Figures and data
Dynein tip-tracks on polymerizing MTs and walks processively at high velocities in interphase HeLa cells. (A)
Representative spinning disc confocal time-lapse of a DHC-EGFP-expressing HeLa cell showing robust tip-tracking. Zoomed views of the numbered boxed regions are shown to the right with kymographs of the tip-tracking events highlighted with yellow arrows in the zoomed panels. (B) Representative TIRFM time-lapse of a DHC-EGFP-expressing HeLa cell showing the tip-tracking population. Zoomed views of the numbered boxed regions are shown with kymographs of the tip-tracking events highlighted with yellow arrows. (C) Still frame from a representative TIRFM time-lapse of a DHC-EGFP-expressing HeLa cell treated with SiR-Tubulin. In the merge image DHC is green and Sir-Tubulin labeled MTs are magenta. (D) Still frame from a representative high temporal resolution (5 fps) TIRFM time-lapse of a DHC-EGFP-expressing HeLa cell treated with SiR-Tubulin. Boxed region is shown as a zoomed inset in the lower panel with the track of a motile DHC puncta highlighted in green. (E) Representative kymographs of motile puncta spanning the range of measured velocities (Vel) and run lengths (RL). (F) Distribution of DHC velocities (n = 100 puncta). (G) Distribution of DHC run lengths (n = 81 puncta). (H) Distribution of DHC run times (n = 81 puncta). Scale bars, 10 μm (A-D); 1 μm (all insets), 10 μm (horizontal); 1 min (vertical) in the kymographs in A and B; and 1 μm (horizontal); 1 second (vertical) in E. Displayed times are min:sec.
The dynactin complex component p50 tip-tracks on polymerizing MTs and walks processively at high velocities in interphase HeLa cells. (A)
Still frames from a representative spinning disc confocal time-lapse of a p50-EGFP-expressing HeLa cell. Zoomed view of the boxed region is shown with a kymograph of the tip-tracking event highlighted with the yellow arrow. (B) Still frames from a representative TIRFM time-lapse of a p50-EGFP-expressing HeLa cell showing the tip-tracking population. Zoomed view of the boxed region is shown with a kymograph of the tip-tracking event highlighted by the yellow arrow. (C) Still frame from a representative TIRFM time-lapse of a p50-EGFP-expressing HeLa cell treated with SiR-Tubulin. In the merge image p50 is green and Sir-Tubulin labeled MTs are magenta. (D) Representative kymographs of motile p50 puncta spanning the range of measured velocities (Vel) and run lengths (RL). (E) Distribution of p50 velocities (n = 44 puncta). (F) Distribution of p50 run lengths (n = 41 puncta). (G) Distribution of p50 run times (n = 41 puncta). Scale bars, 10 μm (A-C); 1 μm (all insets), 5 μm (horizontal); 1 min (vertical) in the kymographs in A and B; and 1 μm (horizontal); 1 second (vertical) in D. Displayed times are min:sec.
Motile DHC and p50 puncta exhibit identical motility parameters but different fluorescent intensities. (A-C)
Box and whisker plots of DHC and p50 velocities (A), run lengths (B), and run times (C). (D-F) Distributions of kinesin-1-EGFP (D), DHC-EGFP (E), and p50-EGFP background corrected fluorescence intensities. The dashed line in each histogram denotes the mean value of the kinesin-1-EGFP data set. (G) Box and whisker plots of the kinesin-1, DHC, and p50 fluorescence intensities. (H) PCR of genomic DNA from the DCH-EGP clone used in this study using PCR primers flanking the integration site of the repair cassette. The upper band was extracted and subjected to sequencing the results of which are shown in Figure 1 – supplemental figure 1. (I) Western blot for p50 of cell lysates from the parental HeLa cell line and the p50-EGFP clone used in this study. The tagged p50 runs ∼30 kDa larger than the untagged p50 and is expressed at ∼5-6-fold lower levels than the endogenous p50. In the box and whisker plots, whiskers indicate min-max, box is 25th-75th percentile, and bar is median. The reported p-values were determined by a randomization method: n.s. is not significant (p > 0.05).
