MTs do not polarize to the contact site in INF2 KD cells. (A and B) Control (A) or INF2 KD Jurkat cells (B) expressing GFP-tubulin were conjugated to SEE-loaded APCs and subjected to time-lapse video microscopy. Numbers indicate time in seconds. (C) Jurkat cells were treated with 0.1% DMSO, 1 µM latrunculin A (LatA), 2.5 µM cytochalasin D (CytD), 0.5 µM jasplakinolide (Jasp), or 10 µm nocodazole (Noc) for 1 h. Cells were conjugated in the presence of the drugs to SEE-loaded APCs and were stained for α- or γ-tubulin as indicated. APCs (B cells) were stained with CMTMR. Note that the CMTMR stain from B cells diffuses into the medium, resulting in some background staining of T cells. The CMTMR images were subjected to nonlinear adjustment to help B cell identification. CMTMR-positive cells are marked with an asterisk. The histogram represents the percentage of T cells with polarized MTOC. (D) Jurkat cells were treated with 1 µM latrunculin A and conjugated in the presence of the drug to SEE-loaded APCs that were stained with CMTMR. Cells were finally stained for Glu- and Tyr-MTs. The histogram represents the percentage of T cells with Glu-MTs. (E) Control or Jurkat cells expressing EB1-C-GFP or EB1-C-KR-GFP were conjugated to SEE-loaded APCs. Cells were then stained for Glu-MT and α-tubulin. The histogram represents the percentage of transfected T cells with Glu-MTs or polarized MTOC. The arrowheads indicate the position of the MTOC of the T cell. At least 40 T cells were analyzed in C–E. Data in C–E are summarized as means ± SEM from three (C and D) or two (E) independent experiments (error bars; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Bar, 5 µm.