Figure 3.

FRAP of mannosidase-GFP shows breakdown of continuity during differentiation. C2 cells that express man-GFP were pretreated with 25 μg/ml cycloheximide to inhibit protein synthesis and examined in the LSM 510 confocal microscope at 37°C. Each series of images shows a view before (pre), immediately after (5 s), and at different times after photobleaching. (A) Fast recovery of a 2-μm-wide band is observed in myoblasts (arrowhead). The fluorescence intensity of the bleached area was measured with NIH Image, and plotted and fitted in Kaleidagraph (see graph) to determine the diffusion coefficient (see MATERIALS AND METHODS and text). (B) In differentiated myotubes, fragments of individual elements (arrows) or whole elements (arrowheads) were photobleached. Recovery was fast for the fragments connected to a GFP source but absent for the whole elements. In C, the entire GC of a myoblast was photobleached and in D all elements of an extended area of myotubes. A slower and less complete recovery was observed, as plotted in the graph. Bars, 10 μm.