexilin complex. Together, these results are consistent with the notion that insulin-elicited actin remodeling dynamically regulates IRS1?nexilin interactions. To study the functional requirement for nexilin in insulindependent signaling in skeletal muscle cells we began by assessing the effects of siRNA knockdown of nexilin (siNex) on the tyrosine phosphorylation status of IRS1 in response to insulin. As shown in Figure 4A disassembly of the IRS1/nexilin signaling complex correlated temporally with induction of IRS1 phosphorylation in response to insulin exposure, however, siRNA-mediated silencing of nexilin in L6 myotubes did not appear to have any discernible effects on the phosphotyrosine levels of IRS1 in cells incubated with maximal concentrations (100 nM) of insulin. We next sought to evaluate IRS1 tyrosine phosphorylation status under a submaximal dose (10 nM) of insulin. Interestingly, we found that silencing of nexilin under these conditions led to enhancedassociation of the p85/IRS1 signaling complex at earlier time points in the absence of any changes in IRS1 tyrosine phosphorylation (Fig. 4B). Thus our data would appear to indicate that the temporal release of IRS1 from nexilin/cytoskeletal scaffolds increases its coupling efficiency to downstream signalling intermediates. To test this idea, we assessed the effect of nexilin knockdown on PI3K activation and phosphatidylinositol-3,4,5triphosphate (PIP3) production using single cell assays. To this end we made use of a fluorescent indicator consisting of green fluorescent protein (GFP)-tag fused to the pleckstrin homol.