Figure 9.
Figure 9. Myosin-Va prepares insulin-responsive GLUT4 vesicles for fusion. (A–C) Myosin-1c (A), myosin II (B), and myosin-Va short tail (ST; C) tagged with mCherry were separately transfected into adipocytes together with GLUT4-EGFP, and their colocalization was examined using dual-color TIRF microscopy. Images displayed were taken before insulin stimulation. (D–F) mCherry-tagged myosin-1c, myosin II, and myosin-Va ST were separately transfected into adipocytes together with IRAP-pHluorin. The association of these myosin proteins with IRAP-pHluorin fusing vesicles was monitored using dual-color TIRF microscopy 3 min after insulin stimulation. (D) An IRAP-pHluorin fusing vesicle with myosin-Va ST associated with it. Fusion site intensities are measured from both channels and plotted in E. Also see Video 8. (F) Summary of myosin proteins’ association with insulin-stimulated IRAP-pHluorin fusing vesicles. Data are represented as mean ± SEM (error bars). Myosin-Va ST, n = 3 cells and 122 fusions; for either of myosin-1c and myosin II, 2 cells and >60 fusions were examined. To capture a sufficient number of fusion events when myosin-Va ST was expressed, cells with low expression levels of myosin-Va ST were specifically chosen. (G and H) EGFP-Rab10 and EGFP-Rab14 were separately transfected into adipocytes together with mCherry-myosin-Va ST, and their overlap was examined using dual-color TIRF microscopy 3 min after insulin stimulation. Inset panels show enlarged views of the boxed regions. See also Fig. S5. (I and J) IRAP-pHluorin was transfected alone (Control) or with mCherry-myosin-Va ST into adipocytes, and insulin-stimulated IRAP-pHluorin translocation was followed using TIRF microscopy. Insulin-stimulated IRAP translocation was indicated by TIRF image intensities (I) at different time points normalized to the intensity measured before insulin perfusion (I0 min). Images of myosin-Va ST were taken using the epifluorescence mode. To obtain the optimal inhibitory effect, cells with myosin-Va ST expressed at high levels were specifically chosen (HE, high expression). In J, data are represented as mean ± SEM (error bars). Control, n = 11 cells; Myo-Va ST HE, n = 14 cells. (K and L) GLUT4-EGFP was transfected alone (K) or with mCherry-myosin-Va ST (L) into adipocytes. TIRF microscopy images taken 3 min after insulin stimulation are displayed on the left. The effects of myosin-Va ST association on GLUT4 vesicle dynamics are presented using kymographs on the right. Cells with myosin-Va ST expressed at high levels were specifically chosen for the myosin-Va ST group. Bars: (A–C) 4 µm; (D) 0.5 µm; (G, H, and I) 4 µm; (K and L, left) 4 µm; (K and L, right) 0.5 µm.

Myosin-Va prepares insulin-responsive GLUT4 vesicles for fusion. (A–C) Myosin-1c (A), myosin II (B), and myosin-Va short tail (ST; C) tagged with mCherry were separately transfected into adipocytes together with GLUT4-EGFP, and their colocalization was examined using dual-color TIRF microscopy. Images displayed were taken before insulin stimulation. (D–F) mCherry-tagged myosin-1c, myosin II, and myosin-Va ST were separately transfected into adipocytes together with IRAP-pHluorin. The association of these myosin proteins with IRAP-pHluorin fusing vesicles was monitored using dual-color TIRF microscopy 3 min after insulin stimulation. (D) An IRAP-pHluorin fusing vesicle with myosin-Va ST associated with it. Fusion site intensities are measured from both channels and plotted in E. Also see Video 8. (F) Summary of myosin proteins’ association with insulin-stimulated IRAP-pHluorin fusing vesicles. Data are represented as mean ± SEM (error bars). Myosin-Va ST, n = 3 cells and 122 fusions; for either of myosin-1c and myosin II, 2 cells and >60 fusions were examined. To capture a sufficient number of fusion events when myosin-Va ST was expressed, cells with low expression levels of myosin-Va ST were specifically chosen. (G and H) EGFP-Rab10 and EGFP-Rab14 were separately transfected into adipocytes together with mCherry-myosin-Va ST, and their overlap was examined using dual-color TIRF microscopy 3 min after insulin stimulation. Inset panels show enlarged views of the boxed regions. See also Fig. S5. (I and J) IRAP-pHluorin was transfected alone (Control) or with mCherry-myosin-Va ST into adipocytes, and insulin-stimulated IRAP-pHluorin translocation was followed using TIRF microscopy. Insulin-stimulated IRAP translocation was indicated by TIRF image intensities (I) at different time points normalized to the intensity measured before insulin perfusion (I0 min). Images of myosin-Va ST were taken using the epifluorescence mode. To obtain the optimal inhibitory effect, cells with myosin-Va ST expressed at high levels were specifically chosen (HE, high expression). In J, data are represented as mean ± SEM (error bars). Control, n = 11 cells; Myo-Va ST HE, n = 14 cells. (K and L) GLUT4-EGFP was transfected alone (K) or with mCherry-myosin-Va ST (L) into adipocytes. TIRF microscopy images taken 3 min after insulin stimulation are displayed on the left. The effects of myosin-Va ST association on GLUT4 vesicle dynamics are presented using kymographs on the right. Cells with myosin-Va ST expressed at high levels were specifically chosen for the myosin-Va ST group. Bars: (A–C) 4 µm; (D) 0.5 µm; (G, H, and I) 4 µm; (K and L, left) 4 µm; (K and L, right) 0.5 µm.

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