MAL⁺ transport vesicles move along microtubule tracks. (A-B) Jurkat cells coexpressing GFP-tubulin and Cherry-MAL (A) or Lck-Cherry (B) were fixed and analyzed by confocal microscopy. Enlargements of the boxed regions are shown on the right. The arrows indicate MAL⁺ (A) or Lck⁺ (B) vesicles associated with microtubules. (C-D) Jurkat cells were cotransfected with plasmids expressing Cherry-MAL and GFP-tubulin (C) or EB3-GFP (D). After 24 hours, cells were subjected to time-lapse videomicroscopy. The image of a representative cell is shown. Selected frames corresponding to events occurring in the boxed regions during the time-lapse experiment are shown. In panel C, the solid and empty arrowheads indicate 2 MAL⁺ vesicles that move to the plasma membrane or to the pericentrosomal region, respectively, along microtubule tracks. In panel D, the arrowhead indicates a MAL⁺ vesicle moving along a microtubule labeled at its plus tip with EB3, which is indicated by an arrow. Two different areas from the same cell are enlarged in panel D. (E) Jurkat cells coexpressing GFP and shINF2a or transfected with the indicated INF2 construct were fixed, permeabilized, and stained with anti–α-tubulin antibodies followed by Alexa-488 secondary antibodies and with TRITC-phalloidin. The cells expressing the exogenous INF2 fragment were identified with anti-INF2 antibodies and secondary antibodies coupled to Alexa-647 (bottom). The staining of exogenous INF2 is presented in gray. (F) Jurkat cells coexpressing the Cherry protein and the indicated shRNA from the same plasmid were transfected with EB3-GFP and processed for time-lapse videomicroscopy. Arrows indicate microtubules labeled with EB3 at their growing end. Numbers in panels C, D, and F indicate time in seconds. Scale bars correspond to 5 μm unless another value is indicated.