Figure 3.
Importin α/β inhibit XCTK2 antiparallel MT cross-linking and sliding.(A) Schematic of XCTK2 antiparallel (left) and parallel (right) MT cross-linking and sliding assay with segmented MTs. (B) Representative images and kymographs of MT cross-linking and sliding with YPet-XCTK2 (left; Video 1) or YPet-XCTK2 with 4–8 molar excess of importin α/β (right; Video 2). The template MT minus (−) and plus (+) ends are indicated on the top kymograph image, and the sliding cargo MT plus end is indicated by an arrowhead. The YPet-XCTK2 kymograph images increment by 30-s intervals, and the YPet-XCTK2 + importin α/β increment by 75-s intervals to illustrate the multiple events over the course of the time lapse. Scale bars: 5 µm. (C–G) Quantification of the indicated conditions plotted as the number of cross-links per experiment, with the mean ± SD indicated as a bar graph, with the dots representing the values of individual experiments (n = 729 YPet-XCTK2 and 352 YPet-XCTK2 + importin α/β cross-linked MTs, n = 63 YPet-XCTK2 and 31 YPet-XCTK2 + importin α/β antiparallel cross-links, n = 81 YPet-XCTK2 and 56 YPet-XCTK2 + importin α/β parallel cross-links from six independent experiments; F test to compare variances and two-tailed Student’s or Welch’s t tests were performed: *, P < 0.05). (H) The number of sliding events per MT cross-link is graphed with the mean ± SD (n = 212 YPet-XCTK2 and 140 YPet-XCTK2 + importin α/β cross-links from three independent experiments; D’Agostino and Pearson normality and log-normal tests and two-tailed Mann–Whitney t test: ****, P < 0.0001). (I) Velocity of MT sliding events for antiparallel and parallel MT cross-links is graphed as a frequency histogram with the best-fit log Gaussian curve (n = 42 YPet-XCTK2 antiparallel and 71 parallel events, and n = 60 YPet-XCTK2 + importin α/β antiparallel and 116 parallel events from three independent experiments; D’Agostino and Pearson log-normal test and extra sum-of-squares F test to compare geometric means of the log Gaussian distribution of events: XCTK2 antiparallel versus parallel, *, P = 0.0362; XCTK2 + importin α/β antiparallel versus parallel, **, P = 0.0050). The frequency histogram is graphed up to 60 nm/s, excluding a single point at 120 nm/s.

Importin α/β inhibit XCTK2 antiparallel MT cross-linking and sliding. (A) Schematic of XCTK2 antiparallel (left) and parallel (right) MT cross-linking and sliding assay with segmented MTs. (B) Representative images and kymographs of MT cross-linking and sliding with YPet-XCTK2 (left; Video 1) or YPet-XCTK2 with 4–8 molar excess of importin α/β (right; Video 2). The template MT minus (−) and plus (+) ends are indicated on the top kymograph image, and the sliding cargo MT plus end is indicated by an arrowhead. The YPet-XCTK2 kymograph images increment by 30-s intervals, and the YPet-XCTK2 + importin α/β increment by 75-s intervals to illustrate the multiple events over the course of the time lapse. Scale bars: 5 µm. (C–G) Quantification of the indicated conditions plotted as the number of cross-links per experiment, with the mean ± SD indicated as a bar graph, with the dots representing the values of individual experiments (n = 729 YPet-XCTK2 and 352 YPet-XCTK2 + importin α/β cross-linked MTs, n = 63 YPet-XCTK2 and 31 YPet-XCTK2 + importin α/β antiparallel cross-links, n = 81 YPet-XCTK2 and 56 YPet-XCTK2 + importin α/β parallel cross-links from six independent experiments; F test to compare variances and two-tailed Student’s or Welch’s t tests were performed: *, P < 0.05). (H) The number of sliding events per MT cross-link is graphed with the mean ± SD (n = 212 YPet-XCTK2 and 140 YPet-XCTK2 + importin α/β cross-links from three independent experiments; D’Agostino and Pearson normality and log-normal tests and two-tailed Mann–Whitney t test: ****, P < 0.0001). (I) Velocity of MT sliding events for antiparallel and parallel MT cross-links is graphed as a frequency histogram with the best-fit log Gaussian curve (n = 42 YPet-XCTK2 antiparallel and 71 parallel events, and n = 60 YPet-XCTK2 + importin α/β antiparallel and 116 parallel events from three independent experiments; D’Agostino and Pearson log-normal test and extra sum-of-squares F test to compare geometric means of the log Gaussian distribution of events: XCTK2 antiparallel versus parallel, *, P = 0.0362; XCTK2 + importin α/β antiparallel versus parallel, **, P = 0.0050). The frequency histogram is graphed up to 60 nm/s, excluding a single point at 120 nm/s.

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