CP110 binds to MT plus ends and blocks their growth. (A) Scheme illustrating the domain organization of GFP-tagged human CEP97, CP110, and CEP97^CP110 chimeric constructs. Domain nomenclature: LRR, leucine-rich region; CC, coiled coil; IQ is the calmodulin-binding domain; GFP, green fluorescent protein; and SII, twin-Strep-tag. (B and D) Representative fields of view from time-lapse movies of in vitro reconstitution of MT growth from GMPCPP-stabilized seeds (blue) in presence of 15 μM tubulin (gray) and 30 nM GFP-CP110 (green) (B) or 80 nM CEP97^CP110-GFP (green) (D); blocked plus ends are indicated with arrowheads. (C and E) Kymographs illustrating a dynamic MT without CP110 or CEP97^CP110 binding, transient pausing, or plus end blocking by CP110 (C) or CEP97^CP110 (E); growth, pauses, or blocking events are indicated by white arrows. The plus and minus ends of the MTs are indicated by “+” and “−,” respectively, and lines below kymographs indicate the position of the GMPCPP-stabilized seed. (F and G) Percentage of MTs displaying no pauses, occasional pauses, or fully blocked seeds (F) and pause duration (G), observed over 10 min with increasing concentrations of CEP97^CP110. Plots show percentage mean-SEM (F) and median ± interquartile range (IQR) of pause duration (G) at different CEP97^CP110 concentrations, with data points showing individual MT plus ends. Nonsignificant (ns), P > 0.05; **P = 0.0036 with Kruskal–Wallis ANOVA with Dunn’s test for multiple comparisons. n = 7, 14, 13, 20, 30, and 34 MT plus ends for 2, 4, 7.5, 10, 20, and 40 nM of CEP97^CP110, respectively. Number of independent assays was 3, 3, 4, 3, 3, 3, 4 for 2, 4, 7.5, 10, 20, 40, and 80 nM CEP97^CP110. (H) Histograms of fluorescence intensities of single molecules of GFP (n = 6,865), GFP-EB3 (n = 14,082), and CEP97^CP110-GFP (n = 6,942) immobilized on coverslips (symbols) and the corresponding fits with lognormal distributions (lines). The inset shows the number of CEP97^CP110-GFP molecules present at a paused or blocked MT plus end. The values were obtained by comparing the fitted mean intensity of CEP97^CP110-GFP at MT tips with the fitted mean intensity of single GFP molecules in parallel chambers. Floating bars represent maximum to minimum intensities of CEP97^CP110-GFP molecules relative to GFP per condition, with the line showing the mean value (n = 23 MTs for paused MTs at 7.5 nM, for blocked MTs n = 15 at 7.5 nM, n = 22 at 40 nM, and n = 28 at 80 nM). (I) Kymographs showing unbleached control and bleached CEP97^CP110-GFP at a blocked MT plus end. White arrow shows the moment of bleaching. (J) Mean + SD of the normalized intensity of CEP97^CP110-GFP at the MT plus end with (n = 28 MTs) and without bleaching (n = 12 MTs) from three independent assays. Frames were acquired at 2 s time interval. (K) Kymographs showing MT plus ends blocked with 2 µM unlabeled DARPin-(TM-3)2 alone (right) or in combination with 3 nM (middle) or 40 nM (right) CEP97^CP110-GFP (green). CEP97^CP110-GFP was bound for a part of the observation time (partial) or for the whole 10 min duration of the movie (full). (L) Percentage of MT plus ends blocked by DARPin-(TM-3)2 that also have CEP97^CP110 bound to them at 3 nM (n = 91 MTs) and 40 nM (n = 110 MTs) in two and four independent assays, respectively. (M) Mean-SEM of fluorescence intensity of CEP97^CP110-GFP on MT plus ends in presence (n = 83 MTs) and absence (n = 76 MTs) of DARPin-(TM-3)2 in two and four independent assays, respectively. Nonsignificant (ns), P = 0.626 with a two tailed Mann–Whitney U test.
