CD2–CD58 microdomains organize signaling molecules. Dual-color images of various signal molecules (red) and CD2–CD58 microdomains (green) at the cell–bilayer interface by TIRF microscopy. (A–F) ∼2 min after interaction with CD58 lipid bilayers, live cell imaging was performed with the GFP-tagged mutant Lck (LckY505F; A) or GFP-LAT (F), and the remainder are immunofluorescence images using antibodies against phosphotyrosine (pY) of activated Src family kinases (including autophosphorylated tyrosine 394 in Lck; B), CD45 (C), phosphotyrosine (D), and phosphorylated ITAM domain of TCR-ζ (E). The white boxes show regions that are magnified in the panels on the right. The intensity ratio of CD45 (outside and inside of microdomains) is ∼3.0 ± 0.3 (mean ± SEM, n = 12). The same molecular patterning was observed by widefield and confocal microscopy as well. (G) Immunostaining of phosphotyrosine at 60 min of cell–CD58 bilayer contact shows concentration of signal in a cSMAC-like cluster and an overall reduction in fluorescence intensity compared with a 2-min time point (quantitation as described in Fig. 1). Data shown are mean ± SEM (n > 20). Bars, 5 µm.