Real-time imaging of spinophilin polarizing at the IS. (a) DCs expressing spinophilin-GFP that were cultured in the presence (+Ag; top, n = 6 conjugates) or absence (−Ag; middle, n = 3 conjugates) of agonist peptide together with antigen-specific CD4+ T cells that had been labeled red by the lipophilic PKH-26 dye (Sigma-Aldrich). In the presence of the antigen, spinophilin was polarized minutes after contact and remained polarized throughout the duration of the contact (30–60 min; a, top; and b). In the absence of antigen, spinophilin was distributed throughout the cytoplasm (a, middle; and b). As a control, the distribution of GFP alone in DCs contacting T cells (n = 3 conjugates) was imaged in the presence of the antigen and localized evenly throughout the cytoplasm (a, bottom). Time is indicated in minutes after imaging began and is rounded to the nearest minute. The bar in section a (middle) applies to the spinophilin-GFP +/− antigen. T, T cell. (b) The distribution of fluorescence signal for GFP alone with an antigen (gray) and spinophilin-GFP both with (green) and without an antigen (black) in live cells was analyzed at 0, 4, 14, and 20 min after imaging began (n = 9, 12, and 22 observations from 3, 3, and 6 conjugates, respectively, for GFP alone, spinophilin-GFP –Ag, and spinophilin-GFP +Ag). Spinophilin-GFP was most polarized toward the T cell in the presence of the antigen (P < 0.05 with respect to spinophilin-gfp –Ag; P < 0.002 with respect to GFP alone +Ag by Student's t test).