Figure 1.
CSPP1 suppresses catastrophes by binding to polymerizing ends where it induces pausing. (A) Schematic representation of the two isoforms expressed by the CSPP1 gene in mammals. Black boxes represent α-helical domains larger than 20 amino acids predicted by AlphaFold. (B) Field of view (left, scale bar 10 µm) and time-lapse images (right, scale bar 3 µm) illustrating MT growth from GMPCPP-stabilized MT seeds in the presence of 15 µM tubulin supplemented with 3% rhodamine-labeled tubulin and 10 nM GFP-CSPP-L. MT polarity is indicated. (C and D) Kymographs illustrating MT growth either with rhodamine-tubulin (C) or mCherry-EB3 (D), supplemented, where indicated, with the indicated concentrations of GFP-CSPP-L. Time and distance axes are indicated with black arrows; growth, shrinkage, and pause events are indicated with bent, white arrows. Scale bars, 2 μm (horizontal) and 60 s (vertical). (E and F) Parameters of MT plus end dynamics in the presence of rhodamine-tubulin alone or together with 20 nM mCherry-EB3 in combination with the indicated GFP-CSPP-L concentrations (from kymographs as shown in C and D). Events were classified as pauses when the pause duration was longer than 20 s. Total number of growth events, pauses, and MTs analyzed (E); tubulin alone, n = 394, 0, 110; tubulin with 10 nM CSPP-L, n = 596, 481, 78; EB3 alone, n = 514, 0, 53; EB3 with 0.5 nM CSPP-L, n = 476, 10, 44; EB3 with 5 nM CSPP-L, n = 564, 241, 47; EB3 with 10 nM CSPP-L, n = 731, 518, 89. Total number of transition events analyzed (F): tubulin alone, n = 194, 0, 0, 0, 15, 0; tubulin with 10 nM CSPP-L, n = 0, 443, 410, 25, 7, 17; 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 10 nM CSPP-L, n = 24, 465, 455, 22, 25, 21. 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. Error bars represent SEM ***, P < 0.001; n.s., not significant; Kruskal–Wallis test followed by Dunn’s post-test. (G) Single frame images of a COS-7 cell overexpressing GFP-CSPP-L and EB3-mCherry, imaged by TIRF microscopy. Scale bars, 5 µm. (H) Kymographs (top) and schematic representation of these kymographs (bottom) of the events indicated with white arrowheads in G. In the schemes, unlabeled MT is visualized in grey, mCherry-EB3 in magenta, and GFP-CSPP-L in green. Bent white arrows indicate growth and pause events; dashed white arrows indicate the direction of movement of the whole MT at that time point. Scale bars, 2 μm (horizontal) and 4 s (vertical). (I) Normalized intensity graphs of EB3-mCherry and GFP-CSPP-L within the white box in H. See also Fig. S1 and Videos 1 and 2.

CSPP1 suppresses catastrophes by binding to polymerizing ends where it induces pausing. (A) Schematic representation of the two isoforms expressed by the CSPP1 gene in mammals. Black boxes represent α-helical domains larger than 20 amino acids predicted by AlphaFold. (B) Field of view (left, scale bar 10 µm) and time-lapse images (right, scale bar 3 µm) illustrating MT growth from GMPCPP-stabilized MT seeds in the presence of 15 µM tubulin supplemented with 3% rhodamine-labeled tubulin and 10 nM GFP-CSPP-L. MT polarity is indicated. (C and D) Kymographs illustrating MT growth either with rhodamine-tubulin (C) or mCherry-EB3 (D), supplemented, where indicated, with the indicated concentrations of GFP-CSPP-L. Time and distance axes are indicated with black arrows; growth, shrinkage, and pause events are indicated with bent, white arrows. Scale bars, 2 μm (horizontal) and 60 s (vertical). (E and F) Parameters of MT plus end dynamics in the presence of rhodamine-tubulin alone or together with 20 nM mCherry-EB3 in combination with the indicated GFP-CSPP-L concentrations (from kymographs as shown in C and D). Events were classified as pauses when the pause duration was longer than 20 s. Total number of growth events, pauses, and MTs analyzed (E); tubulin alone, n = 394, 0, 110; tubulin with 10 nM CSPP-L, n = 596, 481, 78; EB3 alone, n = 514, 0, 53; EB3 with 0.5 nM CSPP-L, n = 476, 10, 44; EB3 with 5 nM CSPP-L, n = 564, 241, 47; EB3 with 10 nM CSPP-L, n = 731, 518, 89. Total number of transition events analyzed (F): tubulin alone, n = 194, 0, 0, 0, 15, 0; tubulin with 10 nM CSPP-L, n = 0, 443, 410, 25, 7, 17; 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 10 nM CSPP-L, n = 24, 465, 455, 22, 25, 21. 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. Error bars represent SEM ***, P < 0.001; n.s., not significant; Kruskal–Wallis test followed by Dunn’s post-test. (G) Single frame images of a COS-7 cell overexpressing GFP-CSPP-L and EB3-mCherry, imaged by TIRF microscopy. Scale bars, 5 µm. (H) Kymographs (top) and schematic representation of these kymographs (bottom) of the events indicated with white arrowheads in G. In the schemes, unlabeled MT is visualized in grey, mCherry-EB3 in magenta, and GFP-CSPP-L in green. Bent white arrows indicate growth and pause events; dashed white arrows indicate the direction of movement of the whole MT at that time point. Scale bars, 2 μm (horizontal) and 4 s (vertical). (I) Normalized intensity graphs of EB3-mCherry and GFP-CSPP-L within the white box in H. See also Fig. S1 and Videos 1 and 2.

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