Figure 6. CSPP1 stabilizes MTs by... | Rockefeller University Press

Figure 6.

$CSPP1 stabilizes MTs by promoting lattice repair. (A) Kymographs of Taxol-stabilized MT seeds in absence or presence of the indicated Taxol, tubulin, and GFP-CSPP-L concentrations. Scale bars, 2 μm (horizontal) and 60 s (vertical). (B) GFP-CSPP-L intensity quantification per MT seed (from kymographs as shown in A). Mean GFP-CSPP-L intensity was measured along the entire length of the seed 2 min after flowing in the protein. The average mean intensity of GFP-CSPP-L in presence of 40 µM Taxol was normalized to the average mean intensity in absence of free Taxol. Total number of Taxol-stabilized MT seeds analyzed: 5 nM CSPP-L alone, n = 102; 20 nM CSPP-L alone, n = 108; 40 µM Taxol, n = 99; 40 µM Taxol together with 5 nM CSPP-L, n = 84; 40 µM Taxol together with 20 nM CSPP-L, n = 114. Bars represent pooled data from two independent experiments. Error bars represent SEM. ***, P < 0.001; Kruskal–Wallis test followed by Dunn’s post-test. (C) Quantification of the percentage of MT seeds that survived 5 min after flow-in of the reaction mix (from kymographs as shown in A). Total number of Taxol-stabilized MT seeds analyzed: control: n = 95; 5 nM CSPP-L alone, n = 120; 20 nM CSPP-L alone, n = 110; 40 µM Taxol, n = 99; 40 µM Taxol together with 5 nM CSPP-L, n = 84; 40 µM Taxol together with 20 nM CSPP-L, n = 120; 2 µM tubulin alone, n = 115; 2 µM tubulin together with 5 nM CSPP-L, n = 124; 2 µM tubulin together with 20 nM CSPP-L, n = 112; 5 µM tubulin alone, n = 122; 5 µM tubulin together with 5 nM CSPP-L, n = 106; 5 µM tubulin together with 20 nM CSPP-L, n = 128. Bars represent pooled data from two independent experiments. Error bars represent SEM. ***, P < 0.001; **, P < 0.01; n.s., not significant; Kruskal–Wallis test followed by Dunn’s post-test. In “control,” conditions with 5 and 20 nM CSPP-L are compared to 0 nM CSPP-L, and for all other bars, comparisons were made to the same CSPP-L concentration in the control condition. (D) Quantification of the fraction of the total GMPCPP seeds that showed MT outgrowth within 10 min at indicated tubulin concentrations, with tubulin alone or together with 5 nM GFP-CSPP-L. Total number of GMPCPP seeds analyzed: 2 µM tubulin alone, n = 74; 5 µM tubulin alone, n = 75; 15 µM alone, n = 69; 2 µM tubulin together with 5 nM CSPP-L, n = 70; 5 µM tubulin together with 5 nM CSPP-L, n = 66; 15 µM tubulin together with 5 nM CSPP-L FL, n = 71. Symbols represent pooled data from two independent experiments. Error bars represent SEM. (E) Kymographs of GMPCPP-stabilized MT seeds in the presence of 5 nM GFP-CSPP-L and the indicated tubulin concentrations. Scale bars, 2 μm (horizontal) and 60 s (vertical). (F) GFP-CSPP-L intensity quantification per µm newly grown MT lattice (from kymographs as shown in E). GFP-CSPP-L integrated intensity was measured on newly grown lattice 5 min after flow-in of the reaction mix. The integrated intensity was normalized to newly grown MT lattice length, and the average mean intensity of GFP-CSPP-L in presence of 15 µM tubulin was normalized to the average mean intensity in presence of 5 µM tubulin. Total number of growth episodes analyzed: 5 µM tubulin, n = 105; 15 µM tubulin, n = 104. Bars represent pooled data from two independent experiments. Error bars represent SEM. ***, P < 0.001, Mann-Whitney test. (G) Denoised tomograms of dynamic MTs polymerized in the presence of 250 µM Taxol, resuspended in buffer containing only 20 nM GFP-CSPP-L with or without free 40 µM Taxol, vitrified on EM grids. Scale bar, 25 nm. (H) Quantification of the percentage of MTs containing luminal densities from total MTs (from tomograms as shown in G). Orange and gray dots (single data points, tomograms), black circle (mean), SD (error bars). **, P < 0.01, Mann–Whitney test. Analysis from two independent experiments. (I) Denoised tomograms of dynamic MTs polymerized in the presence or absence of 250 nM vinblastine with or without 20 nM GFP-CSPP-L, vitrified on EM grids. Scale bar, 25 nm. (J) Quantification of the number of defects per µm MT (from tomograms as shown in I. Orange and gray dots (single data points, tomograms), black circles (mean), SD (error bars). *, P < 0.1, **, P < 0.01, n.s., not significant, Mann–Whitney test. Analysis from two independent experiments. (K) Denoised tomograms of MT ends in the presence or absence of 250 nM vinblastine with or without 20 nM GFP-CSPP-L, vitrified on EM grids. Scale bars, 50 nm. (L) Parameters extracted from manual segmentations of terminal protofilaments. Raggedness is defined as the standard deviation in the coordinate along the MT axis of the first deflection point for each protofilament in a MT end (Gudimchuk et al., 2020). (M) Quantification of plus-end raggedness (from tomograms as shown in K). Blue, orange, and gray dots (single data points, tomograms), black circle (mean), SD (error bars). *, P < 0.1, ***, P < 0.001, n.s., not significant, Mann–Whitney test. Analysis from two independent experiments. See also Fig. S4.$

CSPP1 stabilizes MTs by promoting lattice repair. (A) Kymographs of Taxol-stabilized MT seeds in absence or presence of the indicated Taxol, tubulin, and GFP-CSPP-L concentrations. Scale bars, 2 μm (horizontal) and 60 s (vertical). (B) GFP-CSPP-L intensity quantification per MT seed (from kymographs as shown in A). Mean GFP-CSPP-L intensity was measured along the entire length of the seed 2 min after flowing in the protein. The average mean intensity of GFP-CSPP-L in presence of 40 µM Taxol was normalized to the average mean intensity in absence of free Taxol. Total number of Taxol-stabilized MT seeds analyzed: 5 nM CSPP-L alone, n = 102; 20 nM CSPP-L alone, n = 108; 40 µM Taxol, n = 99; 40 µM Taxol together with 5 nM CSPP-L, n = 84; 40 µM Taxol together with 20 nM CSPP-L, n = 114. Bars represent pooled data from two independent experiments. Error bars represent SEM. ***, P < 0.001; Kruskal–Wallis test followed by Dunn’s post-test. (C) Quantification of the percentage of MT seeds that survived 5 min after flow-in of the reaction mix (from kymographs as shown in A). Total number of Taxol-stabilized MT seeds analyzed: control: n = 95; 5 nM CSPP-L alone, n = 120; 20 nM CSPP-L alone, n = 110; 40 µM Taxol, n = 99; 40 µM Taxol together with 5 nM CSPP-L, n = 84; 40 µM Taxol together with 20 nM CSPP-L, n = 120; 2 µM tubulin alone, n = 115; 2 µM tubulin together with 5 nM CSPP-L, n = 124; 2 µM tubulin together with 20 nM CSPP-L, n = 112; 5 µM tubulin alone, n = 122; 5 µM tubulin together with 5 nM CSPP-L, n = 106; 5 µM tubulin together with 20 nM CSPP-L, n = 128. Bars represent pooled data from two independent experiments. Error bars represent SEM. ***, P < 0.001; **, P < 0.01; n.s., not significant; Kruskal–Wallis test followed by Dunn’s post-test. In “control,” conditions with 5 and 20 nM CSPP-L are compared to 0 nM CSPP-L, and for all other bars, comparisons were made to the same CSPP-L concentration in the control condition. (D) Quantification of the fraction of the total GMPCPP seeds that showed MT outgrowth within 10 min at indicated tubulin concentrations, with tubulin alone or together with 5 nM GFP-CSPP-L. Total number of GMPCPP seeds analyzed: 2 µM tubulin alone, n = 74; 5 µM tubulin alone, n = 75; 15 µM alone, n = 69; 2 µM tubulin together with 5 nM CSPP-L, n = 70; 5 µM tubulin together with 5 nM CSPP-L, n = 66; 15 µM tubulin together with 5 nM CSPP-L FL, n = 71. Symbols represent pooled data from two independent experiments. Error bars represent SEM. (E) Kymographs of GMPCPP-stabilized MT seeds in the presence of 5 nM GFP-CSPP-L and the indicated tubulin concentrations. Scale bars, 2 μm (horizontal) and 60 s (vertical). (F) GFP-CSPP-L intensity quantification per µm newly grown MT lattice (from kymographs as shown in E). GFP-CSPP-L integrated intensity was measured on newly grown lattice 5 min after flow-in of the reaction mix. The integrated intensity was normalized to newly grown MT lattice length, and the average mean intensity of GFP-CSPP-L in presence of 15 µM tubulin was normalized to the average mean intensity in presence of 5 µM tubulin. Total number of growth episodes analyzed: 5 µM tubulin, n = 105; 15 µM tubulin, n = 104. Bars represent pooled data from two independent experiments. Error bars represent SEM. ***, P < 0.001, Mann-Whitney test. (G) Denoised tomograms of dynamic MTs polymerized in the presence of 250 µM Taxol, resuspended in buffer containing only 20 nM GFP-CSPP-L with or without free 40 µM Taxol, vitrified on EM grids. Scale bar, 25 nm. (H) Quantification of the percentage of MTs containing luminal densities from total MTs (from tomograms as shown in G). Orange and gray dots (single data points, tomograms), black circle (mean), SD (error bars). **, P < 0.01, Mann–Whitney test. Analysis from two independent experiments. (I) Denoised tomograms of dynamic MTs polymerized in the presence or absence of 250 nM vinblastine with or without 20 nM GFP-CSPP-L, vitrified on EM grids. Scale bar, 25 nm. (J) Quantification of the number of defects per µm MT (from tomograms as shown in I. Orange and gray dots (single data points, tomograms), black circles (mean), SD (error bars). *, P < 0.1, **, P < 0.01, n.s., not significant, Mann–Whitney test. Analysis from two independent experiments. (K) Denoised tomograms of MT ends in the presence or absence of 250 nM vinblastine with or without 20 nM GFP-CSPP-L, vitrified on EM grids. Scale bars, 50 nm. (L) Parameters extracted from manual segmentations of terminal protofilaments. Raggedness is defined as the standard deviation in the coordinate along the MT axis of the first deflection point for each protofilament in a MT end (Gudimchuk et al., 2020). (M) Quantification of plus-end raggedness (from tomograms as shown in K). Blue, orange, and gray dots (single data points, tomograms), black circle (mean), SD (error bars). *, P < 0.1, ***, P < 0.001, n.s., not significant, Mann–Whitney test. Analysis from two independent experiments. See also Fig. S4.