Figure 4.

TPCs Translocate to an Immunological Synapse upon Stimulation with Anti-CD3

eYFP-tagged HsTPC1 and HsTPC2 (green) were expressed in Jurkat 1G4 cells and added to glass coverslips coated with poly-L-lysine or 10 μg/ml anti-human CD3 antibody mounted in an imaging chamber, as depicted in the cartoon. Cells were labeled with FM4-64 (red) to delineate the plasma membrane edge of the cell.

(A and B) Confocal images of cells attached to poly-L-lysine (Ctrl; left-hand side) or engaged with anti-CD3 antibody (αCD3; right-hand side) showing z stacks through x and y axes (top images) and 3D reconstruction of the z stack (bottom images). TPC localization was assessed through the z stack as a product its area and its fluorescence, as depicted in (C)–(E). A slice depth of 0 indicates the bottom of the cell (i.e., the contact zone between cell and the coverslip).

(C and D) TPC1 (C) and TPC2 (D) localization significantly alters from being present deeper in the cell (slice depth 3–5 μm) toward αCD3, i.e., the bottom of the cells (slice depth 0–2 μm) compared to control.

(E) Comparison of TPC1 and TPC2 movement in cells engaged with anti-CD3. There was no significant difference in the migration of the two isoforms. TPC1 n = 32–43; TPC2 n = 52–69.

Error bars are mean ± SEM. See also Figure S4.