Fig. 5. Quantitative analysis of insulin-containing vesicle movements in KHC^mut-expressing and control cells. (A) Cells were co-transfected with KHC^mut-pAdTrack-CMV and phogrin.DsRed. 48 hours after transfection cells were imaged at a stimulatory glucose concentration (16 mM) on a confocal microscope. The KHC^mut-expressing cells were identified by exciting EGFP at 488 nm and the DsRed fluorescence of the same cells was visualised by exciting at 568 nm. Typical 568 nm in vivo confocal images of insulin-containing vesicles (a,c) in KHC^mut-expressing (a) and control cells (c) are shown. Images were taken every 5 seconds for 4 minutes or 2 frames/s for 30 seconds (for a total of 60 frames). The movements of vesicles were tracked for 60 frames unless the spot was lost from view. (b,d) Tracks of granules in (a) and (c). Bars, 1 µm (a,c); 0.75 µm (b,d). (B) For quantification of motility, vesicles were randomly selected (20 in each cell) in seven dominant-negative kinesin-expressing (open bars) and seven control cells (closed bars) and the location of granules was determined using the image analysis software MethaMorph^TM (see Materials and Methods). The bar diagrams show the probability of vesicles travelling at the indicated velocities. For corresponding movies (Fig. 5Aa,c), see ftp://researcher{at}137.222.66.116/ (long on as `researcher', password c1100cs, directory `kinesin').

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