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Pivoting of microtubules around the spindle pole accelerates kinetochore capture

Abstract

During cell division, spindle microtubules attach to chromosomes through kinetochores, protein complexes on the chromosome1. The central question is how microtubules find kinetochores. According to the pioneering idea termed search-and-capture, numerous microtubules grow from a centrosome in all directions and by chance capture kinetochores2,3,4. The efficiency of search-and-capture can be improved by a bias in microtubule growth towards the kinetochores5,6, by nucleation of microtubules at the kinetochores7,8,9 and at spindle microtubules10,11, by kinetochore movement9, or by a combination of these processes12,13,14. Here we show in fission yeast that kinetochores are captured by microtubules pivoting around the spindle pole, instead of growing towards the kinetochores. This pivoting motion of microtubules is random and independent of ATP-driven motor activity. By introducing a theoretical model, we show that the measured random movement of microtubules and kinetochores is sufficient to explain the process of kinetochore capture. Our theory predicts that the speed of capture depends mainly on how fast microtubules pivot, which was confirmed experimentally by speeding up and slowing down microtubule pivoting. Thus, pivoting motion allows microtubules to explore space laterally, as they search for targets such as kinetochores.

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Figure 1: Kinetics of kinetochore capture and the behaviour of microtubules and kinetochores.
Figure 2: The pivoting motion of microtubules does not depend on ATP.
Figure 3: The model for kinetochore capture based on random angular movement of the microtubule and random movement of the kinetochore.
Figure 4: Comparison between theoretical predictions and experimental data.

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Acknowledgements

We thank K. Sawin, A. Haese, Y. Caldarelli, E. Guarino, S. Kearsey and the Yeast Genetic Resource Center for strains and plasmids; B. Schroth-Diez from the Light Microscopy Facility of MPI-CBG for help with microscopy; I. Šarić for the drawings; W. Zachariae, S. Grill, J. Howard, D. Cimini, J. Gregan, M. Žanić, E. Paluch, N. Maghelli, M. Coelho and V. Ananthanarayanan for discussions and advice; the German Research Foundation (DFG) and the Human Frontier Science Program (HFSP) for financial support. M.R.C. was supported by a Marie Curie Intra-European Fellowship and D.R-J. by a Humboldt Research Fellowship for Postdoctoral Researchers.

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Contributions

I.K. carried out all experiments and data analysis, A.N. performed simulations, P.D., M.R.C. and A.H.K. carried out AMP-PNP and FRAP experiments, D.R-J. analysed the data shown in Supplementary Fig. S3b, A.K. developed the tracking software, B.L. and N.P. developed the theory, and I.M.T-N. and N.P. designed the project and wrote the paper.

Corresponding authors

Correspondence to Nenad Pavin or Iva M. Tolić-Nørrelykke.

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The authors declare no competing financial interests.

Supplementary information

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Supplementary Information (PDF 527 kb)

Supplementary Note

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Supplementary Table 1

Supplementary Information (XLSX 9 kb)

Supplementary Table 2

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Capture of a lost kinetochore by the tip of a polar microtubule (end-on attachment).

Live cell microscopy of an S. pombe mitotic cell, expressing tubulin labeled with GFP (green), and the kinetochore protein Ndc80p labeled with tdTomato (magenta); strain AH01 (Supplementary Table S1). Images were acquired at 2 s intervals. The video is displayed at 15 fps. Time of recovery after cold stress is indicated in minutes:seconds. Scale bar represents 1 μm. The movie corresponds to Fig. 1c. (AVI 1446 kb)

Capture of a lost kinetochore by the lateral side of a polar microtubule (lateral attachment).

Live cell microscopy of an S. pombe mitotic cell, expressing tubulin labeled with GFP (green), and the kinetochore protein Ndc80p labeled with tdTomato (magenta); strain AH01 (Supplementary Table S1). Images were acquired at 2 s intervals. The video is displayed at 15 fps. Time of recovery after cold treatment is indicated in minutes:seconds. Scale bar represents 1 μm. The movie corresponds to Fig. 1d. (AVI 1086 kb)

Pivoting of polar microtubules around the SPB in a cell expressing Mal3-GFP.

Live cell microscopy of an S. pombe mitotic cell, expressing Mal3-GFP and Sid4 (SPB marker) labeled with GFP; strain YC001 (Supplementary Table S1). Note that Mal3-GFP visualizes the movement of the microtubule tip, which allows us to observe the pivoting of a growing microtubule. Mal3p is not present at the end of shrinking microtubules. Images were acquired at 250 ms intervals. The green line marks the position used to make the kymograph shown in Supplementary Fig. S3a. The video is displayed at 15 fps. Time is indicated in seconds. Scale bar represents 1 μm. (AVI 1005 kb)

Pivoting of polar microtubules in a cell treated with AMP-PNP.

Live cell microscopy of an S. pombe mitotic cell treated with 50 mM AMP-PNP (strain KI061, Supplementary Table S1). Images were acquired at 2.2 s intervals. The video is displayed at 15 fps. Time from the beginning of AMP-PNP treatment is indicated in minutes:seconds. Scale bar represents 1 μm. The movie corresponds to Fig. 2a. (AVI 3733 kb)

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Kalinina, I., Nandi, A., Delivani, P. et al. Pivoting of microtubules around the spindle pole accelerates kinetochore capture. Nat Cell Biol 15, 82–87 (2013). https://doi.org/10.1038/ncb2640

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