Quantitative analysis of all trajectories of vesicles containing of BChol. CHO cells pre-incubated in the absence or presence of Cytochalasin D, to disrupt the actin cytoskeleton, or nocodazole, to disrupt microtubule, were pulse-labeled with BChol followed by a chase in the presence or absence of the drugs to obtain the steady-state distribution. The cells were imaged as described in the legend to Figure 3. Vesicles were tracked as described in Materials and Methods and their mobility was analyzed by the average mean square displacement (MSD) averaged over all vesicle trajectories per condition. Disruption of microtubule (panel A, red) or actin filaments (panel A, blue) significantly reduced vesicle mobility compared to the mobility of vesicles in control cells (panel A, black). Vesicle mobility was analyzed by a model describing the transition from anomalous subdiffusion to directed motion (Eq. (1)). Panels B, C, and D show the regression of this model (red line) to the MSD for vesicles in control cells (panel B) and in cells with disrupted microtubule (panel C) or disrupted actin filaments (panel D). Also shown are the contribution from anomalous subdiffusion (dashed lines) and superdiffusion consistent with directed motion (dotted lines). Disruption of both microtubule and actin filaments reduced the anomalous diffusion constant and the velocity of the directed motion. Notice that as the anomalous diffusion constant, Dα, depends on the anomalous exponent the comparison in panel E is only valid as we find α to be constant. Data is given as mean ± SD being calculated as described in Eq. 5 in Materials and Methods.