Feedback control of chromosome separation by a midzone Aurora B gradient

Abstract

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. Here, we identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER.

Fig. 1. Incomplete chromosome separation delays the anaphase-telophase transition

(A) Control, Taxol-treated, and kinesin-like protein KLP10A RNAi S2 cells stably expressing Lamin B–GFP and mCherry–α-tubulin (mCherry-TUB). Time shown as minutes:seconds. (B and C) Quantification (box plots) of anaphase duration in response to experimental attenuation of half-spindle elongation velocity. Whiskers define the maximum and the minimum value. Statistics: t test or Mann-Whitney U test depending on the normality of the samples (***P < 0.001; ** P < 0.01). (D) Linear regression and distribution of anaphase duration versus half-spindle elongation velocity in the different conditions. rs, Spearman correlation coefficient, P < 0.001. Note that there is a minimal anaphase duration time (~5 min).

Fig. 2. NER is delayed on lagging chromosomes and DNA bridges

(A and B) S2 cells stably expressing Nup107-mRFP and H2B-GFP showing a delay in NER on lagging chromosomes [white arrowheads in (A)] and (B) DNA bridges. Time shown as minutes:seconds. (C) Many lagging chromosomes are reintegrated into the main nuclei or form micronuclei. Those cases in which the envelope did not form 30 min after anaphase onset were classified as “No NER.” (D and D’) NER correlates with lower Aurora B activity [p-H3 (S10)] and chromosome separation. Wheat germ agglutinin (WGA) staining was used to reveal the nuclear envelope. A.U., arbitrary units. Scale bars, 5 μm.

Fig. 3. Aurora B at the spindle midzone is required for spatial control of NER

(A) S2 cell stably expressing Nup107-mRFP and H2B-GFP. Time shown as minutes:seconds. Scale bars, 5 μm. (B) Induction of acentric chromosomes with laser microsurgery (arrowhead) generates lagging fragments that delay NER. (C) Subito/Mklp2 RNAi delays NER. (D) Simultaneous NER on lagging chromosomes and main nuclei after Subito/Mklp2 depletion.

Fig. 4. PP1/PP2A phosphatases counteract Aurora B and are required to promote chromosome decondensation and NER

(A) Acute Aurora B (Bi-2) or PP1/PP2A inhibition (OA) at anaphase onset in S2 cells stably expressing Lamin B–GFP and mCherry–α-tubulin. (B) Quantification of anaphase duration until DNA decondensation or NER in control cells (n = 16 cells per condition) and Bi-2–treated cells (n = 10 cells per condition). Time shown as minutes:seconds. (C) Quantification of half-spindle elongation velocity. (D) RNAi against specific PP1 or PP2A subunits (n = 8 cells per condition) in the Lamin B–GFP/mCherry–α-tubulin cell line (Mann-Whitney U test). The whiskers define the maximum and minimum values. (E) Distribution of anaphase duration in control (n = 16 cells), PP1-87B (n = 8 cells), and PP2A-C RNAi (n = 23 cells). Scale bars, 5 μm.