Figure 3. The intact syntaxin SNARE-motif is required for mobility restriction in HepG2 cells.
Comparative fluorescence recovery after photobleaching analysis of GFP-labelled full length syntaxin and the deletion constructs syx-ΔS and syx-ΔCyt (for details see Figure 1). (a) Live cells expressing the indicated constructs are imaged by confocal microscopy at the basal plasma membrane. In a squared region of interest (ROI) fluorescence is bleached (compare pre-bleach and post-bleach images). Then images are taken at 1 Hz for several minutes to monitor the recovery of fluorescence in the ROI whose half-time is inversely proportional to the apparent lateral diffusion coefficient (or molecule mobility). Images are shown at the same scalings. (b) Averaged recovery traces from several cells imaged for one experiment. Values are given as means ± S.D. To better illustrate the traces, only every second point of the measurement is shown. (c) Half-times of recovery obtained by fitting hyperbola functions to traces as shown in (b). Values are given as means ± S.E.M. (n = 3 independent experiments; Kruskal-Wallis one way analysis of variance on ranks (p=0.011; *p<0.05); 8–18 cells per construct and experiment). (d) Half-times of recovery were also determined for individual cells. Values obtained from all experiments were plotted versus the pre-bleach intensity (a relative measure for the expression level). Only for full length syntaxin at higher expression levels a trend is observed towards longer half-times of recovery.