Lck- and Fyn-deficient CTLs cannot polarize their centrosome toward antigen-presenting targets or anti-CD3 beads. (A) Killing assay of F5 WT (diamonds) and Lck^off Fyn^−/− (squares) CTLs using NP68-pulsed EL4 as targets. Error bars show standard deviations from triplicates. (B) Representative immunofluorescence images of centrosome polarization observed in WT and Lck^off Fyn^−/− F5 CTLs with NP68-pulsed EL4 targets. Cells are labeled with antibodies against talin (green), γ-tubulin (red), and CD8 (white). Nuclei were stained with Hoechst (blue). Bars, 10 µm. (C) Quantitation of centrosome position relative to the synapse in conjugates formed between EL4-pulsed targets and F5 Lck^off Fyn^−/− (n = 103) CTLs from 3D reconstructions from the same experiment shown in Fig. 3 C showing distal (blue), proximal (red), or docked (green) centrosomes at the synapse. A two-tailed Student’s t test for loss of centrosome docking in Lck^off samples compared with WT gave a statistical significance of P = 0.007. Lck^off Fyn^−/− formed conjugates with similar frequency (31%; n = 317) to WT CTLs (33%; n = 424). Centrosome polarization was also quantitated in three independent experiments without 3D reconstruction (Fig. S3). (D) Quantitation of centrosome position relative to anti-CD3 antibody–coated latex beads for F5 WT (n = 400), Lck^off Fyn^−/− (n = 400), and Lck^off Fyn^−/− + PP2 (n = 300) CTL conjugates. Error bars show the standard deviation from the means from at least three independent experiments. A two-tailed Student’s t test for loss of centrosome docking in Lck^off and Lck^off Fyn^−/− samples compared with WT gave statistical significances of P = 0.0006 and P = 0.007, respectively. (E) Representative images of F5 CTLs from WT, Lck^off Fyn^−/−, and Lck^off Fyn^−/− + PP2 conjugated to anti-CD3 antibody–coated latex beads labeled with Hoechst (blue) and antibodies against γ-tubulin (red) and anti–rat-FITC (green) viewed by differential interference contrast (DIC) and merged. Also see Fig. S2. Bars, 5 µm.