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Microtubule depolymerization at kinetochores restricts anaphase spindle elongation

Nature Chemical Biology (2026)Cite this article

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Abstract

Anaphase chromosome segregation depends on forces exerted by spindle microtubules. Current models propose two force-generating mechanisms: kinetochore–microtubule (kMT) depolymerization pulls chromosomes toward spindle poles (anaphase A), while antiparallel microtubule sliding in the central spindle further separates sister chromosomes by elongating the spindle (anaphase B). Experimental evidence in cells supports the sliding mechanism but contributions of the depolymerization mechanism remain unclear. We show that kMT depolymerization limits spindle elongation rather than moving chromosomes apart. We developed a chemical optogenetic approach to recruit microtubule depolymerases to kinetochores at anaphase onset, thereby increasing kMT depolymerization rates without perturbing earlier stages of mitosis. We find that increased depolymerization slows the velocity at which spindle poles move apart without changing kinetochore separation velocities. Our findings support a model in which kinetochores selectively couple to central spindle microtubules parallel to their kMTs, such that antiparallel sliding drives chromosome segregation while kMT depolymerization pulls poles inward.

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Fig. 1: Induced kMT depolymerization shortens the metaphase spindle.
Fig. 2: Enhanced kMT depolymerization slows pole separation in anaphase.
Fig. 3: Antipoleward kinetochore motions in monopolar anaphase.
Fig. 4: Suppressed bundle sliding inhibits kMT depolymerization.
Fig. 5: Proposed sort-and-grip processes.

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Data availability

Plasmids generated during the current study are available through Addgene. Source data are provided with this paper.

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Acknowledgements

We thank the Philly ChromoClub, the Penn Center for Genome Integrity and A. Khodjakov (Wadsworth Center) for insightful discussions. This work was supported by the National Institutes of Health (GM122475 and P01-CA265794) and the Basser Center for BRCA (Early Career Award).

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Authors and Affiliations

  1. Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

    Geng-Yuan Chen & Michael A. Lampson

  2. Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA

    Changfeng Deng & David M. Chenoweth

  3. Penn Center for Genome Integrity, University of Pennsylvania, Philadelphia, PA, USA

    Michael A. Lampson

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  1. Geng-Yuan Chen
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  2. Changfeng Deng
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Contributions

All authors contributed to development of chemical optogenetics probes and experimental design. C.D. and D.M.C. synthesized and characterized the chemical optogenetic probes. G.-Y.C. conducted the cell biology experiments and wrote the paper. M.A.L. edited the paper.

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Correspondence to Michael A. Lampson.

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Extended data

Extended Data Fig. 1 Recruitment of an MCAK catalytic mutant to kinetochores modestly shortens the metaphase spindle.

MCAK depolymerase activity was suppressed by generating a mutant (MCAKMUT) with combined mutations at S192E and the hypir triple substitution (H536A/R540A/K543A). Pole-to-pole distances were measured for each cell and averaged at every time point (n = 29 MCAK cells pooled from four independent experiments; n = 8 MCAKMUT cells pooled from two independent experiments). For MCAK cells, data are replotted from Fig. 1f. Error bars: mean ± SEM.

Source data

Extended Data Fig. 2 MCAK overexpression does not affect motions of chromosomes and poles.

Velocities of kMT depolymerization (a), kinetochore separation (b), and pole separation (c) were measured for cells expressing mScarlet-eDHFR (–MCAK, n = 40 cells) or MCAK-mScarlet-eDHFR (+MCAK, n = 58 cells) as in Fig. 2c, d. For +MCAK cells, data were replotted from –dimerizer cells in Fig. 2e–g. Each data point represents a single cell. Lines: mean ± SD. Statistics: two-tailed Student’s t-test.

Source data

Extended Data Fig. 3 Recruitment of an MCAK catalytic mutant to kinetochores modestly restricts spindle elongation.

Initial velocity analyses for kMT depolymerization (a), kinetochore separation (b), and pole separation (c). From left to right in each panel: n = 58 MCAK –dimerizer cells polled from six independent experiments, n = 30 MCAK +dimerizer cells polled from seven independent experiments, n = 18 MCAKMUT –dimerizer cells polled from two independent experiments, and n = 16 MCAKMUT +dimerizer cells polled from two independent experiments. For MCAK –dimerizer and MCAK +dimerizer cells, data are replotted from Fig. 2e–g. Each data point represents a single cell. Lines: mean ± SD.

Source data

Extended Data Fig. 4 Enhanced kMT depolymerization does not affect PRC1 bundle length reduction in anaphase.

(a) Schematic of PRC1 bundle length reduction during antiparallel sliding in anaphase. Sliding reduces the overlap length (blue, Vsliding), while polymerization of central spindle microtubules at plus-ends increases the overlap length (gray, Vpol), leading to the net rate of bundle length reduction (pink, VPRC1 = Vsliding - 2× Vpol). (b–d) Analysis of bundle reduction rate. Representative images (b) show 670nano3-PRC1 in cells also expressing Halo-GFP-SPC25, MCAK-mScarlet-eDHFR, and PACT-GFP. Time stamps (min:s) indicate time after first observing sister kinetochore separation at t = 0. Scale bars, 5 µm. Example snapshots of PRC1 intensity (c) were analyzed by line scans between the two spindle poles. Bundle lengths at t = 0 (black) and t = 2 min (red) are defined by their full widths at half-maximum. Example trace (d) shows changes in bundle length over time, with rate (dashed lines) measured in the time window defined as in Fig. 2d. (e–h) Initial velocities were calculated for kMT depolymerization (e), kinetochore separation (f), pole separation (g), and bundle length reduction (h) (n = 22 cells –dimerizer pooled from three independent experiments, 12 cells +dimerizer pooled from two independent experiments). Each data point represents a single cell. Lines: mean ± SD. Statistics: two-tailed Student’s t-test. We note that kinetochores separate faster than bundle length reduction (VKK > VPRC1) both with and without dimerizer, consistent with microtubule polymerization in the central spindle that increases bundle length during anaphase.

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Extended Data Fig. 5 PRC1 morphology in monopolar spindles.

(a) Representative images of PRC1 bundles before (left) or after (right) anaphase onset. Time stamps indicate the time after uncaging. Scale bars, 5 µm. (b) An example of PRC1 intensity analyzed by line scan (box in a). Bundle length and intensity are defined by the full width at half-maximum and the peak height, respectively. (c, d) Analyses for bundle length (c) and intensity (d). Each dot represents a PRC1 bundle (n = 162 for pre-anaphase bundles and 148 for anaphase bundles from 23 cells). Lines: mean ± SD. Statistics: two-tailed Student’s t-test.

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Extended Data Fig. 6 PRC1 overexpression and formation of brake complexes inhibit kMT depolymerization.

(a) Representative images of cells expressing CenpB-GFP and PACT-GFP, with or without expression of Halo-670nano3-PRC1. Time stamps (min:s) indicate time after first observing sister kinetochore separation at t = 0. Scale bars, 5 µm. (b–d) Dependence of pole separation (b), kinetochore separation (c), and kMT depolymerization (d) velocities on spindle-bound Halo-670nano3-PRC1. Colored data points show control cells without Halo-670nano3-PRC1 or mScarlet-eDHFR-PRC1M (gray), cells expressing Halo-670nano3-PRC1 (orange), cells expressing Halo-670nano3-PRC1 and mScarlet-eDHFR-PRC1M without dimerization (blue), and cells expressing Halo-670nano3-PRC1 and mScarlet-eDHFR-PRC1M with dimerization (pink). All cells express CenpB-GFP and PACT-GFP. Each data point represents a single cell.

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Extended Data Fig. 7 Velocity relationships predicted by the sort-and-grip model and proposed mechanisms of parallel sorting.

(a) Schematic shows kinetochore velocity (VK), pole velocity (VP), and antiparallel sliding in the central spindle (Vsliding). Kinetochore and pole velocities are half of the velocities of kinetochore separation (VKK) and pole separation (VPP), respectively, as measured in Figs. 2 and 4. Kinetochores, poles, and fiducial marks that label antiparallel sliding are shown in green, black, and brown, respectively. In the sort-and-grip model, kinetochore velocity matches sliding velocity, and pole velocity is sliding velocity (moving poles outward) subtracting kMT depolymerization from either end (VPK, moving poles inward). Manipulations of kMT depolymerization affect pole velocity but not kinetochore velocity in this model. (b) Kinetochore-nucleated kMT fragments (red) have their plus-ends at kinetochores. Minus-end directed motors (gray) at the minus-ends of these short kMTs preferentially guide kMT growth toward the minus ends of parallel microtubules in the central spindle (blue) to biorient sister kinetochores. (c) Minus-end directed motors (gray) guide the minus ends of newly-grown microtubules in the central spindle toward spindle poles through kMTs. (d) A fraction of the central spindle is grown by microtubules branching from kMTs (gray). These branching microtubules form antiparallel bundles (bridging fibers) between the two sister kinetochores, bound by antiparallel couplers.

Supplementary information

Supplementary Information (download PDF )

Legends for Supplementary Videos 1–6.

Reporting Summary (download PDF )

Supplementary Video 1 (download AVI )

Max-intensity projection over 1 μm for the cell shown in Fig. 1e. Time stamps (min:s) indicate the time after uncaging at t = 0. White arrows represent the spindle pole. Scale bar, 10 μm.

Supplementary Video 2 (download AVI )

Max-intensity projection over 4 μm for the cells shown in Fig. 2b, with (right) or without (left) dimerization. Time stamps (min:s) indicate the time after uncaging at t = 0. White arrows represent the spindle pole. Scale bar, 5 μm.

Supplementary Video 3 (download AVI )

Max-intensity projection over 2 μm showing the off-bundle sister kinetochore pair (yellow arrow) indicated by the gray box in Fig. 3c. Time stamps (min:s) indicate the time after uncaging at t = 0. Scale bar, 5 μm.

Supplementary Video 4 (download AVI )

Max-intensity projection over 2 μm showing the on-bundle sister kinetochore pair (yellow arrow) indicated by the orange box in Fig. 3c. Time stamps (min:s) indicate the time after uncaging at t = 0. Scale bar, 5 μm.

Supplementary Video 5 (download AVI )

Max-intensity projection over the whole cell for the cells shown in Fig. 4c. Left, –dimerizer; right, +dimerizer. Time stamps (min:s) indicate the time after uncaging at t = 0. White arrows represent the spindle pole. Scale bar, 5 μm.

Supplementary Video 6 (download AVI )

Max-intensity projection over the whole cell for the cells shown in Extended Data Fig. 6a, with (right) or without (left) expression of Halo–670nano3–PRC1. Time stamps (min:s) indicate the time after first observing sister kinetochore separation at t = 0. White arrows represent the spindle pole. Scale bar, 5 μm.

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Chen, GY., Deng, C., Chenoweth, D.M. et al. Microtubule depolymerization at kinetochores restricts anaphase spindle elongation. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-026-02143-y

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