Figure 2.
CSPP1 binds to precatastrophe MT ends, resembling taxane behavior. (A) Kymographs of MT growth with 100 nM Fchitax-3 together with 5 nM mCherry-CSPP-L in the presence of 20 nM dark EB3. Scale bars, 2 μm (horizontal) and 60 s (vertical). (B) Normalized intensity graph of Fchitax-3 and mCherry-CSPP-L along the yellow line in A. (C) Normalized intensity graph of Fchitax-3 and mCherry-CSPP-L within the white box in A. (D) Quantification of the number of GFP-CSPP-L molecules per 8 nm MTs. The integrated intensity of one GFP-CSPP-L accumulation in an in vitro assay was divided by the average intensity of single GFP monomers in a separate chamber on the same coverslip and subsequently normalized to 8 nm accumulation length. The number of GFP-CSPP-L accumulations, n = 215 from two independent experiments. (E and F) Kymographs illustrating MT growth in the presence of 20 nM mCherry-EB3 together with 10 nM GFP-CSPP-L. Scale bars, 2 μm (horizontal) and 2 min (vertical, E) or s (vertical, F). (G) Time plot of the normalized maximum intensity profile of a single mCherry-EB3 comet and the normalized area under the curve (AUC) of a single GFP-CSPP-L accumulation (left) and averaged EB3 and GFP-CSPP-L profiles, normalized and aligned using half-maximum effective intensity values from Hill equation fits as reference points (right; from kymographs as shown in F). Light, thin lines represent SEM. Number of events analyzed, n = 12 from two independent experiments. (H) Kymographs illustrating MT growth in the presence of 20 nM mCherry-EB3 alone or together with the indicated concentrations of GFP-CSPP-L in the presence or absence of 100 nM vinblastine (VBL). Scale bars, 2 μm (horizontal) and 60 s (vertical). (I) Quantification of the mean GFP-CSPP-L intensity at the MT tip per growth event. The average mean intensity of GFP-CSPP-L in the presence of 100 nM vinblastine was normalized to the average mean intensity in absence of vinblastine. Total number of growth events analyzed; 0.5 nM GFP-CSPP-L control, n = 474; 5 nM GFP-CSPP-L control, n = 598; 0.5 nM GFP-CSPP-L with vinblastine, n = 1,363; 5 nM GFP-CSPP-L with vinblastine, n = 897. Bars represent pooled data from three independent experiments. (J and K) Parameters of MT plus end dynamics in the presence of 20 nM mCherry-EB3 together with the indicated GFP-CSPP-L concentrations (from kymographs as shown in G). Events were classified as pauses when the pause duration was longer than 20 s. Total number of growth events, pauses and MTs analyzed (J); EB3 alone, n = 514, 0, 53; EB3 with 0.5 nM CSPP-L, EB3 with 0.5 nM CSPP-L, n = 476, 10, 44; EB3 with 5 nM CSPP-L, n = 564, 241, 47; EB3 with vinblastine, n = 915, 0, 54; EB3 with 0.5 nM CSPP-L and vinblastine, n = 1,204, 33, 40; EB3 with 5 nM CSPP-L and vinblastine, n = 632, 408, 47. Total number of transition events analyzed (K): EB3 alone, n = 461, 0, 0, 0, 4, 0; EB3 with 0.5 nM CSPP-L, n = 309, 8, 10, 0, 216, 2; EB3 with 5 nM CSPP-L, n = 75, 209, 224, 9, 57, 27; EB3 with vinblastine, n = 162, 0, 0, 0, 33, 0; EB3 with 0.5 nM CSPP-L and vinblastine, n = 1,079, 19, 31, 0, 1,002, 14; EB3 with 5 nM CSPP-L and vinblastine, n = 147, 372, 386, 7, 127, 27. Bars for growth rate and pause duration represent pooled data from three independent experiments. For dynamic state and transition frequencies, bars represent the average of the means (symbols) of three independent experiments. Data for conditions without vinblastine is the same as in Fig. 1, E and F. (L) Kymographs illustrating MT growth in the presence of 20 nM mCherry-EB3 alone or together with 5 nM GFP-CSPP-L in presence of 3 nM MCAK. Scale bars, 2 μm (horizontal) and 60 s (vertical). For all plots. Error bars represent SEM. ***, P < 0.001; *, P < 0.1; n.s., Kruskal–Wallis test followed by Dunn’s post-test.

CSPP1 binds to precatastrophe MT ends, resembling taxane behavior. (A) Kymographs of MT growth with 100 nM Fchitax-3 together with 5 nM mCherry-CSPP-L in the presence of 20 nM dark EB3. Scale bars, 2 μm (horizontal) and 60 s (vertical). (B) Normalized intensity graph of Fchitax-3 and mCherry-CSPP-L along the yellow line in A. (C) Normalized intensity graph of Fchitax-3 and mCherry-CSPP-L within the white box in A. (D) Quantification of the number of GFP-CSPP-L molecules per 8 nm MTs. The integrated intensity of one GFP-CSPP-L accumulation in an in vitro assay was divided by the average intensity of single GFP monomers in a separate chamber on the same coverslip and subsequently normalized to 8 nm accumulation length. The number of GFP-CSPP-L accumulations, n = 215 from two independent experiments. (E and F) Kymographs illustrating MT growth in the presence of 20 nM mCherry-EB3 together with 10 nM GFP-CSPP-L. Scale bars, 2 μm (horizontal) and 2 min (vertical, E) or s (vertical, F). (G) Time plot of the normalized maximum intensity profile of a single mCherry-EB3 comet and the normalized area under the curve (AUC) of a single GFP-CSPP-L accumulation (left) and averaged EB3 and GFP-CSPP-L profiles, normalized and aligned using half-maximum effective intensity values from Hill equation fits as reference points (right; from kymographs as shown in F). Light, thin lines represent SEM. Number of events analyzed, n = 12 from two independent experiments. (H) Kymographs illustrating MT growth in the presence of 20 nM mCherry-EB3 alone or together with the indicated concentrations of GFP-CSPP-L in the presence or absence of 100 nM vinblastine (VBL). Scale bars, 2 μm (horizontal) and 60 s (vertical). (I) Quantification of the mean GFP-CSPP-L intensity at the MT tip per growth event. The average mean intensity of GFP-CSPP-L in the presence of 100 nM vinblastine was normalized to the average mean intensity in absence of vinblastine. Total number of growth events analyzed; 0.5 nM GFP-CSPP-L control, n = 474; 5 nM GFP-CSPP-L control, n = 598; 0.5 nM GFP-CSPP-L with vinblastine, n = 1,363; 5 nM GFP-CSPP-L with vinblastine, n = 897. Bars represent pooled data from three independent experiments. (J and K) Parameters of MT plus end dynamics in the presence of 20 nM mCherry-EB3 together with the indicated GFP-CSPP-L concentrations (from kymographs as shown in G). Events were classified as pauses when the pause duration was longer than 20 s. Total number of growth events, pauses and MTs analyzed (J); EB3 alone, n = 514, 0, 53; EB3 with 0.5 nM CSPP-L, EB3 with 0.5 nM CSPP-L, n = 476, 10, 44; EB3 with 5 nM CSPP-L, n = 564, 241, 47; EB3 with vinblastine, n = 915, 0, 54; EB3 with 0.5 nM CSPP-L and vinblastine, n = 1,204, 33, 40; EB3 with 5 nM CSPP-L and vinblastine, n = 632, 408, 47. Total number of transition events analyzed (K): EB3 alone, n = 461, 0, 0, 0, 4, 0; EB3 with 0.5 nM CSPP-L, n = 309, 8, 10, 0, 216, 2; EB3 with 5 nM CSPP-L, n = 75, 209, 224, 9, 57, 27; EB3 with vinblastine, n = 162, 0, 0, 0, 33, 0; EB3 with 0.5 nM CSPP-L and vinblastine, n = 1,079, 19, 31, 0, 1,002, 14; EB3 with 5 nM CSPP-L and vinblastine, n = 147, 372, 386, 7, 127, 27. Bars for growth rate and pause duration represent pooled data from three independent experiments. For dynamic state and transition frequencies, bars represent the average of the means (symbols) of three independent experiments. Data for conditions without vinblastine is the same as in Fig. 1, E and F. (L) Kymographs illustrating MT growth in the presence of 20 nM mCherry-EB3 alone or together with 5 nM GFP-CSPP-L in presence of 3 nM MCAK. Scale bars, 2 μm (horizontal) and 60 s (vertical). For all plots. Error bars represent SEM. ***, P < 0.001; *, P < 0.1; n.s., Kruskal–Wallis test followed by Dunn’s post-test.

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