Dynamic distribution of CD9 during TC-EC interactions.

(A) A375 melanoma cells were transfected with GFP-tagged CD9 and were allowed to invade a confluent EC monolayer. Cellular distribution of CD9-GFP was assessed by time-lapse confocal microscopy every 3 minutes. The focal plane corresponding to TC-EC contact is shown (Z = 0 μm). At 0 and 24 minutes, a projection of the fluorescence signal of the different sections and of the DIC image is shown. (B). ECs were transfected with GFP-tagged CD9 and allowed to grow onto collagen gels to confluence. A375 melanoma cells were allowed to invade the EC monolayer, and the distribution of endothelial CD9-GFP was observed by confocal microscopy. Upper panels show a representative EC-TC interaction confocal section. Reinforcement of the signal because of CD9-GFP relocalization is indicated by arrowheads at EC-EC contact sites by arrows at EC-TC heterotypic junctions. Lower panels show a time-lapse experiment of the TC insertion. Merging of fluorescence and DIC images is shown. Note that no redistribution of CD9 is seen at initial time points where TC adheres to luminal surfaces of ECs but only at the time of insertion (10-20 minutes, arrows). The processes in panels A and B are also depicted in 2 video sequences on the Blood website (see the Supplemental Videos link at the top of the online article).