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Fig. 1. Tetraspanin molecules co-cap with GM1 gangliosides together with F-actin. (Aa-d,Ba-d) Capping and labeling of GM1 gangliosides at the Jurkat T-cell surface induced by FITC-labeled avidin cross-linking (at 37°C) of biotin-labeled CTx. Surface proteins (CD9, CD81, CD82 or CD71) and actin were visualized on capped and fixed cells by labeling with specific mAbs, followed by Cy3-conjugated secondary reagent and rhodamine-conjugated phalloidin. (Be-h) Reciprocal experiments in which surface proteins were first labeled with specific antibodies and induced to cap with Cy3-coupled secondary antibodies. GM1 and actin were stained on capped and fixed cells as previously. (Ae-h) Control experiments in which cells were stained without any capping with indicated antibody and Cy3 secondary reagent, GM1 or rhodamine-phalloidin at 4°C. In all pictures, red represents actin, GM1 is shown in green and surface proteins (tetraspanins or CD71) are in dark blue. (A) Cross-linking of GM1. (Aa-c,e-g) Single color analysis. (Ad,h) Merge of three images. (B) Only merged images are shown. (Ba-d) Cross-linking of GM1. (Be-h) Cross-linking of CD82 (e), CD81 (f), CD9 (g) or CD71 (h). Dark blue indicates CD82 (a,e), CD81(b,f), CD9 (c,g) or CD71 (d,h). White colors and arrows indicate clusters of tetraspanin, actin and GM1.
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