FIGURE 2.

Vesicles formation and secretion differs in WT-Nephrin and 3YF-Nephrin-overexpressing cells. A, cell clones were exposed to the FM dye 4-64 (red) to determine the degree of vesicle internalization at base line (time 0) and to determine the degree of vesicle secretion over time after glucose stimulation with 11 mm glucose. Although WT-Nephrin-overexpressing cells (WT-N) had higher vesicle content at time 0, red fluorescence over time was lost in WT-Nephrin clones upon glucose stimulation. In contrast, 3YF-Nephrin-overexpressing cells (3YF-N) demonstrated an impaired ability to secrete vesicles upon glucose stimulation. The graphic representation of the means ± S.D. of FM4-64 intensity measured every 2 min in four different WT-Nephrin clones when compared with four 3YF-Nephrin clones is shown. WT-Nephrin-overexpressing cells generated more vesicles at base line and were characterized by a more rapid loss of vesicles after glucose stimulation. B, WT-Nephrin- or 3YF-Nephrin-overexpressing cells were analyzed by TIRF microscopy. Changes in GFP-TIRF fluorescence intensity were monitored after either HBSS addition (CTRL) or a 16.7 mm glucose addition (glucose) at 60 s and plotted as the means ± S.D. f/f₀ of three experiments. The time point of addition is marked with an arrow. The bar graph represents the ratio of relative TIRF fluorescence intensity at 300 s between glucose-treated and untreated cells. **, p < 0.001, n = 3.