Figure 2. Full versus partial release of... | Rockefeller University Press

Figure 2.

$Figure 2. Full versus partial release of IRAP-pHluorin vesicles at the PM. (A and B) IRAP-pHluorin was transfected into adipocytes. Vesicle fusions at the PM were captured using TIRF microscopy 3 min after insulin stimulation, and fusion rates were quantified. Adipocytes showing no vesicle fusion before insulin stimulation were preferentially chosen, as they usually responded to insulin very well, producing many fusion events after stimulation. (A) The maximum projections of the subtraction image stacks of two videos acquired from the same cell before and 3 min after insulin stimulation. The subtraction image stacks are generated with an interval of 1 frame; each individual fusion event is, therefore, represented by one bright spot in the projection. Bar, 4 µm. (B) The data are represented as mean ± SEM (error bars), n = 3 cells. (C–H) Adipocytes were cotransfected with IRAP-pHluorin and GLUT4-mCherry, and fusion events were analyzed using dual-color TIRF microscopy 3 min after insulin stimulation. (C) A full release event. Intensities measured from the fusion site and the adjacent annulus (see Materials and methods and Fig. S3) are plotted in E. IRAP and GLUT4 were completely released from the vesicle after fusion, with the fusion site intensities having already returned to the background level when the lateral diffusion stopped, as indicated by the annulus intensities dropping back to the background. Bar, 0.5 µm. (D) A partial release event. Intensities measured from the fusion site and the adjacent annulus are plotted in F. Only a small fraction of IRAP and GLUT4 were released from the vesicle during fusion; therefore, GLUT4 intensity of the vesicle was still above the background when annulus intensities of IRAP and GLUT4 returned to the baseline, which indicates closure of the fusion pore and attenuation of lateral diffusion. IRAP-pHluorin intensity of the vesicle had already dropped back to the background level at the time because of vesicular lumen reacidification while GLUT4-mCherry intensity persisted. Bar, 0.5 µm. (G) Summary of full versus partial releases of IRAP-pHluorin vesicles at the PM in adipocytes under insulin stimulation. Data are represented as mean ± SEM, n = 3 cells and 107 fusions. See also Video 1. (H) The presence of GLUT4-mCherry on insulin-stimulated IRAP-pHluorin fusing vesicles. Data are represented as mean ± SEM (error bars), n = 3 cells and 117 fusions.$

Full versus partial release of IRAP-pHluorin vesicles at the PM. (A and B) IRAP-pHluorin was transfected into adipocytes. Vesicle fusions at the PM were captured using TIRF microscopy 3 min after insulin stimulation, and fusion rates were quantified. Adipocytes showing no vesicle fusion before insulin stimulation were preferentially chosen, as they usually responded to insulin very well, producing many fusion events after stimulation. (A) The maximum projections of the subtraction image stacks of two videos acquired from the same cell before and 3 min after insulin stimulation. The subtraction image stacks are generated with an interval of 1 frame; each individual fusion event is, therefore, represented by one bright spot in the projection. Bar, 4 µm. (B) The data are represented as mean ± SEM (error bars), n = 3 cells. (C–H) Adipocytes were cotransfected with IRAP-pHluorin and GLUT4-mCherry, and fusion events were analyzed using dual-color TIRF microscopy 3 min after insulin stimulation. (C) A full release event. Intensities measured from the fusion site and the adjacent annulus (see Materials and methods and Fig. S3) are plotted in E. IRAP and GLUT4 were completely released from the vesicle after fusion, with the fusion site intensities having already returned to the background level when the lateral diffusion stopped, as indicated by the annulus intensities dropping back to the background. Bar, 0.5 µm. (D) A partial release event. Intensities measured from the fusion site and the adjacent annulus are plotted in F. Only a small fraction of IRAP and GLUT4 were released from the vesicle during fusion; therefore, GLUT4 intensity of the vesicle was still above the background when annulus intensities of IRAP and GLUT4 returned to the baseline, which indicates closure of the fusion pore and attenuation of lateral diffusion. IRAP-pHluorin intensity of the vesicle had already dropped back to the background level at the time because of vesicular lumen reacidification while GLUT4-mCherry intensity persisted. Bar, 0.5 µm. (G) Summary of full versus partial releases of IRAP-pHluorin vesicles at the PM in adipocytes under insulin stimulation. Data are represented as mean ± SEM, n = 3 cells and 107 fusions. See also Video 1. (H) The presence of GLUT4-mCherry on insulin-stimulated IRAP-pHluorin fusing vesicles. Data are represented as mean ± SEM (error bars), n = 3 cells and 117 fusions.