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. 2015 Jul 20;25(14):1835-41.
doi: 10.1016/j.cub.2015.05.013. Epub 2015 Jul 9.

Spatial Regulation of Kinetochore Microtubule Attachments by Destabilization at Spindle Poles in Meiosis I

Lukáš Chmátal  1 , Karren Yang  1 , Richard M Schultz  2 , Michael A Lampson  3
Affiliations

Spatial Regulation of Kinetochore Microtubule Attachments by Destabilization at Spindle Poles in Meiosis I

Lukáš Chmátal et al. Curr Biol. .

Abstract

To ensure accurate chromosome segregation in cell division, erroneous kinetochore-microtubule (MT) attachments are recognized and destabilized . Improper attachments typically lack tension between kinetochores and are positioned off-center on the spindle. Low tension is a widely accepted mechanism for recognizing errors , but whether chromosome position regulates MT attachments has been difficult to test. We exploited a meiotic system in which kinetochores attached to opposite spindle poles differ in their interactions with MTs and therefore position and tension can be uncoupled. In this system, homologous chromosomes are positioned off-center on the spindle in oocytes in meiosis I, while under normal tension, as a result of crossing mouse strains with different centromere strengths, manifested by unequal kinetochore protein levels. We show that proximity to spindle poles destabilizes kinetochore-MTs and that stable attachments are restored by inhibition of Aurora A kinase at spindle poles. During the correction of attachment errors, kinetochore-MTs detach near spindle poles to allow formation of correct attachments. We propose that chromosome position on the spindle provides spatial cues for the fidelity of cell division.

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Figures

Figure 1
Figure 1. Proximity to spindle poles destabilizes kinetochore MTs, dependent on AURKA activity
(A) An Rb fusion metacentric pairs with the two homologous telocentric chromosomes in MI to form a trivalent. (B) The trivalent is typically positioned off-center with the two telocentrics near the spindle pole. (C–I) Rb(6.16) x CF-1 oocytes were fixed at metaphase I and analyzed for cold-stable MTs. Images (C, H) are maximal intensity z-projections showing centromeres (CREST), tubulin, and DNA; arrowheads indicate unattached kinetochores of a trivalent (yellow) and a bivalent (white) positioned near the spindle poles; insets are optical sections showing individual kinetochores. Scale bar, 5 μm. Distance between kinetochores and the nearest spindle pole (D) was measured for telocentrics in trivalents (n=28) and bivalents (n=280, half the data points are displayed). Inter-kinetochore distance (E) was measured between homologous centromeres of the bivalents, between centromeres of telocentrics and Rb metacentrics in the trivalents, and between homologous centromeres of bivalents in monastrol-treated cells (n=220, half the data points are displayed). Schematics show the MT attachment configurations and frequency for trivalents (F) and bivalents (G) positioned off-center on the spindle. Numbers indicate chromosomes counted in each category, from multiple independent data sets. (H, I) For controls or oocytes treated with 5 μM of the AURKA inhibitor MLN8054 for 1 h before fixation, kinetochores from both bivalents and trivalents were binned in 1.5 μm intervals based on distance from the nearest spindle pole, and the fraction of attached kinetochores was calculated in each bin. Lines show the logistic regression curves, based on parameters in Table S1. Numbers above each data point represent total numbers of kinetochores in each bin. Asterisk, p < 0.001; NS, not significant.
Figure 2
Figure 2. Kinetochores accumulate MAD1 as they approach spindle poles
(A) Chromosome composition in CF-1 and CHPO and in CHPO x CF-1 MI oocytes. (B–E) CHPO x CF-1 (B–D) or Rb(6.16) x CF-1 (E) oocytes expressing MAD1-2EGFP and histone H2B-mCherry were imaged live. An optical section (B) shows chromosomes near spindle poles (arrowheads and insets 1–3) and at the metaphase plate (4). Kinetochore MAD1-2EGFP intensity is plotted vs. distance from the nearest spindle pole (C); colors indicate kinetochores (n > 15) from 5 different oocytes; R2, cumulative correlation coefficient for all oocytes for a linear regression model; p < 0.0001. MAD1-2EGFP intensity was tracked on kinetochores of oscillating bivalents (D, E). Images are optical sections; arrowheads indicate kinetochores tracked in the kymographs; dashed ovals indicates spindle outlines. Graphs show MAD1-2EGFP intensity and displacement towards the pole over time course. Scale bars, 5 μm; A.U., arbitrary units.
Figure 3
Figure 3. Kinetochore poleward movement precedes MAD1 accumulation
(A) Schematics showing two models: chromosome poleward movement precedes (i) or follows (ii) MAD1-2EGFP accumulation, and how they can be distinguished graphically. (B–E) CHPO x CF-1 or Rb(6.16) x CF-1 oocytes expressing MAD1-2EGFP and histone H2B-mCherry were imaged live. Images (B–D) are optical sections; arrowheads indicate kinetochores tracked in the kymographs; dashed ovals indicates spindle outlines. MAD1-2EGFP intensity is plotted vs. displacement towards the pole (E) for 13 individual kinetochores. Data points are sequential time points, with the last time point indicated by the arrowhead. Individual traces are horizontally offset by an arbitrary distance for visual clarity. Scale bars, 5 μm; A.U., arbitrary units.
Figure 4
Figure 4. MAD1 accumulates on kinetochores of syntelic chromosomes as they approach the spindle pole during error correction
(A–C) Oocytes expressing MAD1-2EGFP and histone H2B-mCherry were imaged live during correction of syntelic attachment errors. Images (A) are optical sections from a time-lapse; timestamp h:min; arrowheads indicate the bivalent tracked in the kymograph (B). Yellow arrow indicates re-orientation of the kinetochores to face opposite poles. MAD1-2EGFP intensity was summed over both kinetochores in the syntelic and plotted vs. displacement towards the pole (C) for individual bivalents, from Rb(6.16) x CF-1 (n = 4) or SPRET x C57BL/6 (n = 2) oocytes. For clarity, only poleward movements are plotted. Data points are sequential time points, with the last time point indicated by the arrowhead. Individual traces are horizontally offset by an arbitrary distance for visual clarity. Scale bars, 2.5 μm; A.U., arbitrary units. (D) Model for correction of syntelic attachment errors: (i) low tension leads to kinetochore MT disassembly and poleward movement; (ii) MTs detach from kinetochores near the spindle poles due to AURKA activity; (iii–iv) chromosomes re-orient by congressing towards the metaphase plate and capturing MTs from the opposite spindle pole.
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