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Adaptive changes in the kinetochore architecture facilitate proper spindle assembly

Nature Cell Biology volume 17, pages 1134–1144 (2015)Cite this article

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Abstract

Mitotic spindle formation relies on the stochastic capture of microtubules at kinetochores. Kinetochore architecture affects the efficiency and fidelity of this process with large kinetochores expected to accelerate assembly at the expense of accuracy, and smaller kinetochores to suppress errors at the expense of efficiency. We demonstrate that on mitotic entry, kinetochores in cultured human cells form large crescents that subsequently compact into discrete structures on opposite sides of the centromere. This compaction occurs only after the formation of end-on microtubule attachments. Live-cell microscopy reveals that centromere rotation mediated by lateral kinetochore–microtubule interactions precedes the formation of end-on attachments and kinetochore compaction. Computational analyses of kinetochore expansion–compaction in the context of lateral interactions correctly predict experimentally observed spindle assembly times with reasonable error rates. The computational model suggests that larger kinetochores reduce both errors and assembly times, which can explain the robustness of spindle assembly and the functional significance of enlarged kinetochores.

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Figure 1: Changes in the outer kinetochore architecture at various stages of mitosis.
Figure 2: The kinetochore core remains relatively compact throughout mitosis.
Figure 3: Kinetochore morphology at the onset of spindle assembly.
Figure 4: Kinetochore outer layer compaction occurs on the formation of end-on microtubule attachments.
Figure 5: Effects of kinetochore enlargement–compaction on the efficiency and fidelity of capture-driven spindle assembly.
Figure 6: Centromere rotation on the surface of the spindle precedes formation of end-on microtubule attachment.
Figure 7: Computational models that consider centromere rotation due to lateral interactions with microtubules predict experimentally observed parameters of spindle assembly.
Figure 8: Abnormal geometry of the nascent spindle during early prometaphase correlates with erroneous chromosome segregation.

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Acknowledgements

This work was supported by NIH grant GM059363 to A.K. and NSF grant DMS-1118206 to A.M. The Electron Microscopy was enabled by the use of the Wadsworth Center’s Electron Microscopy Core Facility. We thank J. Pines (University of Cambridge, UK) for his generous donation of Mad2–Venus cells and S. Li (Air Worldwide, USA) for assistance with the intensity quantifications.

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Author notes

  1. Valentin Magidson & Christopher B. O’Connell

    Present address: Present addresses: National Cancer Institute, Frederick, Maryland 21702, USA (V.M.); Nikon Instruments, Melville, New York 11747, USA (C.B.O’C.).,

  2. Valentin Magidson and Raja Paul: These authors contributed equally to this work.

Authors and Affiliations

  1. New York State Department of Health, Wadsworth Center, Albany, New York 12201, USA

    Valentin Magidson, Nachen Yang, Jeffrey G. Ault, Christopher B. O’Connell, Irina Tikhonenko, Bruce F. McEwen & Alexey Khodjakov

  2. Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India

    Raja Paul

  3. Courant Institute and Department of Biology, New York University, New York, New York 10012, USA

    Raja Paul & Alex Mogilner

  4. Rensselaer Polytechnic Institute, Troy, New York 12180, USA

    Alexey Khodjakov

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Contributions

A.K. and B.F.M. designed the experiments, V.M., N.Y., C.B.O’C. and I.T. performed the experiments. J.G.A., B.F.M., A.K. and I.T. conducted correlative LM/EM. A.M. and R.P. developed the computational models. R.P. designed computer code and performed simulations. The manuscript was written by A.K., B.F.M. and A.M.

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Integrated supplementary information

Supplementary Figure 2 Experimental approach used to characterize the ‘virgin’ kinetochore architecture at spindle assembly onset.

(a) Selected differential interference contrast (DIC) images (individual planes) illustrating a prophase cell immediately before the addition of 3-μM nocodazole (0 min) and at nuclear envelope breakdown (NEB, 6 min). The cell was fixed with 1% glutaraldehyde immediately after NEB. (b) A single optical plane near the middle of the cell shown after fixation in DIC and fluorescence. Notice that chromosomes (blue, Hoechst 33343) appear to be still aligned along the remnants of the nuclear envelope (arrows) indicating that the cell was fixed at the onset of mitosis. (c) Maximal intensity projection of the entire cell. (d) Higher magnification view of kinetochores from the boxed area in (c). The outer layer (red, CenpF) is enlarged and largely encircles the centromere. Inner kinetochores (green, CenpA-GFP) remain compact. Maximal intensity projections of a local sub-volume and surface-rendered reconstruction segmented at 25% of maximal intensity. (e,f) Treatment history of the cell shown in Fig. 3e, f. (e) Selected DIC and corresponding fluorescent images (CenpA-GFP and Centrin-GFP) depicting a different cell before the addition of 3-μM nocodazole (0 min), during late prophase (4 min), at (NEB, 10 min), and immediately after fixation (11 min). Asterisks indicate the location of the centrioles. The boxed area in panels D and E is shown in Fig. 2B at higher magnification. (f) Electron-microscopy image of the same cell. Remnants of the nuclear envelope are clearly visible (yellow arrows). The box denotes the centromere presented in Fig. 2b.

Supplementary Figure 3 Kinetochore outer layer compaction correlates with the formation of end-on microtubule attachment.

(a) In earlier prometaphase cells, kinetochores with an enlarged outer layer are present throughout the cell (insets 1, 2). However, a few kinetochores that display prominent end-on attachment to microtubule bundles are compact (insets 3, 4). (b) In later prometaphase, at least one kinetochore displays an enlarged outer layer on each monooriented chromosome and these enlarged kinetochores lack end-on microtubule attachments (insets 1, 2). In contrast, both sister kinetochores on bioriented chromosomes are compact (insets 3, 4). Whole-cell images are maximal-intensity projections that include all kinetochores in the cell. Individual kinetochores are shown as maximal-intensity projections of local sub-volumes.

Supplementary Figure 4 Distribution and amounts of various kinetochore proteins in the absence of microtubules.

(a) Maximal intensity projections (include all kinetochores) depicting RPE1 cells after 15-min exposure to 3-μM nocodazole. Notice that the analyses were only on cells that had formed a metaphase plate before the addition of nocodazole as evident from the pattern of chromosome distribution and positions of centrosomes on the opposite sides of the plates. (b) Examples of individual kinetochores from the boxed areas in (a), shown at higher magnification. CenpF forms large crescents that can completely encircle the centromere. The distributions of Hec1 and Mis12 also appear to broaden albeit to lesser extents than CenpF. (c) Relative fluorescence intensities of kinetochores at various times after addition of nocodazole. The amount of the outer layer protein CenpF (red) remains at the level typical for untreated metaphase (compare with. Figure 1c, p > 0.3, two-tailed Student’s t-test for both blue versus blue and yellow versus yellow bars.) and then increases approximately threefold. The amount of Hec1 instantly increases approximately twofold over the levels typical for kinetochores during metaphase (compare with Fig. 1c, p < 0.0001, two-tailed Student’s test for both blue versus blue and yellow versus yellow bars.). The amount of Mis12 in nocodazole-treated cells is not significantly different from the metaphase level (compare with Fig. 1c, p > 0.09, two-tailed Student’s test for both blue versus blue and yellow versus yellow bars). Blue bars in (c) are mean kinetochore intensity calculated as mean of mean values for multiple kinetochores in individualcells (n values above the bars, Cs; cells) Error bars represent s.e.m. Yellow bars are mean values calculated for all kinetochores pooled from all cells in that class (n values above the bars, Ks; kinetochores). Error bars represent s.d.

Supplementary Figure 5 Effects of kinetochore enlargement-compaction on the efficiency and fidelity of capture-driven spindle assembly.

(a–a′) Architecture of the virgin (unattached) centromere considered in the previous computational models of spindle assembly (a) versus the current model (a′). dKt, diameter of the discoid kinetochore in previous models; wKt and hKt, width and height of the expanded crescent-like kinetochore; Gap, segment of the centromere not covered by the kinetochore outer layer. (b) Diagram showing the changes in the centromere architecture considered in the minimalistic computational model. τcapt, time from the onset of spindle assembly to end-on attachment; τcomp, duration of the conversion from the expanded crescent to compact architecture of the kinetochore. (c) Sequence of events envisioned in the minimalistic model. Attachment triggers kinetochore compaction but does not affect orientation of the centromere. Green lines represent properly attached microtubules, red lines—potential erroneous attachments. (d,d′) Duration of spindle assembly and frequency of errors predicted for various final gap sizes, and various durations of kinetochore compaction. Notice that both efficiency and accuracy of spindle assembly remain nearly constant at τcomp > 60 s. (e) Frequency of errors and duration of spindle assembly predicted for centromeres with various final gap sizes for specific values of τexpand and τcomp.

Supplementary Figure 6 Rotation of the centromere due to lateral interactions between kinetochores and microtubules.

(a) Opposition of inward-directed forces generated at the kinetochore (dynein) and outward forces acting on chromosome arms (chromokinesins) rotate the centromere positioned on the surface of the spindle. (b) Centromeres with a small gap between sister kinetochores can rotate significantly while maintaining constant contact with microtubules. In contrast, rotation of centromeres with a large gap between sister kinetochores is sterically limited due to small kinetochores losing direct contact with microtubules. As the result, after rotation large sister kinetochores are primarily exposed to their proximal poles (green arrows) and shielded from the distal poles by the centromere. Due to the lesser angular improvement, smaller sister kinetochores remain exposed to both proximal and distal poles (red arrows).

Supplementary Figure 7 Conventional (not rotationally aligned) views of the cells presented in Fig. 8.

(a) Normal spindle assembly in an RPE1 cell (see Fig. 7a). (b) An untreated RPE1 cell with lagging chromosomes (Fig. 7b). (c) An RPE1 cell that assembled its spindle after nocodazole washout. Notice that drug washout is initiated soon after NEB when remnants of the nuclear envelope are still present in the cell (arrowheads). Each time point is shown in DIC (medial slices from 3D volumes) and GFP-fluorescence (maximal intensity projections). Asterisks denote mother centrioles (labeled with centrin-GFP). Arrows point at NEB remnants in DIC images and lagging chromosomes in fluorescent images. Time in minutes: seconds from NEB (a,b) or from completion of nocodazole washout (c).

Supplementary Table 1 Parameters used in the computational models.
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Incorporation of individual chromosomes into the spindle. Related to Fig. 6.

Mad2 fluorescence (green) is overlaid on phase contrast (grey). The video starts with 3 centromeres on the lower left side of the spindle showing Mad2 fluorescence. The chromosome nearest the spindle equator has already initiated congression at the start of filming and rapidly moves to the equator where it loses Mad2 fluorescence. The other two centromeres show one or more rapid rotations (lasting no more than two consecutive frames at 30-s intervals) before initiating congression and loss of Mad2 fluorescence. Time in minutes: seconds from the start of the video. (MOV 1632 kb)

Axial and transverse views of spindle assembly in control and nocodazole treated cells. Related to Fig. 8.

A typical control cell is shown in the left panels, a control cell with lagging chromosomes in the middle panels, and a cell subjected to nocodazole treatment followed by washout of the drug in the right panels. Note that the typical control cell forms the clear middle zone with all centromeres confined to the periphery (frames 3–13). The fluorescent dots in the middle of the transverse views are the centrioles. Time in minutes : seconds from NEB (left and central panels) or from completion of nocodazole washout (right panel). (MOV 3653 kb)

A typical control cell as conventionally seen in the light microscope (not rotationally aligned).

The video shows the medial plane of DIC and the maximal-intensity projection of GFP-fluorescence for the cell shown in Fig. 8a and the left panels of Supplementary Video 2 . Note that clear zone formation in the central spindle is not evident in this view. Time in minutes: seconds from NEB. (MOV 5145 kb)

A control cell with lagging and lost chromosomes as conventionally seen in the light microscope (not rotationally aligned).

The video shows the medial plane of DIC and the maximal-intensity projection of GFP-fluorescence for the cell shown in Fig. 8b and the middle panels of Supplementary Video 2 . Note that the difference in clear zone formation between a typical control cell (Supplementary Video 3) and one leading to lost chromosomes (this video) is not detected. Hence, detection of the lack of clear zone formation requires that data sets be rotationally aligned. Time in minutes: seconds from NEB. (MOV 4934 kb)

A cell treated with nocodazole in prophase followed by drug washout after NEB as conventionally seen in the light microscope (not rotationally aligned).

The video shows the medial plane of DIC and the maximal-intensity projection of GFP-fluorescence for the cell shown in Fig. 8c and the right panels of Supplementary Video 2 . As in Supplementary Video 4, the lack of a clear zone is not detected unless data sets are rotationally aligned. Time in minutes: seconds from completion of nocodazole washout. (MOV 6000 kb)

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Magidson, V., Paul, R., Yang, N. et al. Adaptive changes in the kinetochore architecture facilitate proper spindle assembly. Nat Cell Biol 17, 1134–1144 (2015). https://doi.org/10.1038/ncb3223

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