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XMAP215 activity sets spindle length by controlling the total mass of spindle microtubules

Nature Cell Biology volume 15, pages 1116–1122 (2013)Cite this article

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Abstract

Metaphase spindles are microtubule-based structures that use a multitude of proteins to modulate their morphology and function. Today, we understand many details of microtubule assembly, the role of microtubule-associated proteins, and the action of molecular motors1,2. Ultimately, the challenge remains to understand how the collective behaviour of these nanometre-scale processes gives rise to a properly sized spindle on the micrometre scale. By systematically engineering the enzymatic activity of XMAP215, a processive microtubule polymerase3,4, we show that Xenopus laevis spindle length increases linearly with microtubule growth velocity, whereas other parameters of spindle organization, such as microtubule density, lifetime and spindle shape, remain constant. We further show that mass balance can be used to link the global property of spindle size to individual microtubule dynamic parameters. We propose that spindle length is set by a balance of non-uniform nucleation and global microtubule disassembly in a liquid-crystal-like arrangement of microtubules.

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Figure 1: Spindle length is directly proportional to in vitro XMAP215 microtubule polymerase activity.
Figure 2: Spindle length scales with spindle microtubule growth velocity.
Figure 3: XMAP215 activity limits upper spindle length.
Figure 4: In Xenopus spindles, microtubule lifetime, density and spindle shape do not depend on XMAP215 activity.

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Acknowledgements

We are grateful to A. Bird, C. Brangwynne, M. Braun, N. Goehring, J. Gopalakrishnan, O. Gruss, G. Salbreux, M. Zanic and D. Zwicker for critical evaluation of the manuscript. We thank all members of the Howard, Hyman and Jülicher laboratories for continuous discussions, H. Andreas for frog care, O. Gruss for the XKCM1 antibody, and Y. Kalaidzidis and M. Chernykh for help with the tracking software. P.O.W. was supported by an EMBO long-term fellowship. J.B. is supported by the European Commission’s 7th Framework Programme grant Systems Biology of Mitosis (FP7-HEALTH-2009-241548/MitoSys). S.R. is supported by the European Commission’s 7th Framework Programme grant Systems Biology of Stem Cells and Reprogramming (HEALTH-F7-2010-242129/SyBoSS).

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  1. Jonathon Howard

    Present address: Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA,

Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Str. 108, 01307 Dresden, Germany

    Simone B. Reber, Per O. Widlund, Andrei Pozniakovsky, Jonathon Howard & Anthony A. Hyman

  2. Max Planck Institute for the Physics of Complex Systems, Nöthnitzer Str. 38, 01187 Dresden, Germany

    Johannes Baumgart & Frank Jülicher

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This work represents a truly collaborative effort. Each author has contributed significantly to the findings and regular group discussions guided the development of the ideas presented here.

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Integrated supplementary information

Supplementary Figure 1 XMAP215 TOG1-5AA binds to spindle microtubules and XTACC3.

(a) Xenopus egg extract was MOCK or XMAP215 depleted, recombinant GFP-tagged XMAP215 wildtype (WT) or TOG1-5AA protein was added back to 120 nM, which is the endogenous concentration. Tubulin was used as a loading control. (b) A spindle assembled in XMAP215-depleted Xenopus egg extract in the presence of recombinant XMAP215-GFP (WT) or TOG1-5AA-GFP to 120 nM. Although the full mutant XMAP215 TOG1-5AA no longer binds tubulin, it still localizes to spindle microtubules. Note that spindles assembled in the presence of TOG1-5AA are much smaller than wildtype spindles, also at high concentrations. Scale bar: 10 μm (c) Pulldown of recombinant proteins from Xenopus egg extract on anti GFP-beads, beads eluate was analyzed by Western Blot for the presence of XTACC3. (d) Spindles were assembled in the presence of XMAP215 or the full mutant XMAP215 TOG1-5AA at the indicated concentrations. The upper row shows representative spindles (red: microtubules, blue: DNA), the lower row shows an average image of all spindles (n = 40, assessed over three independent experiments). Scale bar: 10 μm (e) Spindle length (Fig. 1c) plotted versus the in vitro microtubule growth velocities promoted by different XMAP215 mutants at 100 nM (as compared to the maximum growth promotion at 400 nM, shown in Fig. 1h). Error bars indicate SE (ny = 40 spindles assessed over 3 independent experiments; nx = WT 11(2), 5AA 15(2), 3&4AA 11(2), 1AA 11 (2), 1&2AA 15 (2), 1-5 AA no growth. Nx = independent measurements of individual growth events. Number of independent experiments for each condition is given in parentheses.). Grey area indicates the 95% confidence interval of the fitted linear function. (f) Microtubule growth velocities measured at different XMAP215 concentrations in a TIRF assay. The mean growth velocity is indicated by open circles, individual data points by dots (n = 10 independent measurements of individual growth events), which were fitted with Michaelis–Menten kinetics. Grey area indicates the 95% confidence interval of the fitted function.

Supplementary Figure 2 Depleting XKCM1 from Xenopus egg extracts increases microtubule lifetime.

(a) CSF-extract was immuno-depleted of XKCM1 (2ndΔ), one volume of fresh CSF-extract was added back to obtain 50% of endogenous XKCM1 concentration. (b) Spindles were assembled in the presence of Fluor647-tubulin and imaged with an Olympus IX81 spinning disc confocal microscope. Distribution of tubulin lifetimes in spindles assembled in control extracts (blue) with τ = 5.1±0.3 s (SE, n = 60 bins, 13 745 tracks from 12 spindles assessed from 3 independent experiments) and XKCM1-depleted extracts (orange) with τ = 7.8±0.4 s (SE, n = 60 bins, 63 583 tracks from 13 spindles assessed from 3 independent experiments). All data were fitted with a sum of two exponentials, the grey areas indicate the 95% confidence intervals. Note that the setup of the microscope used to determine the lifetimes in Fig. 4b was different (see Methods).

Supplementary Figure 3 Reduction in microtubule density at the spindle centre is due to volume exclusion by DNA.

Microtubule density (bold line) along the longitudinal spindle axis was quantified by the incorporated Cy3-tubulin fluorescence. The variation in intensity along the axis is due to volume exclusion by the DNA, as quantified by the DAPI signal (thin line). The graphs show an average fluorescence intensity of 15 spindles per XMAP215 concentration. The chromatin width was estimated by fitting a Gaussian to the DAPI intensity profiles (SE, n = 15 spindles per concentration).

Supplementary Figure 4 Spindle volume.

(a) Individual data points of spindle volume versus spindle length at different XMAP215 concentrations fitted by a power law (exponent: 1.96±0.18 (SE, n = 60 spindles assessed over 3 different experiments). (b) Individual data points of spindle height versus spindle length at different XMAP215 concentrations fitted by a linear function. (c) Spindle volume versus microtubule growth velocity by combing fits from Supplementary Fig. S4a and Fig. 2f. Grey areas indicate the 95% confidence interval of the fitted functions.

Supplementary Figure 5 Uncropped Western Blots.

Supplementary Table 1 Spindle parameters.
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Reber, S., Baumgart, J., Widlund, P. et al. XMAP215 activity sets spindle length by controlling the total mass of spindle microtubules. Nat Cell Biol 15, 1116–1122 (2013). https://doi.org/10.1038/ncb2834

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