Figure 2. SKAP55 is required for... | Rockefeller University Press

Figure 2.

$Figure 2. SKAP55 is required for microcluster persistence and movement, and for contact stability. (A) Confirmation of the efficacy of SKAP55 knockdown and add-back in stable SKAP55 knockdown cells (JSKAP.SY) with or without transient reconstitution (n = 3). (B) J14.SY and JSKAP.SY cells expressing mRFP1 or SKAP55.mRFP1 were stimulated on coverslips and imaged for at least 5 min. Representative MOT images and kymographs are shown. Bars: (MOT images) 10 µm; (kymographs) 5 µm × 60 s. See Table 2 for microcluster properties and experiment numbers. (C) Composite microcluster traces for the conditions examined in B. Numbers in parentheses indicate the total number of cells examined. Line intensity corresponds to the fraction of microclusters surviving; arrowheads identify points of half-maximal microcluster dissociation. (D) Fraction of the contact area engaged in fluctuation (see Fig. S1, F and G; n = 3). (E) Fraction of cells scored as displaying unstable contacts (see Fig. S1, I and J; n = 5). (F) Surface expression of CD69 in J14.SY and JSKAP.SY cells, after normalization to a TCR-stimulated J14.SY control (n = 4). (G) Kinetics of Erk1/2 phosphorylation in J14.SY and JSKAP.SY stimulated for the indicated time points (n = 3). Error bars indicate mean ± SEM. From parental J14.SY cells (with or without mRFP1): **, P < 0.01. From JSKAP.SY (with or without mRFP1): ##, P < 0.01.$

SKAP55 is required for microcluster persistence and movement, and for contact stability. (A) Confirmation of the efficacy of SKAP55 knockdown and add-back in stable SKAP55 knockdown cells (JSKAP.SY) with or without transient reconstitution (n = 3). (B) J14.SY and JSKAP.SY cells expressing mRFP1 or SKAP55.mRFP1 were stimulated on coverslips and imaged for at least 5 min. Representative MOT images and kymographs are shown. Bars: (MOT images) 10 µm; (kymographs) 5 µm × 60 s. See Table 2 for microcluster properties and experiment numbers. (C) Composite microcluster traces for the conditions examined in B. 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. (D) Fraction of the contact area engaged in fluctuation (see Fig. S1, F and G; n = 3). (E) Fraction of cells scored as displaying unstable contacts (see Fig. S1, I and J; n = 5). (F) Surface expression of CD69 in J14.SY and JSKAP.SY cells, after normalization to a TCR-stimulated J14.SY control (n = 4). (G) Kinetics of Erk1/2 phosphorylation in J14.SY and JSKAP.SY stimulated for the indicated time points (n = 3). Error bars indicate mean ± SEM. From parental J14.SY cells (with or without mRFP1): **, P < 0.01. From JSKAP.SY (with or without mRFP1): ##, P < 0.01.