Extended Data Fig. 6: Pac1-microtubule binding behavior, and its contribution to dynein activity.
(a and b) Representative images (a) and intensity scatter plots (b; bars depict mean ± standard deviation) of microtubule-bound Pac1 in different buffers. Pac1-SNAP647 diluted in motility buffer (50 nM, dimer concentration) with indicated salts was introduced into a chambers with coverglass-adhered microtubules, and images were acquired (yellow and magenta circles represent data acquired from each independent experiments; n = 38, 49, 41, 49, and 45 microtubules that span 911 µm, 1074 µm, 1077 µm, 906 µm, 1017 µm in length for each condition, left to right). (c) Pac1-microtubule binding is reduced after enzymatic removal of the unstructured tubulin carboxy-terminal tails (see Methods; similar results were obtained from 2 independent experiments). (d–g) Addition of cell extracts reduces Pac1-microtubule binding, and attenuates Pac1-mediated dynein velocity reduction. Representative fluorescence images of Pac1-SNAP647 on microtubules (d; Pac1-SNAP647 shown as a heat map) and scatter plots depicting intensity values (e; bars depict mean ± standard deviation; n= 48 and 47 microtubules that span 890 µm and 841 µm in length for each condition, left to right; similar results were obtained from 2 independent experiments). (f and g) Plots depicting the motility properties for GST-dyneinMOTOR in the absence and presence of 25 nM Pac1 (dimer concentration) in low ionic strength buffer (50 mM potassium acetate) in the presence of cell extracts (0.96 mg/ml final; 275 and 258 motors, left to right, from 2 independent experiments were quantitated). Note the small Pac1-mediated GST-dyneinMOTOR velocity reduction in the presence of cell extracts (22.1%, compared to 69.5% in the absence of extracts). (h) Additional representative kymograph of GST-dyneinMOTOR comigrating with Pac1 in buffer with 150 mM KCl (see Fig. 6d and Extended Data Fig. 7E – G for quantitation and statistics). Note the diffusive behavior of Pac1 on microtubules. Scale bars in panels a, c and d, 4 µm.
