The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules
- Department of Anatomy and Cell Biology, McGill University, Canada
Figures
Figure 1 with 1 supplement
Cytoskeleton-Associated Protein 2 (CKAP2) is an intrinsically disordered protein that increases microtubule formation.
(a) Schematic of mmCKAP2 protein domains and disorder prediction (Dosztányi et al., 2005; Obradovic et al., 2003). (b) Circular dichroism of 4.5 μM CKAP2. (c) Coomassie Blue-stained sodium dodecyl …
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Figure 1—source data 1
Protein purification gel.
- https://cdn.elifesciences.org/articles/72202/elife-72202-fig1-data1-v2.zip
Figure 1—figure supplement 1
Cytoskeleton-Associated Protein 2 (CKAP2) is an intrinsically disordered protein that increases microtubule assembly.
(a) Comparison of amino acid sequence conservation for CKAP2, TPX2, and chTOG between human (hs, Homo sapiens), mouse (ms, Mus musculus), and African clawed frog (xl, Xenopus laevis) and amino acid …
Figure 2 with 1 supplement
Cytoskeleton-Associated Protein 2 (CKAP2) lowers the critical concentration of microtubule growth and speeds up microtubule assembly rates.
(a) Dynamic microtubule growth assay schematic (upper panel). Microtubule dynamics for tubulin control and in the presence of CKAP2-mNeonGreen (mNG) are analyzed from space–time plots (kymographs, …
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Figure 2—source data 1
The data and analysis for microtubule growth rates for different concentrations of Cytoskeleton-Associated Protein 2 (CKAP2).
- https://cdn.elifesciences.org/articles/72202/elife-72202-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Cytoskeleton-Associated Protein 2 (CKAP2) lowers the critical concentration of microtubule growth and speeds up microtubule assembly rates.
(a) Representative kymograph of microtubule growth in the presence and absence of CKAP2-mNeonGreen (mNG). (b) Schematic of linear polymer growth equation used to derive apparent on-rate constant ka, …
Figure 3 with 1 supplement
Cytoskeleton-Associated Protein 2 (CKAP2) increases templated and spontaneous microtubule nucleation.
(a) Representative field of view of guanosine-5′-[(α,β)-methyleno]triphosphate (GMPCPP)-stabilized microtubule seeds nucleating microtubules from 8 µM tubulin. (b) Plot of the cumulative probability …
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Figure 3—source data 1
The data and analysis for templated and spontaneous microtubule nucleation for different concentrations of Cytoskeleton-Associated Protein 2 (CKAP2).
- https://cdn.elifesciences.org/articles/72202/elife-72202-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Cytoskeleton-Associated Protein 2 (CKAP2) increases templated and spontaneous microtubule nucleation.
(a) Plots of the cumulative probability distributions for time to nucleate as a function of tubulin concentration, same n as Figure 3C.
Figure 4 with 1 supplement
Cytoskeleton-Associated Protein 2 (CKAP2) suppresses catastrophe.
(a) Kymographs of representative microtubule growth in the absence and presence of CKAP2-mNeonGreen (mNG). (b) Plot showing the microtubule catastrophe frequencies as a function of CKAP2-mNG …
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Figure 4—source data 1
The data and analysis for catastrophe frequency measurements.
- https://cdn.elifesciences.org/articles/72202/elife-72202-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Cytoskeleton-Associated Protein 2 (CKAP2) suppresses catastrophe.
(a) Plot of tubulin growth rate versus catastrophe frequency. (b) Plot of the % of microtubules still growing (cumulative microtubule survival) as a function of time, same n as Figure 4B.
Figure 5 with 1 supplement
Cytoskeleton-Associated Protein 2 (CKAP2) recruits tubulin, recognizes lattice curvature, but does not catalyze microtubule depolymerization.
(a) Size exclusion chromatography traces for 4 µM CKAP2, 4 µM tubulin, and a combination of both. (b) Field of view showing 1 µM CKAP2-mNeonGreen (mNG) recruiting Atto-633 tubulin to guanosine-5′-[(α…
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Figure 5—source data 1
The data and analysis for tubulin recruitment by Cytoskeleton-Associated Protein 2 (CKAP2) and shrinkage rate for guanosine-5′-[(α,β)-methyleno]triphosphate (GMPCPP) microtubules in the presence and absence of CKAP2.
- https://cdn.elifesciences.org/articles/72202/elife-72202-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Cytoskeleton-Associated Protein 2 (CKAP2) recruits tubulin, recognizes lattice curvature, but does not catalyze microtubule depolymerization.
(a) Guanosine-5′-[(α,β)-methyleno]triphosphate (GMPCPP) ‘seed’ depolymerization kymograph for 1 µM CKAP2-mNeonGreen (mNG) over 10 hr.
Author response image 1
Tables
Author response table 1
| Published concentration for Xenopus (Table S2/S3, Wuehr et al. 2014) | |||||
|---|---|---|---|---|---|
| Α Tubulins | ng | Β Tubulins | ng | ||
| TUBA4A | Tubulin α-4A chain | 4741.14 | TUBB | Tubulin β chain | 5931.21 |
| TUBA3C | Tubulin α-3C/D chain | 1686.89 | TUBB2B | Tubulin β-2B chain | 1357.19 |
| TUBA1B | Tubulin α-1B chain | 1143.46 | TUBB4B | Tubulin β-4B chain | 1173.38 |
| TUBA1A | Tubulin α-1A chain | 790.93 | |||
| TUBA1C | Tubulin α-1C chain | 439.50 | |||
| 8801.91 | 8461.78 |
Additional files
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Supplementary file 1
Available plasmids used for Cytoskeleton-Associated Protein 2 (CKAP2) protein expression.
- https://cdn.elifesciences.org/articles/72202/elife-72202-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/72202/elife-72202-transrepform1-v2.docx
