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Microtubule-associated proteins control the kinetics of microtubule nucleation

Nature Cell Biology volume 17, pages 907–916 (2015)Cite this article

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Abstract

Microtubules are born and reborn continuously, even during quiescence. These polymers are nucleated from templates, namely γ-tubulin ring complexes (γ-TuRCs) and severed microtubule ends. Using single-molecule biophysics, we show that nucleation from γ-TuRCs, axonemes and seed microtubules requires tubulin concentrations that lie well above the critical concentration. We measured considerable time lags between the arrival of tubulin and the onset of steady-state elongation. Microtubule-associated proteins (MAPs) alter these time lags. Catastrophe factors (MCAK and EB1) inhibited nucleation, whereas a polymerase (XMAP215) and an anti-catastrophe factor (TPX2) promoted nucleation. We observed similar phenomena in cells. We conclude that GTP hydrolysis inhibits microtubule nucleation by destabilizing the nascent plus ends required for persistent elongation. Our results explain how MAPs establish the spatial and temporal profile of microtubule nucleation.

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Figure 1: Microtubule nucleation from templates is a sigmoid function of tubulin concentration.
Figure 2: Microtubule plus ends elongate at tubulin concentrations where templates do not.
Figure 3: There is a time lag associated with templated nucleation.
Figure 4: Catastrophe factors slow down templated nucleation.
Figure 5: TPX2 is an anti-catastrophe factor that accelerates nucleation.
Figure 6: A polymerase and GMPCPP–tubulin both accelerate nucleation.
Figure 7: Depletion of tubulin by nocodazole reduces the rate of nucleation in cells.

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Acknowledgements

We thank H. Higgs for suggesting the hysteresis experiment (Fig. 2) during the question period of an American Society for Cell Biology mini-symposium. We thank L. Cassimeris (Lehigh University, USA) for providing the LLCPK1:EB1–GFP cell line, A. Bird (Max Planck Institute of Molecular Physiology, Germany) for providing the U2OS:EB3–mCherry cell line, and R. Ohi (Vanderbilt University, USA) for providing the LLCPK1:GFP–tubulin cell line. We thank the Cell Imaging and Analysis Network for microscopy support. We thank C. Rocha for help with immunoblot sample preparation. We thank A. Bird, C. Friel, J. Howard, L. Rice and M. Zanic for comments on the manuscript. This work was supported by the Canadian Institutes of Health Research (CIHR, MOP-111265 to G.J.B.), by the Natural Sciences and Engineering Research Council of Canada (NSERC, no. 372593-09 to G.J.B.), and by McGill University. M.W. is supported by an NSERC CGS-D award and a Fonds de Recherche du Quebec—Nature et Technologie Bourse de Doctorat en Recherche. S.C. is supported by the NSERC CREATE training program in the Cellular Dynamics of Macromolecular Complexes and an NSERC CGS-D. G. Brouhard is a CIHR New Investigator.

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Authors and Affiliations

  1. Department of Biology, McGill University, 1205 ave Docteur Penfield Montréal, Québec H3A 1B1, Canada,

    Michal Wieczorek, Susanne Bechstedt, Sami Chaaban & Gary J. Brouhard

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  1. Michal Wieczorek
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  2. Susanne Bechstedt
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Contributions

M.W., S.B. and G.J.B. conceived the project. M.W. performed nucleation experiments, characterized the MAPs, and imaged cells. S.B. established infrastructure for molecular biology and protein expression. S.C. performed electron microscopy experiments. M.W. and G.J.B. analysed data, developed models, and wrote the paper.

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Correspondence to Gary J. Brouhard.

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Integrated supplementary information

Supplementary Figure 1 Controls for centrosome, axoneme and GMPCPP nucleation experiments in Fig. 1.

(A) Immunoblot against γ-tubulin for a purified centrosome fraction. (B) Immunofluorescence of purified centrosomes after microtubule nucleation showing αβ-tubulin (left) and γ-tubulin (middle). In the merged image (right), the γ-tubulin signal is at the centre of the microtubule aster. (C) Overlay of our centrosome nucleation data (Fig. 1c) onto data from Fig. 4A of ref. 29, NPG. (D) Overlay of our axoneme nucleation data (Fig. 1f) onto data from Fig. 6 of ref. 30, Rockefeller Univ. Press. (E) Images from two consecutive nucleation experiments performed on the same set of GMPCPP seeds. In experiment #1, 33% of the seeds produced microtubules (left, white arrows). In experiment #2, after thorough rinsing, 25% of the seeds produced microtubules (right, white arrows). The theoretical probability that a seed produced microtubules twice agrees well with the measured value (see text at right). n = 214 GMPCPP seeds measured consecutively in the same experiment.

Supplementary Figure 2 Controls for hysteresis experiments in Fig. 2 and in-house spontaneous nucleation measurements.

(A) Plot of the average background intensity from fluorescent tubulin against time for the experiment in Fig. 2d. The intensity increases when the tubulin concentration is increased from 4 μM to 15 μM. When the solution is exchanged from 15 μM back to 4 μM, the intensity returns to the baseline, 4 μM tubulin level, indicating that the solution exchange is complete. Error bars represent the s.d. of intensity values from a 20 × 20 pixel box from one experiment. (B) Plot of the absorbance at 350 nm (A350), or turbidity, against time for solutions containing the indicated concentration of tubulin. An increase in A350 indicates polymer formation. (C) Plot of the A350 values at t = 60 min against tubulin concentration. A fit to data for which A350 > 0.05 (red line) gives an x-intercept of 21 ± 4.2 μM tubulin, which is the critical concentration for spontaneous nucleation. (D) Images of Coomassie-stained SDS-PAGE gels for the spin-down spontaneous nucleation assay, showing the total tubulin (top gel) and the polymeric tubulin in the pellet (bottom gel). (E) Plot of the concentration of pelleted tubulin against total tubulin concentration. A fit of the data for which the concentration of tubulin in the pellet ≥ 1 μM (red line) gives an x-intercept of 21 ± 7.9 μM. For increasing tubulin concentrations, n = 2, 4, 5, 5, 5, 5, 5, 5, 5, 4 and 2 independent experiments, respectively.

Supplementary Figure 3 Controls for nucleation time distribution experiments in Fig. 3.

(A) Plot of the first nucleation time against the second nucleation time for two consecutive experiments in which the same set of GMPCPP seeds were exposed to 12 μM tubulin for t = 15 min. If a seed did not produce a microtubule during the experiment, its nucleation time was recorded as >15 min. A red line shows the result predicted by the hypothesis that the seeds will have identical nucleation times in both experiments. The data clearly do not fall on the line. n = 118 GMPCPP seeds. (B) Plot of the background intensity from fluorescent tubulin against time averaged from several experiments. The intensity increases quickly as tubulin is introduced. The red line shows a fit of the data to an exponential function, y(t) = y0 + aekt. From this fit, the time at which the intensity reaches 95% of its plateau was calculated (t95% = 4 s, black dotted line). Error bars represent s.d. n = 3 independent flow-in experiments. (C) Plot of the flow cell temperature against time during a typical experiment. The red line is a fit of the temperature data to an exponential function, y(t) = y0 + aekt. From this fit, the time at which the temperature reaches 95% of its plateau was calculated (t95% = 17 s, black dotted line). Error bars represent s.d. n = 3 independent experiments.

Supplementary Figure 4 Controls for the effects of MCAK and EB1 on nucleation in Fig. 4.

(A) Image of a Coomassie-stained SDS-PAGE gel showing the purified protein fractions for MCAK, EB1, and EB1-GFP used in this study. (B) Plot of the depolymerization rate of GMPCPP microtubules against MCAK concentration. Error bars represent the s.d. For increasing MCAK concentrations, n = 10, 11, 10, 11 and 7 GMPCPP microtubules analysed in one experiment. (C) Kymographs showing double-cycled GMPCPP seeds used in our nucleation assays without (left) and with (right) 10 nM MCAK. Double-cycled GMPCPP seeds are resistant to depolymerization at these MCAK concentrations. (D) Cumulative frequency distribution of the time until catastrophe in the presence of 200 nM EB1 (red) and in control buffers (blue) at 10 μM tubulin. The solid lines are fits to the Gamma distribution, as described in Gardner et al.(2011). n = 186 (with 200 nM EB1) and n = 111 (without EB1) catastrophe events collected from different experiments. (E) In the absence of added salt, EB1-GFP binds along the GMPCPP seeds, the GDP lattice and the tip of growing microtubules. Adding 100 mM KCl to the imaging solution reduces the affinity of EB1-GFP to the seed and lattice, but end-binding persists. The tubulin concentration in these experiments was 20 μM. The EB1-GFP concentration in these experiments was 200 nM. (F) In high salt conditions, EB1 still makes nucleation difficult (green squares), arguing that EB1 lattice binding does not contribute to nucleation inhibition. The solid green line is the sigmoidal equation fit. The fit from the control data is shown in light blue for comparison. For increasing tubulin concentrations, n = 102, 92, 105 and 140 GMPCPP seeds pooled from 3 experiments. Error bars represent s.e.m.

Supplementary Figure 5 Controls for the effects of TPX2, XMAP215 and GMPCPP–tubulin on nucleation in Figs 5 and 6.

(A) Image of a Coomassie-stained SDS-PAGE gel of a microtubule cosedimenation assay done with 200 nM TPX2. The control lane indicates a calculated amount of TPX2 that would represent 100% cosedimentation. The solid triangle points to the cropped area of the original gel provided in Supplementary Fig. 7B. (B) Image of a Coomassie-stained SDS-PAGE gel of the SUMO-tagged TPX2 truncation constructs NT, CT1 and CT2 expressed and purified from bacteria. The solid triangle points to the cropped area of the original gel provided in Supplementary Fig. 7C. (C) (Top) Image of a Coomassie-stained SDS-PAGE gel of a cosedimentation assay done with 200 nM of NT, CT1 or CT2 in the presence (+) and absence (−) of 1 μM taxol-stabilized microtubules. (Bottom) Image of a Western blot against the 6xHis-tag confirming that only NT cosediments with microtubules. (D) Image of a Coomassie-stained SDS-PAGE gel showing the purified protein fractions for XMAP215 and rKin430-GFP. The solid triangle points to the cropped area of the original gel shown in Supplementary Fig. 7D. (E) Plot of microtubule growth rates against tubulin concentration in the presence of 200 nM XMAP215 (red) and in control buffers (blue). For increasing tubulin concentrations, n = 3, 29, 41 and 53 microtubules, respectively with 200 nM XMAP215 and 31, 47, 21 and 18 microtubules, respectively without XMAP215. Data were pooled across 3 experiments. Error bars represent s.e.m. (F) Plot of the nucleation probability in the presence of 200 nM rKin430-GFP (kinesin-1). The solid red line is the sigmoidal equation fit. The fit from the control data is shown in light blue for comparison. For increasing tubulin concentrations, n = 15, 10, 10, 30, 30, 30, 30, 30, 20 and 20 GMPCPP seeds, respectively. Data were pooled across 1–3 experiments. Error bars represent s.e.m.

Supplementary Figure 6 Controls and model for effects of tubulin depletion by nocodazole on nucleation in cells in Fig. 7.

(A) Plot of the microtubule growth rate against nocodazole concentration in two cell lines (U2OS, blue; LLCPK1, red). The microtubule growth rates were inferred from the velocity of EB comets. For increasing nocodazole concentrations, n = 25(2 independent experiments), 13(2), 13(2) and 10(2) (for LLCPK1 cells) and 20(2), 17(2), 12(2) and 12(2) (for U2OS cells) EB comets. Error bars represent the s.d. (B) Plot of the percentage of free soluble tubulin as a function of nocodazole concentration. Assuming a 1:1 stoichiometry and equilibrium conditions, we model the percentage of free tubulin as % Free Tubuli n = 100/(Keq−1[Nocodazole] + 1), where Keq is the equilibrium constant. The range of measured equilibrium constants is indicated. (C) Theoretical plot of the centrosomal nucleation rate (comets ⋅ min−1 emerging from the centrosome) against predicted tubulin concentration in two cell lines (U2OS, blue; LLCPK1, red). The plot assumes an equilibrium constant for tubulin:nocodazole binding of 1 μM and a baseline soluble tubulin concentration of 10 μM. (D) Plot of the centrosomal nucleation rate against colchicine concentration in two cell lines (U2OS, blue; LLCPK1, red). For increasing colchicine concentrations, n = 12, 19, 18 and 19 U2OS cells, respectively and n = 29, 17, 20, 22 and 22 LLCPK1 cells, respectively. Data were pooled across 2 experiments. Error bars represent the s.d. (E) Kymographs of EB1-GFP end-tracking in vitro in control buffers (left), in the presence of 200 nM nocodazole (middle), or in the presence of 1 μM colchicine (right). (F) Images of Western blots performed against EB1 (left) and tubulin (right) on LLCPK1:EB1-GFP cells. The EB1 blot shows a band at ∼55 kDa in the LLCPK1:EB1-GFP cell line that is absent in the control LLCPK1 cells. We estimate EB1-GFP overexpression at ∼30% relative to the endogenous level based on the intensity of this band measured from two independent blots.

Supplementary Figure 7 Uncropped SDS-PAGE gels.

(A) Uncropped scanned image file of the TPX2 purification SDS-PAGE gel shown in Fig. 5a. (B) Uncropped scanned image file of the TPX2 cosedimentation assay SDS-PAGE gel shown in Supplementary Fig. 5A. (C) Uncropped scanned image file of the SUMO-tagged TPX2 truncation construct purification SDS-PAGE gel shown in Supplementary Fig. 5C. (D) Uncropped scanned image file of the MAP purification gel shown in Supplementary Fig. 5D.

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Microtubule nucleation from GMPCPP seeds at 12 μM tubulin.

Epifluorescence images of GMPCPP microtubule seeds (magenta) combined with total-internal-reflection fluorescence images of elongating microtubules (green) were recorded at 10 s intervals for 15 min. Some GMPCPP seeds are observed to produce microtubules immediately while others produce microtubules after a time lag. Some GMPCPP seeds do not produce microtubules during the experiment. Video playback is 100× real-time (see time stamp). (AVI 12756 kb)

Hysteresis in microtubule elongation experiments.

Epifluorescence images of GMPCPP microtubule seeds (magenta) combined with total-internal-reflection fluorescence images of elongating microtubules (green) were recorded at 10 s intervals. At the start of the experiment, the GMPCPP seeds are exposed to 4 μM tubulin (indicated). After a short period, the solution is exchanged with 15 μM tubulin (indicated). At this concentration, the GMPCPP seeds produce microtubules readily. The solution is exchanged back to 4 μM tubulin (indicated). Microtubules continue to elongate until they undergo catastrophe, after which the GMPCPP seed is dormant. Video playback is 100× real-time (see time stamp). (AVI 2544 kb)

LLCPK1 cells expressing GFP-EB1.

Spinning-disk confocal images of LLCPK1 cells constitutively expressing GFP-EB1. Images were taken every 2 s. Playback is 20× real-time (see time stamp). (AVI 19090 kb)

LLCPK1 cells expressing GFP-EB1 in the presence of 40 nM nocodazole.

Spinning-disk confocal images of LLCPK1 cells constitutively expressing GFP-EB1 in the presence of 40 nM nocodazole. Fewer ‘comets’ emerge from the centrosome. Images were taken every 2 s. Playback is 20× real-time (see time stamp). (AVI 19090 kb)

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Wieczorek, M., Bechstedt, S., Chaaban, S. et al. Microtubule-associated proteins control the kinetics of microtubule nucleation. Nat Cell Biol 17, 907–916 (2015). https://doi.org/10.1038/ncb3188

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