Pivoting of microtubules around the spindle pole accelerates kinetochore capture

Journal name:
Nature Cell Biology
Volume:
15,
Pages:
82–87
Year published:
DOI:
doi:10.1038/ncb2640
Received
Accepted
Published online

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.

At a glance

Figures

  1. Kinetics of kinetochore capture and the behaviour of microtubules and kinetochores.
    Figure 1: Kinetics of kinetochore capture and the behaviour of microtubules and kinetochores.

    (a) Experimental protocol. Mitotic cells were cooled to 2 °C to depolymerize microtubules (see Methods). Consequently, some kinetochores were lost in the nucleoplasm. After the temperature was increased to 24 °C, microtubules grew from the SPBs and captured lost kinetochores. (b) Normalized average fraction of lost kinetochores as a function of time after relieving cold stress (mean±s.e.m., n = 13, see also Supplementary Fig. S1). The numbers inside the bars represent the total number of metaphase cells (strains AH01 and KI061, Supplementary Table S1). (c,d) Time-lapse images and the corresponding drawings of 2 examples of kinetochore capture, where the kinetochore was captured close to the microtubule tip (c) or away from the tip (d). The cells (strain AH01) expressed α-tubulin–GFP, shown in green, and Ndc80–tdTomato, shown in magenta. Kinetochores overlapping with the spindle appear white. In the drawings, microtubules are represented in green, and the lost kinetochore in magenta. Microtubule orientations and kinetochore positions from the previous images are marked with white dashed lines and white circles, respectively. The time after relieving cold stress is shown in minutes:seconds; scale bars, 1 μm. (e) Mean squared angular displacement (MSAD) of the microtubule. A linear fit with weights 1/s.e.m., MSAD = 2DMTΔt+offset, yields DMT = 3.3±0.1 degrees2 s−1. Note that with the measured DMT, microtubules cover on average Δα≈35° during their lifetime (3 min), but in principle, Δα can take values above 360° if a microtubule performs more than a full revolution. Microtubules of length 1–2 μm were used, n = 106. Grey denotes the area corresponding to subpixel movement of the plus end of a 1.5-μm-long microtubule. One-minute-long time series of α were used; error bars represent s.e.m. (strain AH01). The scheme indicates the angle used for the MSAD calculation. (f) Mean squared displacement (MSD) of the kinetochore. A linear fit, similar to that in Fig. 1e, yields DKC =  (5.9±0.3)×10−4 μm2 s−1 (n = 92). Kinetochores were tracked with subpixel precision (Methods). Grey denotes the area corresponding to subpixel movement of the kinetochore. One-minute-long tracks were used; error bars represent s.e.m. (strains KI061 and AH01). The scheme represents a lost kinetochore trajectory. DKC and DMT refer to kinetochores and microtubules, respectively.

  2. The pivoting motion of microtubules does not depend on ATP.
    Figure 2: The pivoting motion of microtubules does not depend on ATP.

    (a) Time-lapse images and the corresponding drawings of microtubules in cells treated with 50 mM AMP-PNP (permeabilized by Triton X-100, see Methods; strain KI061 from Supplementary Table S1). In the drawings, microtubule orientations from the previous images are marked with white dashed lines. The time from the beginning of the AMP-PNP treatment is given in minutes:seconds. (b) Mean squared angular displacement (MSAD) of the microtubules in cells treated with AMP-PNP (strain KI061). A linear fit with weights 1/s.e.m., MSAD = 2DMTΔt+offset, yields DMT = 4.4±0.7 degrees2 s−1 (black line). Thirty-second-long time series of α of microtubules of length 0.75–2 μm were used (microtubule length was 1.2±0.3 μm, mean±s.e.m., n = 27); error bars represent s.e.m. The fit from Fig. 1e for untreated cells is redrawn for comparison (grey line). (c) Image of the AMP-PNP-treated cell from a, taken 41 min after the beginning of the AMP-PNP treatment, when the spindle was 3 μm long, showing that spindle elongation was inhibited (left). Image of a control cell (without AMP-PNP), taken 15 min after the time when the spindle was 3 μm long, showing normal spindle elongation (right). The control cell was treated with Triton X-100 (see Methods) 30 min before the image was taken. The time is given in minutes:seconds; scale bars, 1 μm.

  3. The model for kinetochore capture based on random angular movement of the microtubule and random movement of the kinetochore.
    Figure 3: The model for kinetochore capture based on random angular movement of the microtubule and random movement of the kinetochore.

    (a) A polar microtubule (green) explores the space by pivoting around the SPB (grey cone). At the same time, a kinetochore (magenta) diffuses. Darker colours represent the moment of capture and lighter colours depict previous positions. (b) Geometry of the model. The microtubule (green) is a thin stiff rod freely jointed to the SPB (grey cone), which is on the nuclear envelope (grey half-sphere). The microtubule and the kinetochore (magenta) coordinates are explained in Supplementary Note S2. (c) Comparison of theoretical predictions and experimental measurements for the fraction of lost kinetochores as a function of time. Five theoretical curves are shown for n = 1–5 microtubules; parameters DMT = 10−3 rad2 s−1, DKC = 6×10−4 μm2 s−1 and L = 1.5 μm were measured here; rKC (t = 0) is taken to be 1.2 μm (mean value of all experiments); R = 1.5 μm and a = 0.2 μm are taken from the literature (Table 1). The experimental data (points with error bars) are redrawn from Fig. 1b. (d) The effect of microtubule and kinetochore diffusion on the capture process. All parameters are as in c including n = 3 microtubules, termed original parameters (black). The green and magenta curves show results for the original parameters except DMT = 0 and DKC = 0, respectively. (e) The effect of microtubule length on the capture process. The curve for L = 1.5 μm corresponds to the original parameters, whereas the other curves correspond to different microtubule lengths (see legend). (f) The effect of low-temperature parameters. Results are shown: in black, for the original parameters; in green, magenta and grey for the original parameters except a single parameter, which is specified in the legend; in blue, for the original parameters except 3 parameters, see legend. The blue curve corresponds to low-temperature parameters, measured at 14 °C (Table 1). (g) The effect of high-temperature parameters. Results are shown: in black, for the original parameters; in green and magenta, for the original parameters except a single parameter, which is specified in the legend; in orange, for the original parameters except 2 parameters, see legend. The orange curve corresponds to high-temperature parameters, measured at 32 °C (Table 1).

  4. Comparison between theoretical predictions and experimental data.
    Figure 4: Comparison between theoretical predictions and experimental data.

    (a,b) Top, theoretical curves for 2 sets of parameters: DMT = 0.5×10−3 rad2 s−1, DKC = 3×10−4 μm2 s−1, L = 1.2 μm (a) and DMT = 1.2×10−3 rad2 s−1, DKC = 15×10−4 μm2 s−1, L = 1.5 μm (b), which were experimentally measured at 14 and 32 °C, respectively (Table 1). The remaining parameters are as in Fig. 3c. In each panel, 5 theoretical curves are shown for n = 1–5 microtubules. Points with error bars (mean±s.e.m.), calculated as in Fig. 1b, represent the experimental data. The number of experiments was 8 and 11 at 14 and 32 °C, respectively (strains KI061 and AH01 from Supplementary Table S1; see also Supplementary Fig. S1). Bottom, drawings showing the orientations of a single microtubule during 4 min at 3-s intervals obtained by numerically solving equations (1) and (2) (Supplementary Note S2), using the same DMT and L values as in the respective panels above. The initial microtubule orientation is marked by the arrowhead; the trace of the plus end is depicted by the black line. Note that the microtubule on the right explored more space and thus had a higher chance to capture the kinetochore.

Videos

  1. Capture of a lost kinetochore by the tip of a polar microtubule (end-on attachment).
    Video 1: 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.
  2. Capture of a lost kinetochore by the lateral side of a polar microtubule (lateral attachment).
    Video 2: 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.
  3. Pivoting of polar microtubules around the SPB in a cell expressing Mal3-GFP.
    Video 3: 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.
  4. Pivoting of polar microtubules in a cell treated with AMP-PNP.
    Video 4: 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.

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Author information

Affiliations

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

    • Iana Kalinina,
    • Petrina Delivani,
    • Mariola R. Chacón,
    • Anna H. Klemm,
    • Damien Ramunno-Johnson,
    • Alexander Krull,
    • Nenad Pavin &
    • Iva M. Tolić-Nørrelykke
  2. Max Planck Institute for the Physics of Complex Systems, 01187 Dresden, Germany

    • Amitabha Nandi,
    • Benjamin Lindner &
    • Nenad Pavin
  3. Department of Physics, Faculty of Science, University of Zagreb, 10002 Zagreb, Croatia

    • Nenad Pavin
  4. Present address: European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany (I.K.); Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA (A.N.); Bernstein Center for Computational Neuroscience Berlin and Physics Department of Humboldt University Berlin, 10115 Berlin, Germany (B.L.)

    • Iana Kalinina,
    • Amitabha Nandi &
    • Benjamin Lindner

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.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Video

  1. Video 1: Capture of a lost kinetochore by the tip of a polar microtubule (end-on attachment). (1,447 KB, Download)
    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.
  2. Video 2: Capture of a lost kinetochore by the lateral side of a polar microtubule (lateral attachment). (1,086 KB, Download)
    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.
  3. Video 3: Pivoting of polar microtubules around the SPB in a cell expressing Mal3-GFP. (1,005 KB, Download)
    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.
  4. Video 4: Pivoting of polar microtubules in a cell treated with AMP-PNP. (3,734 KB, Download)
    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.

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