HAP1 activates dynein motility. (A) Peroxisome recruitment assay wherein peroxisomes (PEX-GFP-DHFR) were tethered to Halo-mCh-HAP1 via dimerizer and imaged for 25 min to show relocalization to the cell center. Left: Initial (0 min) and concluding (25 min) stills of peroxisomes. Right: Maximum time projection pseudo-colored by frame (scale below). Yellow line indicates cell outline. Scale bar, 10 µm. (B) Example micrographs from single-molecule motility TIRF assay showing TMR-labeled Halo-HAP1 traversing an MT from the dynamic plus end to the minus end, indicated by the 647-labeled MT seed. Single stills from both tubulin channels are shown (488 and 647) as well as the first and last frames merged (all three channels). Scale bar, 2 µm. (C and ;D) Example kymographs showing dynein-directed motility of HAP1 and BICD2N puncta in the lysate-based motility assay. Kymographs span the length of each MT with the plus end (+) and minus end (−) labeled at the bottom. Arrowheads represent motile minus end–directed events (teal), diffusive events (yellow), and sustained MT binding events (pink). (E–G) Quantifications of single-molecule motility assay showing the average velocity (E), run length (F), and stationary event duration (G).n = 30–51 runs (E and F), 457–471 events (G) in three independent trials on 57 total MTs. Welch’s two-tailed t test (E: P = 0.1969), Mann-Whitney U test (F: P = 0.0568; G: P < 0.0001). Y-axes in F and G represent 1 - the cumulative distribution function (CDF); x-axis in F begins at 1 µm because only runs ≥1 µm were analyzed; x-axis in G ends at 120 s, the length of videos acquired. (H and I) Blotting and quantification of cell extract MT pelleting assay. n = 3 independent assays; one-way ANOVA followed by Tukey’s multiple comparisons test (ATP addition, P = 0.6691; KCl without ATP, P = 0.0075; KCl with ATP, P = 0.0011). Bars throughout show mean ± SEM. *, P < 0.05; ****, P < 0.0001.
Figure 5.

HAP1 activates dynein motility. (A) Peroxisome recruitment assay wherein peroxisomes (PEX-GFP-DHFR) were tethered to Halo-mCh-HAP1 via dimerizer and imaged for 25 min to show relocalization to the cell center. Left: Initial (0 min) and concluding (25 min) stills of peroxisomes. Right: Maximum time projection pseudo-colored by frame (scale below). Yellow line indicates cell outline. Scale bar, 10 µm. (B) Example micrographs from single-molecule motility TIRF assay showing TMR-labeled Halo-HAP1 traversing an MT from the dynamic plus end to the minus end, indicated by the 647-labeled MT seed. Single stills from both tubulin channels are shown (488 and 647) as well as the first and last frames merged (all three channels). Scale bar, 2 µm. (C and ;D) Example kymographs showing dynein-directed motility of HAP1 and BICD2N puncta in the lysate-based motility assay. Kymographs span the length of each MT with the plus end (+) and minus end (−) labeled at the bottom. Arrowheads represent motile minus end–directed events (teal), diffusive events (yellow), and sustained MT binding events (pink). (E–G) Quantifications of single-molecule motility assay showing the average velocity (E), run length (F), and stationary event duration (G).n = 30–51 runs (E and F), 457–471 events (G) in three independent trials on 57 total MTs. Welch’s two-tailed t test (E: P = 0.1969), Mann-Whitney U test (F: P = 0.0568; G: P < 0.0001). Y-axes in F and G represent 1 - the cumulative distribution function (CDF); x-axis in F begins at 1 µm because only runs ≥1 µm were analyzed; x-axis in G ends at 120 s, the length of videos acquired. (H and I) Blotting and quantification of cell extract MT pelleting assay. n = 3 independent assays; one-way ANOVA followed by Tukey’s multiple comparisons test (ATP addition, P = 0.6691; KCl without ATP, P = 0.0075; KCl with ATP, P = 0.0011). Bars throughout show mean ± SEM. *, P < 0.05; ****, P < 0.0001.

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