TCR activation increases stathmin phosphorylation and alters microtubule organization.
T cells were plated on anti-CD3 antibody or control IgG for 45 min. (A) Cells were lysed and the levels of phospho-stathmin (pStathmin) and total stathmin assessed by western blotting. The graph shows relative levels of phospho-stathmin (normalized to total stathmin) of 3 independent experiments +/− S.E.M. in arbitrary units. (B) Antibody pre-treated T cells were collected, plated on ICAM-1 (IC-1) and lysed at the indicated time points. The levels of phospho-stathmin and total stathmin were assessed by western blotting. Representative blots are shown; the graph shows mean levels of phospho-stathmin (normalized to total stathmin) of 3 independent experiments +/− S.E.M. relative to IgG-treated cells at t = 0. (C) Antibody pre-treated cells expressing EB3-GFP were plated on ICAM-1-coated coverslips. Images were collected every 5 sec. Images shown correspond to five consecutive frames, from 130 sec after the start of the movie. Greyscale images (256 greyscale) were converted to indexed color (scale on right) to improve the visualization of small differences in intensity value. Arrows indicate individual microtubule tips moving towards the leading edge. Tracking images (right panels) show the movement of EB3-GFP in the 5 frames (see Methods for detail). Scale bars, 10 µm.