Which phase are sister chromatids separated?

. 2022 Mar 8;119[10]:e2123363119.

doi: 10.1073/pnas.2123363119. Epub 2022 Mar 2.

Affiliations

• PMID: 35235450
• PMCID: PMC8915976
• DOI: 10.1073/pnas.2123363119

Free PMC article

Sister chromatids separate during anaphase in a three-stage program as directed by interaxis bridges

Lingluo Chu et al. Proc Natl Acad Sci U S A. 2022.

Free PMC article

Abstract

During mitosis, from late prophase onward, sister chromatids are connected along their entire lengths by axis-linking chromatin/structure bridges. During prometaphase/metaphase, these bridges ensure that sister chromatids retain a parallel, paranemic relationship, without helical coiling, as they undergo compaction. Bridges must then be removed during anaphase. Motivated by these findings, the present study has further investigated the process of anaphase sister separation. Morphological and functional analyses of mammalian mitoses reveal a three-stage pathway in which interaxis bridges play a prominent role. First, sister chromatid axes globally separate in parallel along their lengths, with concomitant bridge elongation, due to intersister chromatin pushing forces. Sister chromatids then peel apart progressively from a centromere to telomere region[s], step-by-step. During this stage, poleward spindle forces dramatically elongate centromere-proximal bridges, which are then removed by a topoisomerase IIα–dependent step. Finally, in telomere regions, widely separated chromatids remain invisibly linked, presumably by catenation, with final separation during anaphase B. During this stage increased separation of poles and/or chromatin compaction appear to be the driving force[s]. Cohesin cleavage licenses these events, likely by allowing bridges to respond to imposed forces. We propose that bridges are not simply removed during anaphase but, in addition, play an active role in ensuring smooth and synchronous microtubule-mediated sister separation. Bridges would thereby be the topological gatekeepers of sister chromatid relationships throughout all stages of mitosis.

Keywords: TopoII decatenation; anaphase; cohesin; interaxis bridges; mitosis.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

Sister chromatids are linked by interaxis bridges until the end of anaphase. [A] Structure of metaphase chromosomes: Axes [green], loops [black and purple], and interaxis bridges with cohesin [red lines]. [B] Bridges are built on catenations between sister chromatids. [C] Three morphological stages of sister chromatid separation. From Top to Bottom: From close association at metaphase, sister chromatids undergo: global separation with modest bridge elongation; peeling apart with dramatic bridge extension at separation forks followed by bridge disappearance; and finally delayed bridge removal at telomeres. [A, Bottom and B are adapted from ref. 6]. [Scale bars: 1 μm.].

Fig. 2.

Three morphological stages of anaphase sister separation defined by time-lapse imaging of chromosome axes illuminated with EGFP-TopIIα in LLC-PK cells. [A–C] Three-dimensional images illustrate global parallel separation, peeling apart, and final resolution of telomere linkages. [A and B] Images processed by Imaris software. [B] Two individual chromosomes were traced after extraction from the entire complement [A] and shown individually in random relative dispositions with centromeres marked by cyan dots. The Right-most image shows the chromosomes at t = 7 min: The two sisters are in the final stages of peeling apart [single asterisk] or have completed peeling apart but still appear connected at their ends [double asterisk]. Importantly, at t = 7 min, the nucleus is at anaphase B, as indicated by the increased distance between the two poles as compared to t = 6 min [A]. [C] Quantitative description of the first two stages of sister separation. Global separation is defined by the distance between sister axes in regions where those axes remain parallel [dashed green and purple lines in B]. Onset and progression of peeling apart is defined by changes in intercentromere distance [yellow lines]. Each curve corresponds to 3D distances obtained from one of the two chromosomes in B. Data for interarm distances are the average and SD for all distances along still-parallel regions: Entire chromosome at earlier times or remaining parallel regions during peeling apart [dashed lines in B]. Each intercentromere distance comprises a single value for each given chromosome [thus without error bars]. [D and E] Finer kinetics of separation revealed by 2D images and distance quantifications. Note tendency for sister centromere regions to approach one another before onset of peeling apart; basis unknown. See also

Movie S1

. Each parallel interarm distance data point is an average of at least 10 different distances; error bars denote SD. [Scale bars: 1 μm, solid line; 5 μm, dashed line.].

Fig. 3.

Three morphological steps of bridge removal during anaphase sister separation defined by live cell imaging. Chromosomes illuminated with EGFP-TopIIα [A–G] or EGFP–kleisin-γ or H2B-GFP [H] in time-lapse series [A–E] or selected snapshots [F–H]. Images illustrate whole nucleus [A and E, Upper, and G] and detailed [B, D, E, Bottom, F, and H] bridge morphogenesis during the three stages of sister separation [Fig. 1C]. [C] Each interarm distance data point is an average of at least 10 different distances along the measured chromosome. Each intercentromere distance is a single data point. Error bars denote SD. [Scale bars: 1 μm, solid line; 5 μm, dashed line.].

Fig. 4.

Global separation is licensed by cohesin cleavage. Global parallel separation [blue arrows] and peeling apart [orange arrows] are both dependent upon the proteasome, as shown by addition of inhibitor MG132 at metaphase [A and B] [

SI Appendix, Fig. S6

]. Both are also dependent upon proteasome-mediated cohesin removal [C and D]. Oppositely, proteasome-independent removal of cohesin by auxin-induced degradation allows global separation [E and F], with peeling apart blocked due to activation of the SAC. Vertical white arrows indicate time of drug addition [defined as t = 0]. Each interarm distance data point for each chromosome is an average of at least 10 different distances. Error bars denote SD. [Scale bars: 1 μm.].

Fig. 5.

Global separation is independent of spindle forces. Addition of nocodazole at metaphase blocks global separation [blue arrows] and peeling apart [yellow arrows] [A and B, Second row]. Concomitant inhibition of the SAC [by AZ3146] alleviates the block to global separation [by allowing cohesin cleavage] but not microtubule-dependent peeling apart [A and B, Bottom row]. Inhibition of the SAC alone [A and B, Third row from Top] [

SI Appendix, Fig. S12

] results in accelerated onset of both global separation and peeling apart. Vertical white arrows indicate time of drug addition [defined as t = 0]. Each interarm data point is an average of at least 10 different interaxis distances; each intercentromere data point is one distance. Error bars denote SD. [Scale bars: 1 μm, solid line; 5 μm, dashed line.].

Fig. 6.

Global separation is independent of TopIIα-mediated decatenation. [A–D] Global separation of sister chromatids [blue arrows] occurs despite the presence of ICRF193 added at prometaphase [thus well before onset of anaphase]. Separation occurs with a long delay due to activation of the SAC [A–D] and in a timely fashion if the SAC is inactivated by simultaneous addition of AZ3146 [E]. Plots of parallel interarm distances in

SI Appendix, Fig. S11 B and C

. Peeling apart does not initiate because it requires decatenation in centromere regions during mid/late metaphase [Fig. 7]. White arrows indicate time of drug addition, defined as t = 0. [Scale bars: 5 μm.].

Fig. 7.

Initiation of peeling apart requires TopIIα-mediated decatenation. Addition of ICRF193 to cells in early or late metaphase results in two different phenotypes: Type I [A and B] or type II [C and D]. The type I phenotype is the same as when ICRF193 is added at prometaphase [Fig. 6]; global separation occurs but peeling apart is not initiated. In the type II phenotype, in contrast, peeling apart initiates but progresses for only a limited distance before arresting with the same fork morphology seen throughout normal anaphase. Additional evidence suggests that type I and type II cells are at early metaphase and late metaphase, respectively, at the time of inhibitor addition and that the difference reflects the occurrence of decatenation within centromere regions at mid/late metaphase [

SI Appendix, Fig. S11D

]. The same two phenotypes occur with a delay if ICRF193 is added alone [A and C] and in a timely fashion if SAC inhibitor reversine is added at the same time [B and D]. Thus, inhibition of decatenation during metaphase activates the SAC. SAC activation is more or less severe in type I and type II cells [

SI Appendix, Fig. S11D

]. White arrows indicate time of drug addition, defined as t = 0. [Scale bars: 1 μm, solid line; 5 μm, dashed line.].

Fig. 8.

TopIIα-mediated decatenation is the rate-limiting step in peeling apart. [A and B] When ICRF193 is added when peeling apart is in progress, that process is stopped in its tracks [except that eventually, after many minutes, forks open up to give hyperelongated bridges]. White arrows indicate time of drug addition, defined as t = 0. [C–E] Model for peeling apart. [C] Spindle forces promote separation of axes proximal to the centromeres, causing bridge elongation. Since the axes are stiff, this effect is propagated for some distance along the chromosome. [D] Full scenario: During peeling apart, spindle forces cause extensive bridge elongation, thereby making the component sister chromatid catenations subject to decatenation by TopIIα. [E] Morphologies corresponding to the steps of peeling apart outlined in D. [Scale bars: 1 μm, solid lines; 5 μm, dashed lines.].

Fig. 9.

Global separation is driven by intersister chromatin pushing forces. [A] Three-dimensional time-lapse images acquired simultaneously for chromatin [Left] and axis markers [Middle] of a chromosome in a living LLC-PK cell as it progresses from metaphase through midanaphase and corresponding intensity-weighted centroids [Right, obtained as in ref 6]. Adjacent balls along a centroid path corresponds to adjacent positions along the horizontal [y] axis. Centroid relationships were analyzed both for the entire chromosome and for a selected internal region [white dashed rectangle]. [Scale bars, 1 µm.] [B–D] At each centroid position, a plane perpendicular to the long axis of the chromosome was defined [gold box], and, within that plane, for each chromatid, the vector that links the TopIIα centroid to its partner H2B centroid [green and pink arrows] was defined. The lengths of the two chromatid loop/axis vectors [DTH1 and DTH2] and the distances between the corresponding centroids of the two sisters [DTT and DHH] were then determined. [E] Values of the indicated parameters, averaged over all positions along the internal chromosome segment selected in A, were plotted as a function of imaging time. Analogous plots for the entire chromosome and for both selected segments and entire chromosomes in four other samples are shown in

SI Appendix, Figs. S14 and S15

. [F] The relationships between DTT and DHH as defined in E, i and E, ii can be explained if the rotational relationships between sister loop/axis arrays change relative to one another over time, in two transitions [I–II and II–III], which correspond, respectively, to the onset of global separation [Middle] and the onset of peeling apart [Right]. [G] The scenario of F was evaluated by defining, at each position along a chromosome, the projections of each chromatid TopIIα-H2B vector onto the bridge/axis/bridge plane with corresponding signs defined with mirror symmetry for the two sisters [a1 and b1]. In this coordinate system, the tendency for the two vectors to point away from one another, to be parallel, or to point toward one another [as in F] is given by their sum [a1 + b1], which will be negative, zero, or positive in the three cases. [H] Model for how loop/axis shapes and intersister relationships might evolve during global separation. Top Left: Prior to the onset of global separation, sister loop/axis arrays point away from one another because bridges hold axes close together, forcing chromatin to the outside so as to minimize chromatin pressure. Top Right: Prior to cohesin cleavage, a global volume increase will force elongation of chromatin loops away from the constraints of the bridge-linked axes. Bottom Left and Right: Cleavage of cohesin will weaken bridges such that chromatin pressure will be strong enough to push axes farther apart, with concomitant bridge elongation [black line], thus producing a more energetically favorable chromatin configuration that now dictates axis/bridge/axis relationships. This effect will be more efficient and dramatic if it is preceded by a global volume increase [Right versus Left].

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