HPK1 reduces the persistence of SLP76 microclusters. (A) J14-SLP76-YFP cells transfected with HPK1-mCherry were activated on anti-CD3–coated coverslips and imaged with a spinning-disk confocal microscope at one image/20 s. Frames from Video 1 at different time points are shown. One untransfected control (CTRL) cell (closed arrowhead) and one cell expressing HPK1-mCherry (open arrowhead) are visible in each frame. (B). Lifetime of single SLP76-YFP microclusters was determined by manual tracking using time-lapse images of J14-SLP76-YFP cells, acquired as described in A. The plot shows SLP76 microcluster lifetime in untransfected cells and in cells expressing HPK1-mCherry or HPK1-KD-mCherry. (C, top) Z projection of the HPK1-mCherry–expressing cell shown in A, imaged after 80 s of activation. The boxed area highlights a microcluster containing both SLP76-YFP (green) and HPK1-mCherry (red). (bottom) Magnification of the boxed area shown in the top image at different time points. Time 0 corresponds to the first detection of this SLP76-YFP microcluster (i.e., 40 s in the time lapse displayed in A). Similar results were obtained in three independent experiments. (D) J14-SLP76-YFP cells transfected with control (siCont) or HPK1-specific (siHPK1) siRNAs were stimulated on anti-CD3 coverslips and analyzed as described in A. Knockdown efficiency was assessed by flow cytometry (Fig. S1). (E) SLP76 microcluster lifetime assessed in cells treated as in D. The plot shows pooled data of three experiments. Horizontal lines and error bars show means and SEM. Bars: (A, C [top], and D) 5 µm; (C, bottom) 1 µm.