Fig. 4. I-A^k mediated actin reorganization is DIG dependent and requires the intracytoplasmic domains. (A) SaI/A^k and SaI/A^k tr cells were stimulated with 10.2.16 mAb-coated beads (Anti-I-A^k) or irrelevant mouse Ig-coated beads (nonstimulated, NS) at a ratio of one bead per cell for 30 minutes. Cells were cytospun, fixed and permeabilized. Polymerized actin was detected with Alexa488-phalloidin. The bright-field images of single cell/bead conjugates (a,c,e,g,i,k) and the corresponding fluorescent image of the actin cytoskeleton are presented (b,d,f,h,j,l). Polarization of the actin network was observed only at the site of SaI/A^k cell/10.2.16-coated bead contact (d) and was prevented by pre-treatment with MßCD (f). By contrast, no reorganization of the actin cytoskeleton was detected after engagement of SaI/A^k tr cells with 10.2.16-coated beads (j). (B) The percentage of cells undergoing actin reorganization was determined. At least 200 cell-bead interactions from each condition were scored and cells in which actin relocalization had occurred (to at least the same degree as shown in A, panel d) were counted as positive events. Data from one of three typical experiments is shown.

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