Figure 4. Trafficking of CD3-bearing vesicles at the IS is impaired by Aurora A chemical inhibition or gene ablation.

(a) Maximum Z projections of confocal stacks of Jurkat T cells pretreated with vehicle (DMSO) or Aurora A inhibitor (MLN8237) and conjugated with SEE-pulsed Raji B cells. Cells were incubated for 30 min, fixed and stained for α-tubulin (green) and TRC/CD3ɛ (magenta). The right-hand image shows CMAC cell tracker labelling of Raji B cells (cyan) and bright field. Scale bar, 10 μm. (b) Graph shows quantification of TRC/CD3ɛ clustering at the IS from as in a. Means±s.d. is shown; t-test was used to compare means (n=101 in DMSO and in MLN8237). Map of the trajectories of CD3ζ-cherry-bearing vesicles in human CH7C17 T cells (c) or Aurora-A-deficient and control CD4⁺ T cells (e) pretreated with vehicle (DMSO) or MLN8237 inhibitor and settled on corresponding anti-human or anti-mouse stimulating anti-CD3/CD28-coated glass-bottom chambers. Images were taken every 100 (c) or 110 ms (e) under a TIRF microscope at a penetrance of 200 nm with 561 nm laser; vesicles were tracked with Imaris software over 60 (c) or 30 s (e) and maximal projections of the time lapse are shown for tracks. A representative cell is shown for each case. Fluorescence images from CD3ζ-mCherry (c,e) and EB3-GFP are also shown (e). (d,f) Quantification of the number of vesicle tracks and the speed of vesicles from cells analysed in c and e from three independent experiments (d, n=28 in DMSO, n=39 in MLN8237; f, n=16 in WT, n=17 in KO, n=19 in WT MLN8237, n=12 in KO MLN8237). Data represent means±s.d. Means were compared with a Mann–Whitney test. n.s., nonsignificant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.