Figure 6.
The interaction between CPAP and CP110 promotes extremely slow and processive MT growth in vitro. (A) Scheme illustrating the domains of full-length human CPAP and the shorter versions called CPAP-NWT-mCh and CPAP-NMUT-mCh. CPAP-NWT includes the first 607 amino acids of CPAP followed by a leucine zipper for dimerization and a mCherry (mCh) fluorescent tag. PN2–3, tubulin-binding domain; MBD, MT-binding domain. Two substitution mutations L149A and K150A in CPAP-NMUT disrupt the interaction with CP110. (B) Still images and kymographs illustrating MT dynamics in the presence of CPAP-NWT-mCh and CPAP-NMUT-mCh; arrowheads indicate CPAP on the plus ends of dynamic MTs (gray). (C) Top plot: Mean ± SEM of intensity of the CPAP-NWT-mCh (n = 15 MTs) and CPAP-NMUT-mCh (n = 14 MTs) present on the MT plus end. Bottom plot: Catastrophe frequencies for CPAP-NWT-mCh (n = 151 MTs) and CPAP-NMUT-mCh (n = 129 MTs); nonsignificant (ns); P > 0.05, Mann–Whitney U test. (D) Kymographs showing slow and processive growth of MT plus end (gray) in presence of both CPAP-NWT-mCh (magenta, white open arrowhead indicates plus end accumulation of CPAP-NWT-mCh) and CEP97^CP110-GFP (green, white arrowhead indicates plus end accumulation of CEP97^CP110-GFP). CPAP-NWT is not visible as it is in the same channel as the bright GMPCPP-stabilized seed (magenta). (E) Kymographs representing MTs (gray) growing from the GMPCPP seed (magenta) in presence of CPAP-NMUT-mCh (magenta, white open arrowhead shows plus end localization) and CEP97^CP110-GFP (green, binding event indicated by a white arrowhead). (F) Bar plot with mean-SEM of the percentage of total time that CEP97^CP110 is either bound (green bars) or unbound (gray bars) to the MT plus end with CEP97^CP110 alone (n = 3 independent assays) or in combination with CPAP-NWT-mCh (n = 4 independent assays) or CPAP-NMUT-mCh (n = 3 independent assays). Nonsignificant (ns), P = 0.073, 0.156; **P = 0.0051, one-way ANOVA with Holm–Šídák’s multiple comparisons test. Normality tested using Shapiro–Wilk test; P = 0.88. (G) MT plus-end growth rates in the indicated conditions. Upper panel, mean + SD; bottom panel, a cumulative histogram showing % of the total time spent by MT plus end growing at different growth rates, with X axis in a log scale. n, number of growth events analyzed, is indicated in the figure. Nonsignificant (ns), P = 0.108; ***P = 0.0004; ****P < 0.0001, Kruskal–Wallis ANOVA with Dunn’s test for multiple comparisons. (H) MT plus-end growth rates for the samples 20 nM CEP97^CP110 alone, or in combination with 50 nM CPAP-NWT or CPAP-NMUT, also shown in panel G, but with the values for the events where CEP97^CP110 is present at the tip shown in green and the events where it is absent in gray. The bottom part of the plot shows magnified view for the growth rate values between 0 and 0.4 µm/min; n, number of growth events analyzed, is indicated in the figure.

The interaction between CPAP and CP110 promotes extremely slow and processive MT growth in vitro. (A) Scheme illustrating the domains of full-length human CPAP and the shorter versions called CPAP-NWT-mCh and CPAP-NMUT-mCh. CPAP-NWT includes the first 607 amino acids of CPAP followed by a leucine zipper for dimerization and a mCherry (mCh) fluorescent tag. PN2–3, tubulin-binding domain; MBD, MT-binding domain. Two substitution mutations L149A and K150A in CPAP-NMUT disrupt the interaction with CP110. (B) Still images and kymographs illustrating MT dynamics in the presence of CPAP-NWT-mCh and CPAP-NMUT-mCh; arrowheads indicate CPAP on the plus ends of dynamic MTs (gray). (C) Top plot: Mean ± SEM of intensity of the CPAP-NWT-mCh (n = 15 MTs) and CPAP-NMUT-mCh (n = 14 MTs) present on the MT plus end. Bottom plot: Catastrophe frequencies for CPAP-NWT-mCh (n = 151 MTs) and CPAP-NMUT-mCh (n = 129 MTs); nonsignificant (ns); P > 0.05, Mann–Whitney U test. (D) Kymographs showing slow and processive growth of MT plus end (gray) in presence of both CPAP-NWT-mCh (magenta, white open arrowhead indicates plus end accumulation of CPAP-NWT-mCh) and CEP97^CP110-GFP (green, white arrowhead indicates plus end accumulation of CEP97^CP110-GFP). CPAP-NWT is not visible as it is in the same channel as the bright GMPCPP-stabilized seed (magenta). (E) Kymographs representing MTs (gray) growing from the GMPCPP seed (magenta) in presence of CPAP-NMUT-mCh (magenta, white open arrowhead shows plus end localization) and CEP97^CP110-GFP (green, binding event indicated by a white arrowhead). (F) Bar plot with mean-SEM of the percentage of total time that CEP97^CP110 is either bound (green bars) or unbound (gray bars) to the MT plus end with CEP97^CP110 alone (n = 3 independent assays) or in combination with CPAP-NWT-mCh (n = 4 independent assays) or CPAP-NMUT-mCh (n = 3 independent assays). Nonsignificant (ns), P = 0.073, 0.156; **P = 0.0051, one-way ANOVA with Holm–Šídák’s multiple comparisons test. Normality tested using Shapiro–Wilk test; P = 0.88. (G) MT plus-end growth rates in the indicated conditions. Upper panel, mean + SD; bottom panel, a cumulative histogram showing % of the total time spent by MT plus end growing at different growth rates, with X axis in a log scale. n, number of growth events analyzed, is indicated in the figure. Nonsignificant (ns), P = 0.108; ***P = 0.0004; ****P < 0.0001, Kruskal–Wallis ANOVA with Dunn’s test for multiple comparisons. (H) MT plus-end growth rates for the samples 20 nM CEP97^CP110 alone, or in combination with 50 nM CPAP-NWT or CPAP-NMUT, also shown in panel G, but with the values for the events where CEP97^CP110 is present at the tip shown in green and the events where it is absent in gray. The bottom part of the plot shows magnified view for the growth rate values between 0 and 0.4 µm/min; n, number of growth events analyzed, is indicated in the figure.

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