FIG.3.

GFP-PH-GRP1 protein translocates from the cytosol to the periphery of the cell and concentrates in newly formed structures revealed by time-lapse live-cell microscopy. (A) L6 myoblasts were seeded on 35-mm-diameter glass bottom microwell dishes and transiently transfected with 0.5 μg of GFP-PH-GRP1 (a to c) or GFP-PH(K273A)-GRP1 (d to f), as described in Materials and Methods. The cells were then continuously visualized at 37°C by confocal fluorescent microscopy, and representative time images taken immediately prior to the addition of insulin and 2 and 10 min thereafter are presented. The complete time-lapse movie is provided at http://www.sickkids.ca/research/custom/profiles/klip.asp. Bar, 10 μm. (B) Fluorescent signal intensities in two different regions (as shown in panel A [□ and ▵, perinuclear cytosol; ○ and ⋄, peripheral structures]) within the transfected cell were obtained and quantified. To account for possible bleaching, all measurements were normalized to the signal in the nucleus within each cell. The fluorescent intensities in the perinuclear cytosol and the periphery from two representative cells expressing the wild-type and mutant PH domains were then plotted over time. The y scale represents fluorescent intensity relative to that in the nucleus. (The dotted and solid lines represent quantifications from the left and right cells in each panel, respectively.) (C) The fluorescent intensities from 10 GFP-PH-GRP1- and 19 GFP-PH(K273A)-GRP1-expressing cells at 0, 2, and 10 min following insulin stimulation were obtained. The intensities at 2 and 10 min were then expressed relative to fluorescent intensity at time zero. *, P < 0.001 compared to time zero.