Figure 4.

The SKAP55 PH domain and linker tyrosines are dispensable for recruitment into and stabilization of SLP-76 microclusters, and for T cell adhesion. (A) Domain structures of the ΔPH, 3YF, R131M, and PH-alone SKAP55 chimeras. (B) J14.SY cells were transiently transfected with the indicated SKAP55.mRFP1 chimeras; expression levels were determined by Western blotting (n = 3). (C) Cells from B were stimulated, imaged, and presented as in Fig. 2 B. See Table 1 for microcluster properties and experiment numbers. (D) Composite microcluster traces for the conditions examined in C; numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (E) Fraction of cells scored as displaying unstable contacts (n = 4). (F) JSKAP.SY cells transiently transfected with the indicated SKAP55.mRFP1 chimeras were stimulated, imaged, and presented as in Fig. 2 B (n = 3). (G) JSKAP.SY cells transfected with Akt.PH.mCFP and SKAP55.mRFP1 chimeras were imaged as in Fig. 2 B. Still images acquired at and 4 µm above the coverslip are shown (n = 3). (H) Fractional retention of unstimulated or TCR-stimulated cells on fibronectin-coated coverslips was calculated by dividing the post-wash signal by the prewash signal and normalizing to the corresponding PMA control (n = 3). (I) Fractional retention of T cells on uncoated or anti-CD3 coated coverslips was calculated by dividing the post-wash signal by the prewash signal (n = 3). Bars: (C and F, MOT images; and G) 10 µm; (C and F, kymographs) 5 µm × 60 s. Error bars indicate mean ± SEM. From parental J14.SY cells (with or without mRFP1): *, P < 0.05. From JSKAP.SY (with or without mRFP1): #, P < 0.05.