Pharmacological inhibition of dynamin GTP-activity reduces GLUT4 insertion in the plasma membrane of RCMH myoblasts. Cultured RCMH myoblasts were transfected with the GFP-GLUT4-HA construct and 48 ± 2 h later were stimulated for 5 min with 20 µM ionomycin in the presence of 20 µM dynole 31-2, 20 µM dynole 34-2, 4 µM CytoD or the vehicle DMSO at 37 °C, fixed, immunolabeled with an anti-HA antibody without permeabilization, incubated with a Cy3-conjugated antibody and visualized by TIRF microscopy. The plasma membrane insertion of GLUT4 was quantified as a ratio between Cy3 (red) and GFP (green) signals in the evanescent field. ( a ) Schematic representation of the GFP-GLUT4-HA construct; GFP is located in the N-terminal intracellular loop and the exofacial hemaglutinin (HA)-tag in the first extracellular loop. ( b ) Examples of TIRF images of non-treated cells under resting (upper panels) or stimulated (bottom panels) conditions (250 × 250 pixels images are shown). Note that ionomycin induces GLUT4 translocation, as the HA (Cy3) signal in the evanescent field becomes detectable (red spots). ( d ) 250 × 250 pixels representative images of RCMH cells stimulated in the presence of DMSO, CytoD, dynole 31-2 or dynole 34-2. Cell periphery is drawn in white. ( e ) HA (Cy3)/GFP ratio in the evanescent field is plotted for each experimental condition. Note that, as CytoD does, dynole 34-2 significantly inhibits the stimulus-dependent plasma membrane insertion of GLUT4. Data are means ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbol * denote significance compared to the respective resting condition; and # symbols denote significance compared to ionomycin-stimulated cells treated with DMSO and Dynole 31-2, respectively. N is between 20 and 29 cells, from at least three different cultures per experimental condition. Scale bar = 10 µm.
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