FIGURE 2:

Directional movement of MAP2c-induced short microtubules along the cell cortex after nocodazole washout. (A) COS7 cells were imaged soon after nocodazole washout via wide-field or TIRF microscopy. The yellow arrow points to a more intensely labeled microtubule structure in a peripheral region that was formed during the accumulation of multiple weakly labeled short microtubules in the cell periphery (see also Supplemental Movie S1). (B) Example of a short microtubule that moves directionally near the cell cortex within the evanescent wave of the TIRF microscope (see also Supplemental Movie S1). (C) Automated tracking of short microtubules via TIRF microscopy (see Supplemental Figure S3 and Materials and Methods for details). The trajectory of microtubule movement (red) was overlaid onto the last video frame used for tracking. Blue, final position of tracked short microtubule. Yellow, tracked short microtubule endpoint. (D) Microtubule speed plotted against time reveals saltatory, rapid movements with intermittent pauses characterized by slow directional movements and Brownian motion. (E) Average speed of short microtubules in nocodazole-washout experiments in control Neuro2A cells and Neuro2A cells treated with shRNA targeting Dync1h1 and/or with EGFP-Dync1h1 (mean ± SEM; n_experiments = 8 for control, Dync1h1 shRNA, and Dync1h1 shRNA + EGFP-Dync1h1; n_experiments = 4 for EGFP-Dync1h1 alone). Dynein heavy-chain silencing resulted in a significant decrease in average short microtubule speeds, which was largely recovered by simultaneous expression of shRNA-resistant EGFP-Dync1h1 construct. *p < 0.05; **p < 0.01; one-way analysis of variance.