The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46 Involves Its Recruitment to Lipid Rafts
Figure 2
CD46 alters membrane organization and is recruited to ordered membranes. (A) The GP histograms (open squares) of ~50 cells for each cell type (untransfected cells shown, (A)(i)) were normalized and the distribution fitted onto two Gaussian populations: a fluid population Pf (dotted line) and a raft-like ordered population Po (dashed line). The table (A)(ii) summarizes a similar analysis of GP histograms in cells transfected as shown, with or without CD46 ligation. The table gives the mean GP value that was derived by fitting two populations as per the graph above. Also shown, in brackets, is the proportion of membranes associated with either of the two populations (termed coverage). (B)(i) Laurdan-labeled MD45 cells expressing CD46Cyt1 (a)–(f) or CD46Cyt1.C-A (g)–(l) were left untreated (a)–(c) and (g)–(i) or incubated with CD46-ligating antibodies (d)–(f) and (j)–(l) before fixation and imaging. Left column shows transmission images with overlaid CD46 confocal staining in red. The middle column shows pseudocolored GP images of the identical focal depth as the CD46 confocal images. GP color scale is indicated in (f) with GP values ranging from −1 (blue) to +1 (yellow). The right column shows the masked image, in which the GP values are shown only for those pixels that were defined as positive for CD46 (red coloring in (a), (d), (g), and (j)). Table (B)(ii) summarizes the GP mean ± SD of CD46-positive membranes of 40–50 images. GP values with and without ligation are significantly different () for both CD46Cyt1 and CD46Cyt1.C-A, and CD46Cyt1 is significantly different to CD46Cyt1.C-A (), both with and without ligation. The data is representative of 3 independent experiments.
