The SKAP55 DM is required for binding to RapL and RIAM, and governs the recruitment of talin into TCR-induced contacts. (A and B) 3×Flag.TRT-tagged SKAP55 wild-type (WT), DM deleted (ΔDM), and TD chimeras were expressed in J14.SY cells and immunoprecipitated via the Flag epitope. Blots of total lysates and immunoprecipitates are shown (n = 3). (C and D) Images of SLP-76.YFP (blue) and endogenous talin immunofluorescence (green) in J14.SY or JSKAP.SY cells stimulated on anti-CD3–coated plates and fixed after 10 min. Boxed regions were magnified 4× (shown below) to show microclusters at higher resolution (n = 3 or more). (D) JSKAP.SY cells were transfected with wild-type (WT) or TD SKAP55.3×Flag.TRT chimeras (red) before stimulation. (E) The frequency of cells exhibiting talin clustering and the central accumulation of talin were scored manually, using the images acquired above. (F and G) Cells were prepared as in C and D, but after fixation and before immunofluorescent staining cell bodies were sheared away, leaving cellular material only within the areas of tight contact between the cell and stimulatory substrate. Bars: (main panels) 10 µm; (enlarged panels) 2.5 µm. Error bars indicate mean ± SEM. From parental J14.SY cells: **, P < 0.01. From JSKAP.SY cells: ##, P < 0.01. From JSKAP.SY reconstituted with SKAP55.WT.3×Flag.TRT: ††, P < 0.01.