In Vitro MT Gliding Assays.

Polarity marked X-rhodamine–labeled MTs were polymerized as described (26, 27, 69) and stabilized with 20 μM paclitaxel. MT•motor complexes were performed with a final concentration of 0.5 μM tubulin (paclitaxel-stabilized α,β-tubulin) and 2.5 μM kinesin dimer. Perfusion chambers were constructed using acid-washed coverslips and incubated first with 40 μg/mL Penta-His antibodies (Qiagen) for 10 min followed by a 5-min incubation of blocking buffer (PME80; 1.5 mM magnesium acetate, 500 μg/mL casein, 25 mM glucose, 0.2 mg/mL glucose oxidase, 175 μg/mL catalase, 0.3 mg/mL creatine phosphokinase, 2 mM phosphocreatine, 0.5% β-mercaptoethanol, 15 μM paclitaxel). MT•motor complexes were then introduced into the chamber and allowed to bind for 5 min. Unbound complexes were removed with excess blocking buffer. Gliding via the surface-bound motors was activated by introducing Activity Buffer (blocking buffer supplemented with 1 mM MgATP and either 3% DMSO or 5 μM propofol at 3% DMSO). MT gliding was imaged by TIRF microscopy at 564 nm using a 180-ms exposure time and a 10- (K560) or 20-s (KIF3) interval for 20 min at 25 °C. The velocity of gliding MTs (Fig. 1) was determined by tracking the leading edge of the moving polarity-marked MTs using the tracking algorithm on AxioVision 4.8.2 software (Carl Zeiss Microscopy, Inc.). MTs were scored that moved at least eight frames (≥80 s for K560 and ≥160 s for KIF3AC and KIFAB) and met the following criteria: MTs were entirely in the field of view, were ≥3 but ≤8 μm in length, and traveled in a clearly defined path. MT gliding velocity data were then compiled, and a Gaussian fit of the histogram provided the mean velocity ±SEM. Persistence of MT gliding was defined as the percentage of scorable MTs that remained attached to the surface-bound kinesins and continued to glide. The data reported in Fig. 1 G–I represent a minimum of 3 different experimental days, with six to eight movies collected per day. Within each field of observation, ∼10–60 MTs were scored for persistence of gliding.