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. 2019 Jan 7;218(1):190–205. doi: 10.1083/jcb.201805047

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© 2018 Lindeboom et al.

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Figure 4. — CLASP signal intensities at crossover sites. (A and B) Example of MT and CLASP signal distribution in a 3-d-old dark-grown hypocotyl epidermal cell of a clasp mutant that is complemented by a YFP-CLASP construct and in addition expresses mCherry-TUA5 (see Video 4). A single frame is shown in A, and B shows a maximum-intensity projection of the first 31 frames at a 5-s time interval. Scale bar is 5 µm. (C) Kymograph of MT plus end stabilization and regrowth after severing at a microtubule crossover, showing both MT and CLASP signal (see Video 6). Cyan arrowhead indicates the point of stabilization and regrowth of the new plus end as observed in the MT channel. (D) Histograms of the distribution of CLASP-to-MT signal ratio along the microtubules, excluding crossover sites. The top panel shows the distribution for free MT lattice, and the bottom panel shows the distribution exclusively for MT crossovers (n = 33,742 and n = 1,893 pixels for MT lattice and MT crossovers, respectively, with n = 396 crossovers in six cells). The distribution of CLASP-to-MT signal ratio for the crossovers is significantly different from that on the free MT lattice (P < 0.05, Mann–Whitney U test, one-tailed), with the crossover distribution depleted in the high-ratio values measured along the free lattice. (E) Bar graph depicting the frequency of high CLASP-to-MT signal ratio (>1.5) at MT lattice versus MT crossovers (n = 33,742 and n = 1,893 pixels for MT lattice and MT crossovers, respectively). The frequency of high CLASP-to-MT signal ratio is significantly lower at MT crossovers compared with MT lattice (***, P < 0.001, Fisher’s exact test, one-tailed; error bars show 95% confidence interval). (F) Boxplot of CLASP-to-MT signal intensity ratios at microtubule crossovers in the frame just before the lagging plus end was observed either growing (n = 100) or shrinking (n = 551). MTs observed growing immediately after severing showed modest but significantly higher CLASP-to-MT signal ratios than those observed shrinking after severing (*, P < 0.05, Mann–Whitney U test, one-tailed). Boxplots show the 25th and 75th percentile as box edges, the line in the box indicates median value, and the whiskers show the 2.5th and 97.5th percentile. (G) MT and CLASP signal intensities during crossover formation. The top panel shows the relative MT signal intensity, the middle panel the relative CLASP signal intensity, and the bottom panel shows the ratio of CLASP-to-MT signal intensity (n = 1,532 crossovers in six cells in six plants). The white background indicates the time before the crossovers are formed, and the gray background indicates the time at which the crossover is formed.