Figure 6: Expression of CLCa–DsRed1 or CLC depletion causes loss of G-clathrin structures and CLCa–DsRed1 reduces recycling and cell migration.

(a–c) HeLa-M cells transfected with YFP-GGA1, and with either (a) control siRNA, or (b) CLCab siRNA or with (c) CLCa–DsRed1 were imaged using continuous 30 ms exposures (~\n1 s total). A single image from each sequence is shown on the left. Combined maximum projections (red), which reveal both dynamic and stationary GGA1 structures and sum projections (green), which report stationary structures, are shown at the right, with overlap in yellow (54 μm square panels). Dynamic G-clathrin appears red. Boxed areas are magnified (right) and pixel intensities binarized for clarity. (d) Percent of G-clathrin relative to controls in HeLa-M cells transfected with CLCab siRNA or CLCa–DsRed1 (CLCa-DsR1) in a–c. G-clathrin for each condition was calculated as the difference between the maximum projection and sum projection of YFP-GGA1 signals (see Methods for details) (mean±s.e.m.; n=4 for CLCab, knockdown (KD) n=2 for CLCa-DsR1, ****P<0.0001, Student’s unpaired t-test). (e) Recycling of β1-integrin (left) and TfR (right), assessed by biotinylation as in Fig. 4d, in DsRed1 (DsR)- or CLCa–DsR1-transfected HeLa cells (mean±s.e.m.; n=7; *P<0.05, **P<0.01, two-way analysis of variance followed by Bonferroni post hoc test). (f) Representative immunoblots from one recycling experiment in e. Lysate samples are shown in Supplementary Fig. 5a. (g,h) Dunn chamber migration tracks of H1299 cells transfected with (g) DsR or CLCa–DsR1 or (h) mCherry plus control or CLCab siRNA in the presence of an EGF gradient (source at 90°). Tracks in the absence of a gradient are shown in Supplementary Fig. 5b,c. All tracks were set to a common origin (intersection of x [red] and y axes). Circular histograms depict proportion of cells whose final position lies within each of 18 20° sectors (mean and 95% confidence interval indicated by black line; at least 50 cells analysed from four independent experiments for each condition, P-values, Raleigh uniformity test). (i) Average speed and (j) displacement of DsR- and CLCa–DsR1-transfected gradient-exposed cells. (k) Average speed and (l) displacement of control-treated and CLCab-depleted gradient-exposed cells. (i–l: mean±s.e.m.; n=4 *P<0.05, **P<0.01, Student’s paired t-test).