Insights & Perspectives Hypotheses

A chromosome separation checkpoint A midzone Aurora B gradient mediates a chromosome separation checkpoint that regulates the anaphase-telophase transition Helder Maiato1)2)*, Olga Afonso1)2) and Irina Matos1)2)a Here we discuss a chromosome separation checkpoint that might regulate the anaphase-telophase transition. The concept of cell cycle checkpoints was originally proposed to account for extrinsic control mechanisms that ensure the order of cell cycle events. Several checkpoints have been shown to regulate major cell cycle transitions, namely at G1-S and G2-M. At the onset of mitosis, the prophase-prometaphase transition is controlled by several potential checkpoints, including the antephase checkpoint, while the spindle assembly checkpoint guards the metaphase-anaphase transition. Our hypothesis is based on the recently uncovered feedback control mechanism that delays chromosome decondensation and nuclear envelope reassembly until effective separation of sister chromatids during anaphase is achieved. A central player in this potential checkpoint is the establishment of a constitutive, midzone-based Aurora B phosphorylation gradient that monitors the position of chromosomes along the spindle axis. We propose that this surveillance mechanism represents an additional step towards ensuring mitotic fidelity.

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Keywords: anaphase; Aurora B; checkpoint; gradient; mitosis; nuclear envelope; telophase

Introduction The establishment of a nuclear envelope that compartmentalizes genomic DNA represents a major evolutionary leap

that distinguished eukaryotes from prokaryotes more than 1.7 billion years ago. In metazoans, nuclear envelope breakdown (NEB) takes place soon after the onset of mitosis, and marks the

DOI 10.1002/bies.201400140 1)

2)

Chromosome Instability & Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Cell Division Unit, Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto, Porto, Portugal

*Corresponding author: Helder Maiato E-mail: [email protected]

a Present address: Laboratory of Mammalian Cell Biology and Development, Howard Hughes Medical Institute, The Rockefeller University, New York, USA

Abbreviations: APC/C, anaphase promoting complex/cyclosome; NEB, nuclear envelope breakdown; NER, nuclear envelope reassembly; SAC, spindle assembly checkpoint.

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beginning of prometaphase, while nuclear envelope reformation (NER) coincides with the resolution of mitosis at the anaphase-telophase transition. Mechanistically, NEB involves the hierarchical disassembly of the nuclear pore complexes (NPCs), depolymerization of lamins and the dissociation of inner nuclear membrane proteins from chromatin. All these events are regulated by steady activation of Cdk1 at the G2M transition, resulting in the phosphorylation of a plethora of substrates at the nuclear envelope, including lamins and nucleoporins. In contrast, NER involves stepwise recruitment of membranes to the decondensing chromatin and insertion of NPCs, which conversely require Cdk1 inactivation and the action of PP1 and PP2A phosphatases (reviewed in [1, 2]). Importantly, NER is not simply the reversal of NEB, and additional challenges are imposed by the individuality of the chromosomes, which must be brought together before complete reconstruction of the daughter nuclei. Mitotic fidelity is essential for genomic stability, and relies on several actions that maximize the chances that all chromosomes are successfully distributed during cell division, such as the formation of a metaphase plate (so that all chromosomes initiate poleward movement from the same starting line); the synchronous segregation of chromosomes to focused spindle poles during anaphase [3]; individual chromosome compaction by axial shortening during anaphase [4, 5]; and cell elongation [6, 7].

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In addition to these empirical mechanisms, faithful chromosome segregation is ensured by the action of several checkpoints that regulate key transitions. For instance, the commitment to progress into mitosis and completion of NEB are guarded by a p38-dependent checkpoint (see Box 1 and Table 1), also known as the “antephase checkpoint” (reviewed in [8–10]; Fig. 1). Additional checkpoints have been proposed to operate at the G2/M transition, such as the DNA damage and decatenation/ topoisomerase checkpoints, which share many features with the antephase checkpoint (reviewed in [8, 11]). However, it remains controversial whether the decatenation checkpoint has its own identity or it is simply part of a more global control mechanism that monitors physical and chemical stresses, by communicating with other checkpoints [12–15]. Curiously, exit from mitosis is believed to occur at the metaphase-

Insights & Perspectives

anaphase transition, under surveillance of the spindle assembly checkpoint (SAC), which monitors the attachment of kinetochores to spindle microtubules (reviewed in [8, 16, 17]; Fig. 1 and Table 1). Here we argue that another mitotic checkpoint operates after SAC satisfaction to delay NER (and real exit from mitosis) in response to incompletely separated chromosomes at the anaphase-telophase transition (Fig. 1 and Table 1). We refer to this surveillance mechanism as the “chromosome separation checkpoint”.

Telophase depends on the completion of anaphase Incomplete chromosome separation during a normal anaphase in both Drosophila- and human-cell cultures’ delays the transition to telophase, indicating that cells normally care about

Box 1 What is a checkpoint? The concept of cell cycle checkpoints was originally proposed by Hartwell and Weinert as control mechanisms that ensure the order/dependency of cell cycle events [18]. They proposed that “…the dependence of a late event in the cell cycle on an early event is due to a checkpoint” and “…the existence of a [checkpoint] is suggested when one finds chemicals, mutants, or other conditions that…permit a late event to occur even when an early, normally prerequisite event is prevented”. For instance, mitosis in eukaryotes is normally dependent on the completion of DNA replication, but some mutations allow mitosis to occur before completion of DNA replication. These mutations or ‘conditions’ constitute the “relief of dependence” principle and represent the rationale that led to the identification of many checkpoint genes [19–21] and chemicals that target them [22, 23]. Consequently, checkpoints normally delay cell cycle progression until completion of a critical early event, providing time for the correction of potential errors. The conceptual advance behind the idea of checkpoints was their extrinsic nature, i.e., they should not take an active role in the process being monitored, but the elimination of checkpoints may compromise the fidelity of the process and be deleterious to the cell, eventually leading to cell death. As so, a checkpoint is normally not essential and is only detectable in the event of errors or perturbations of specific events (such as the “relief of dependence”) [24].

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safeguarding sister chromatid separation before triggering NER [26]. The inverse correlation between anaphase duration and chromosome velocity has long been recognized in both plant and animal cells in response to low temperatures [27, 28]. In yeast, which undergo a closed mitosis, experimental reduction of spindle elongation velocity delays the mitotic exit network-dependent release of Cdc14 phosphatase [29]. Moreover, it has been proposed that a checkpoint mechanism delays spindle elongation in the presence of lagging chromosomes (likely caused by mechanical resistance due to merotelic attachments) [30, 31], or DNA damage [32]. Although the biological significance of these observations has remained unclear for decades, they suggest a common evolutionary route. One legitimate question is whether such feedback control mechanisms imply the existence of a bona fide mitotic checkpoint that regulates the

Another important criterion is the constitutive nature of checkpoints [24], in the sense that they must be active before a problem arises to allow its detection. Importantly, checkpoint adaptation or slippage allows the cell cycle to progress without satisfaction of the conditions monitored by a given checkpoint [25], clearly demonstrating that checkpoints delay, rather than arrest, cell cycle progression. Biochemically, checkpoints may consist of the following components: 1) a signal or signaling pathway that delays cell cycle progression until completion of an earlier event or in the presence of errors; 2) a sensor that detects the signal or monitors the process, thereby ensuring the completion of the event or the correction of errors; 3) a target that sustains the cell cycle delay by inhibiting an effector that promotes the transition into the subsequent cell cycle stage. In summary, checkpoints: • ensure the order of dependent cell cycle events; • delay, rather than arrest, cell cycle progression to allow completion of a dependent event; • provide time for the correction of errors; • are extrinsic to the process being monitored; • are constitutively active by default; • can be inhibited to relieve the dependence between successive cell cycle events; • are normally not essential; • may be detected in the event of errors; • are not satisfied in case of permanent or irreversible damage/errors, leading to adaptation or slippage; • involve sensors, signals, targets and/or effectors.

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Table 1. Comparative analysis of mitotic (and cytokinesis) checkpoints

NoCUT Cytokinesisa

Chromosome position along the division axis

DNA at cleavage furrow

Midzone Aurora B gradient Phosphorylation (?) Condensin I; Cyclin B1/Cdk1 (?) PP1, PP2A

Aurora B at midzone and/or near chromatin Chromatin acetylation (?) Anilin-related proteins (?); Mklp1 (?) Anilin-related proteins (?); Mklp1 (?); ESCRT-III (CHMP4C) No Polyploidy; DNA damage (?)

Sensor

p38 kinase; CHFR

Signal Target(s)

(?)b Condensin I/II (?);

Mad2, BubR1 Cdc20

Effector

Cdc25B

APC/C

Essential?c Consequence if inhibited

No DNA damage

No Aneuploidy; Cell death

Dependent relationship? Relief of dependence? Extrinsic to the process being monitored? Constitutively active? Cell cycle delay

Yes Yes (?)

Yes Yes No

No Polyploidy; Aneuploidy; Cell death (?) Yes Yes Yesd

No >10 h

Yes Up to 20 he

Yes > 6 hf

Event(s) being monitored

Yes Yes No No (?)g

a

Coordinates anaphase with cytokinesis. Likely multiple, due to sensitivity to multiple stresses. c For cell division. Cell and organism viability might be compromised. d When Aurora B is prevented to localize to the midzone, but not when its kinase activity is globally inhibited. e After microtubule depolymerisation (e.g. with nocodazole or cold). f After expression of non-degradable Cyclin B1 or PP1/PP2A inhibition at anaphase onset with Okadaic Acid. g Chromosome bridges persist >5 h but it is unclear whether this prevented progression into G1. b

anaphase-telophase transition in animal cells (Box1). The main condition is whether telophase depends on the completion of anaphase. To address this issue, one has to go back more than a century, to the original description of mitosis by Flemming, and the terms “anaphase” and “telophase” introduced by Strasburger and Heidenhain [33–35]. In these classic studies, mitotic stages were originally defined to reflect visible changes in structure and behavior from rudimentary microscopy analysis of fixed material stained by textile dyes, but whether these changes accurately reflect the molecular pathways that define a general state of the cell and underlie key mitotic transitions has been challenged [36]. When does anaphase end and telophase begin? Is it when few chromosomes decondense and start to reform the envelope or only when all chromosomes complete these processes? Our results support the

latter, and simply follow the same rationale behind other mitotic stages: a cell is only in metaphase when all chromosomes align at the equator; a cell is only in anaphase when all sister chromatids have disjoined (cohesion fatigue for example, leads to uncoordinated separation of some sister chromatids after SAC satisfaction, but, physiologically, cells show several hallmarks of prometaphase/metaphase [37]). Accordingly, we found that, during a normal mitosis, telophase only takes place when all sister chromatids have separated a given distance (
8 mm in Drosophila S2 cells [26]), suggesting the existence of a constitutive feedback control mechanism. Moreover, experimental treatments that slow down spindle elongation (and consequently the velocity of chromosome separation), trigger a cellular response that extends the duration of anaphase. This implies the co-existence of mechanisms that

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regulate the anaphase-telophase transition in time and space.

Cdk1 and Aurora B temporally and spatially regulate the anaphasetelophase transition Late mitotic events that represent hallmarks of telophase, such as chromosome decondensation and NER, together with the completion of cytokinesis, can be delayed by several hours in response to experimental conditions (e.g. expression of non-degradable Cyclin B1 or B3) that preserve some Cdk1 activity after anaphase onset [38–41]. This implies that the anaphase-telophase transition requires degradation of B-type Cyclins. Cyclin B1 degradation begins after SAC satisfaction as soon as the last chromosome bi-orients and 3

Hypotheses

Chromosome separation checkpoint Anaphase-Telophase

Spindle assembly checkpoint MetaphaseAnaphase Unattached kinetochores; tension (?) Aurora B (?)

Mitotic transition

Antephase checkpoint ProphasePrometaphase Stress

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Cdk1, Aurora B and the APC/CCdh1 is critical for the spatiotemporal regulation of the anaphase-telophase transition. The recent discovery that Cyclin B is an Aurora B target important for cytokinesis in Drosophila germ cells [52] might help explain how a feedback response delays NER in the presence of incomplete chromosome separation.

PP1 and PP2A phosphatases trigger the anaphase-telophase transition by counteracting Cdk1 and Aurora B activities

Figure 1. Mitotic transitions and checkpoints. The kymograph depicts a living Drosophila S2 cell stably expressing GFP-a-tubulin (which labels spindle microtubules) and CID-mCherry (which labels the kinetochores and allows chromosome tracking) undergoing mitosis and the respective transitions. Two different dimensions are represented space (L) and time (T). We propose that three distinct checkpoints (STOP signs) control the faithful progression of mitosis: the antephase checkpoint, the spindle assembly checkpoint and the chromosome separation checkpoint. The antephase checkpoint guarantees that nuclear envelope breakdown at the prophase-prometaphase transition occurs in the absence of any physical and chemical stress. As cells progress through prometaphase and metaphase, the spindle assembly checkpoint prevents anaphase onset in the presence of unattached kinetochores. Finally, during anaphase, the chromosome separation checkpoint guarantees effective separation of sister chromatids before nuclear envelope reassembly (and real mitotic exit) takes place.

aligns on the metaphase plate but, once started, the kinetics of Cyclin B1 degradation can be slowed down by certain conditions that regulate the activity of the anaphase promoting complex or cyclosome (APC/C), for example by experimentally reactivating the SAC [42, 43]. Thus, Cyclin B1 degradation appears to be a tunable process, and different thresholds likely control distinct mitotic transitions [41]. Alternatively, some organisms such as Drosophila embryos regulate late mitotic events and Cdk1 activity by controlling the degradation of Cyclin B3 [39]. In addition to this temporal control, we showed recently that a constitutive spindle midzone-associated Aurora B phosphorylation gradient [44] works as a sensor for chromosome separation 4

during anaphase (Fig. 2) [26]. Although this spatial control is present by default in a normal mitosis, it becomes more evident on lagging chromosomes, which are subject to higher Aurora B activity and delay envelope reformation relative to the main daughter nuclei (Figs. 2 and 3A). In the same line, Ipl1 (the single Aurora orthologue in yeast), has been shown to restrict chromosome decondensation to telophase [45, 46]. Interestingly, several studies in Drosophila embryos support that Cyclin B1 degradation and Cdk1 activity are also spatially regulated [47] and that a small pool of Cyclin B1 is only degraded during anaphase by the APC/CCdh1 [48]. In humans, Cdk1 and the APC/CCdh1 regulate midzone localization of Aurora B during anaphase [49–51], suggesting that coordination between

The anaphase-telophase transition relies also on the activity of PP1 and PP2A phosphatases that counteract the effect of the Aurora B gradient (Fig. 2). PP1 and PP2A phosphatase activities are modulated by a wide range of regulatory subunits that define the phosphatases’ substrate specificity and respective intracellular localization [53]. During anaphase, two of these regulatory subunits, Repo-Man and Sds22 target PP1 to chromatin and to kinetochores, respectively, to locally counteract Aurora B activity [54–57]. More recently, RepoMan was shown to associate with both PP1 and PP2A phosphatases to oppose Aurora B activity on chromosomes [58]. Repo-Man and the PP2A regulatory subunit B55a have also been implicated in NER in vertebrates [59, 60], and we have shown that PP1 and PP2A activities required for this process lie downstream of both Aurora B and Cdk1 [26]. Thus, PP1 and PP2A can be regarded as effectors that counteract Aurora B and Cdk1 to trigger chromosome decondensation and NER (Table 1).

Condensin I and lamin B receptor are critical targets involved in the spatial and temporal control of the anaphasetelophase transition A critical emerging question relates to the targets of Aurora B and Cdk1 that are

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counteracted by PP1/PP2A phosphatases and ultimately determine chromosome decondensation and NER. These two processes are inherently coupled, and NER has long been established to depend on the presence of chromatin [61]. We therefore reasoned that targets of Aurora B (a chromosomal protein) regulating the anaphase-telophase transition might be involved mainly in chromosome condensation.

Histone H3 phosphorylation at S10 has long been associated with chromosome condensation and is known to be governed by the counteracting activities of Aurora B and PP1 [56, 57, 62, 63]. However, ectopic expression of nonphosphorylatable or phosphomimetic Histone H3 at S10 (and S28) has no impact on chromosome condensation and the anaphase-telophase transition in Drosophila S2 cells [26]. Moreover,

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The dependence of telophase on anaphase chromosome separation can be relieved Another key condition for the existence of a chromosome separation checkpoint is whether the dependence of telophase completion on anaphase chromosome separation can be relieved (Box 1). If a constitutive midzone associated Aurora B activity gradient is the critical sensor of chromosome position along the division axis during anaphase, then inhibition of Aurora B kinase activity should abrogate the dependence of telophase on chromosome separation. Indeed, this was the case in both cultured Drosophila and human cells, often leading to polyploidy [26], a highly deleterious condition associated with chromosomal instability and cancer [76]. Additionally, functional perturbation of the Aurora B-regulatory 5

Hypotheses

Figure 2. Proposed model of the chromosome separation checkpoint. The beginning of Cyclin B1/Cdk1 degradation at the metaphase-anaphase (met-ana) transition regulates Subito/Mklp2 localization at the spindle midzone, to recruit the CPC and establish an Aurora B gradient. The phosphorylation (P) activity of Cdk1 (which gradually decreases as cells exit mitosis) and Aurora B is counteracted by PP1 and PP2A to regulate the anaphase-telophase transition in time and space. A key phosphorylation/dephosphorylation target of Cdk1 is lamin B receptor (LBR), which must be dephosphorylated to trigger NER. In contrast, Condensin I, whose association with chromosomes is spatially regulated by the midzone Aurora B gradient is required for spatial control of NER. In a normal mitosis, NER only takes place when all chromosomes separate away from the effect of a constitutive Aurora B phosphorylation gradient and PP1/PP2A take over. It remains unclear how Aurora B communicates with Cyclin B/Cdk1. In the exceptional event of errors that lead to lagging chromosomes, chromosome decondensation and envelope reformation are locally delayed. This allows the lagging chromosome to be corrected. Because envelope reformation in the main nuclei is not uniform, the side facing Aurora B at the midzone is also delayed, providing an opportunity for re-integration of corrected lagging chromosomes. Occasionally, micronuclei may form if the lagging chromosome is not efficiently corrected during anaphase.

in human cells, there is poor correlation between Histone H3 phosphorylation and chromosome condensation [64, 65], suggesting alternative targets. Nevertheless, it remains possible that endogenous phosphorylation of Histone H3 still accounts for a normal mitosis. Genome editing techniques, such as the CRISPR/Cas9 or TALEN systems [66], will be ideal for clarifying this issue. Another bona fide Aurora B substrate is Condensin I [67–69]. The association of Condensin I with chromosomes also depends on the coordinated activities of Aurora B and PP1/PP2A [54, 69–71], and is sensitive to chromosome positioning along the division axis during anaphase [26] (Figs. 3B, B’). Moreover, Aurora B-mediated recruitment of Condensin I to chromosomes was required to spatially regulate the anaphase-telophase transition [26]. In contrast, Cdk1 has been shown to directly regulate the timing of NER by phosphorylating lamin B receptor [72], an inner nuclear membrane protein that links chromatin with the nuclear envelope through interaction with HP1 and Histones H3/H4 [73–75]. In the future, it will be important to identify additional potential targets on anaphase chromosomes and determine whether Aurora B is able to directly regulate substrates on the nuclear envelope.

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Figure 3. Spatial control of nuclear envelope reassembly and chromosome decondensation at the anaphase-telophase transition. A: Control Drosophila S2 cell stably expressing Nup107mRFP/H2B-GFP with lagging chromosomes. The reformation of the nuclear envelope is inhibited on the lagging chromosomes ( ) while it starts to reform in the main nuclei. Note that the region of the nuclear envelope remains open (arrowheads) increasing the probability of reintegration of lagging chromosomes in the main nuclei. Insets represent higher magnification views of the Nup107-mRFP signal. B: Live cell imaging of a Drosophila S2 cell stably expressing Condensin I/Barren-GFP and H2B-mCherry illustrating the spatial regulation of Condensin I association with chromosomes at the anaphase-telophase transition. Note that as chromosomes segregate to the poles Condensin I is lost from chromosomes that start decondensing. Scale bars ¼ 5 mm. Time is in min:sec. Time zero was set at anaphase onset. B’: Compressed kymograph of the division axis derived from the time-lapse sequence shown in B.

subunit INCENP by antibody microinjection during anaphase allows decondensation of incompletely separated chromosomes [77]. Thus, the dependence of telophase on anaphase chromosome separation can be experimentally relieved by inhibiting Aurora B activity. One important caveat is that Aurora B kinase activity is itself required for spindle elongation (and consequently for chromosome separation), breaking another golden rule that defines a checkpoint – to be extrinsic to the process that is being monitored (Box 1). In fact, taken to one extreme, all SACrelated proteins (Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, and Aurora B) play a checkpoint-independent role in kinetochore-microtubule attachments [22, 78–85]. The key is to find experimental conditions that isolate both functions. Accordingly, by specifically preventing Aurora B localization at the spindle midzone (which per se does not prevent spindle elongation and general chromosome separation), we found that spatial control of NER is lost: envelope 6

reformation now takes place at exactly the same time on lagging chromosomes and main nuclei [26]. Interestingly, it has been previously suggested that drug treatments that compromise the formation of the central spindle (with consequent mislocalization of Aurora B) and give rise to shorter pole-to-pole distance may favor the reformation of a single membrane around telophase chromosomes, including lagging chromosomes [86, 87]. Importantly, spatial control of NER is independent of Aurora B localization on centromeres, since lagging acentric fragments generated by laser microsurgery still delay envelope reformation relative to the main nuclei [26]. Finally, Cdk1 inhibition with RO-3306 also breaks the dependence of envelope reformation in the main nuclei on chromosome separation, and accelerates Aurora B deposition at the spindle midzone, but lagging chromosomes still delay envelope reformation [26]. Thus, spatial control of NER is dependent on Aurora B at the spindle midzone and independent of Cdk1 activity.

A chromosome separation checkpoint promotes mitotic fidelity by allowing the correction of errors invisible to the SAC One could argue that a delay of just few minutes during a normal mitosis cannot be explained by a robust checkpoint (we stress that anaphase in cultured Drosophila or human cells only takes around 10 min to complete). It should be noted, however, that checkpoints are often revealed only in the event of errors. For instance, cultured human cells normally take
30 min from NEB until anaphase onset, and the existence of a SAC is only revealed by careful observation of cells, which appear to wait for the last chromosome to align at the cell’s equator before separating their sister chromatids [88, 89]. Importantly, this period can be experimentally extended up to
20 h in the presence of chronically unattached kinetochores (e.g. due to the presence of microtubule poisons), clearly uncovering the existence of a checkpoint [25]. Similarly, both Drosophila and human cells in culture can remain in anaphase with condensed and separated chromosomes without NER for several hours after experimental perturbation of Cyclin B degradation or specific inhibition of PP1/PP2A activities at anaphase onset [26, 40]. These results predict that if one finds conditions that prevent further separation of sister chromatids after anaphase onset, while simultaneously ensuring that Aurora B

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time, which creates an opportunity for reintegration of lagging chromosomes into the main nuclei after being corrected (Figs. 2 and 3A-B’). Further testing of this mechanism would include ectopic targeting of Aurora B to specific compartments or organelles that lie in the vicinity of the nucleus. Taken together, these data favor the idea that the anaphase-telophase transition should not be regarded as a global response that occurs simultaneously in all chromosomes, but should rather be seen as a gradual process that is regulated at the individual chromosome level, and normally depends on the separation of sister chromatids along the division axis (Fig. 2). Therefore, a chromosome separation checkpoint allows, in the first place, that all separated sister chromatids end up in the corresponding daughter nuclei during a normal mitosis; secondly, it allows eventual errors such as lagging chromosomes to be corrected; and finally, increases the probability of re-integration of corrected lagging chromosomes into the main nucleus, thereby promoting mitotic fidelity.

The chromosome separation checkpoint and NoCut are conceptually and mechanistically distinct The presence of chromosomal bridges during anaphase locally activates Aurora B during cytokinesis, resulting in the inhibition of abscission, as part of a pathway known as NoCut, or “abscission checkpoint”, that monitors clearance of chromatin from the spindle midzone [99, 100]. A putative chromosome separation checkpoint cannot be confused with NoCut for the following two reasons: First, NoCut was proposed as a mechanism that prevents the completion of cytokinesis in the presence of lagging chromosomes/chromatin. The checkpoint that we propose ensures that chromosomes have properly separated apart before reassembling the nuclear envelope. Mechanistically, these are different events, likely involving different targets (Table 1). Second, NoCut was proposed to be activated only when lagging chromosomes are present, and does not take into account the role of an

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Aurora B gradient at the spindle midzone. In fact, it was proposed that Aurora B on lagging chromosomes (at the midzone or chromatin) triggers the NoCut, while in our case a midzone Aurora B gradient is active by default, and monitors chromosome separation even in a normal unperturbed mitosis. This is a key conceptual difference between the two potential checkpoints. Nevertheless, it is conceivable that an Aurora B activity gradient at the spindle midzone plays important surveillance roles that ensure effective separation of sister chromatids prior to NER and completion of cytokinesis. Finally, it is worth mentioning that defects in NPC assembly activate the Aurora B-mediated abscission checkpoint [101], suggesting the existence of additional levels of complexity related to feedback control mechanisms during late mitosis/cytokinesis that remain to be explored.

Is there a link between chromosome separation, aging and cancer? No checkpoint is able to block cell cycle progression indefinitely, and there might be errors that escape their supervision. Notably, while many lagging chromosomes are able to be reincorporated in the main daughter nuclei by the action of a chromosome separation checkpoint, some lagging chromosomes eventually fail to re-integrate and form micronuclei [26], which are well-established biomarkers of genotoxicity, chromosomal instability and cancer [102, 103]. Despite the fact that micronuclei derived from anaphase lagging chromosomes and chromosome bridges often re-integrate into the main nucleus in the subsequent mitosis [104, 105], they are defective in lamin and NPC recruitment, show condensed chromatin and have reduced transcriptional activity. Micronuclei are also more prone to DNA damage and chromosome rearrangements, which may be due to incapacity to assemble a protective envelope [106–108]. It is noteworthy that Aurora B activity is highest on midbodies until late stages of cytokinesis and daughter cell re-adhesion to the extra-cellular matrix [109]; and envelope reformation remains inhibited on 7

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remains associated with the spindle midzone, NER will not take place. Although a robust SAC response makes sense at the metaphase-anaphase transition to prevent massive chromosome missegregation and consequent loss of cell/organism viability [90, 91], this checkpoint is not exempt from failure. In fact cells may progress into anaphase with just one or a few chromosomes that lag behind due to merotelic attachments (when a single kinetochore is attached to microtubules from both spindle poles) [92], which are invisible to the SAC. Despite being quite rare (
1% in Ptk1 cells [93] and
0.1% in human chromosomally stable colorectal cancer HCT116 cells [94]), merotelic attachments have been proposed as a major route to chromosomal instability, a hallmark of human cancers [95]. However, lagging chromosomes rarely missegregate [94], implying the existence of yet poorly understood correction mechanisms. Indeed, merotelic attachments on lagging chromosomes are normally corrected by the action of anaphase spindle forces [96, 97]. This would only be possible if envelope reformation is locally delayed on the lagging chromosome [26] (Figs. 2 and 3A). A related question is whether a local event, such as a single lagging chromosome, is able to impose a global cellular response by delaying the anaphasetelophase transition. We argue that, as a single unattached kinetochore is sufficient to delay sister chromatid disjunction (i.e. anaphase onset) in all chromosomes, the completion of envelope reformation in all chromosomes is what defines the transition into telophase, which can be delayed in the presence of lagging chromosomes or DNA bridges. It should be remarked that we have no evidence that a lagging chromosome per se imposes any delay in envelope reassembly in the main nuclei, consistent with the idea that there is no inhibitory signal coming from chromosomes. Interestingly, however, envelope reassembly in the main nuclei is an asymmetric process, starting near the poles and closing at the side of chromatin away from the poles [1, 98], a phenomenon that can now be understood in light of an inhibitory action imposed by an Aurora B gradient from the spindle midzone [26]. Thus, the nuclear envelope remains “open” for a short window of

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Hypotheses

from a compromised chromosome separation checkpoint. In order to validate this hypothesis, it would be useful in the future to compare the frequency and fate of lagging chromosomes in young vs. older cells, as well as in normal vs. transformed cells, especially those with chromosomal instability. It will also be important to evaluate whether Aurora B inhibitors that are currently in clinical trials impact on a potential chromosome separation checkpoint and how failure of this checkpoint could be explored therapeutically. Figure 4. Lagging chromosomes remain condensed and do not reform the envelope until late cytokinesis. Sextuple immunofluorescence of a pig LLC-PK cell showing a lagging chromosome (arrow) highly enriched for phosphorylated Histone H3 (S10) in the proximity of Aurora B at the midbody during cytokinesis. Microtubules, actin, DNA and nuclear envelope are also depicted. Note the lack of nuclear envelope in the lagging chromosome, in contrast to the main nuclei. Scale bar ¼ 10 mm.

lagging chromosomes (Fig. 4). This suggests that a local inhibitory effect imposed by Aurora B can prevent chromatin decondensation and envelope reformation on lagging chromosomes, which may give rise to micronuclei and explain their unusual features. The prevalence of micronuclei with condensed chromatin and without a surrounding envelope throughout the subsequent interphase might reflect “slippage” from a chromosome separation checkpoint, similar to pre-anaphase cells that cannot satisfy the SAC, and exit mitosis with SAC proteins at kinetochores [110]. Another interesting observation is related to the preferential formation and eventual loss of X chromosome-derived micronuclei and the bias for re-integration of autosome chromosomes in older women, as a function of the distance relative to the spindle poles [111, 112]. Moreover, spindle elongation efficiency was shown to decrease exponentially as women age, and has been proposed to compromise the correction of anaphase lagging chromosomes that could lead to aneuploidy and polyploidy [113]. It is tempting to speculate that the increased propensity of micronuclei formation with aging and in cancer might result 8

Conclusions Here we have proposed and challenged the hypothesis of a “chromosome separation checkpoint” that constitutively operates during late mitosis to regulate the anaphase-telophase transition. The first conclusion is that in light of the current definition of checkpoint, all mitotic (and cytokinesis) checkpoints, including the SAC, have pitfalls. The second conclusion is that mitotic stages, as we know them, do not reflect the individuality of chromosomes. The key question is whether biology should be held hostage to man-made concepts/ definitions or whether those concepts/ definitions have to be broadened in light of new paradigms. In either case, it appears that the spatiotemporal regulation of mitosis involves a series of feedback mechanisms that ultimately control entry, progression through and exit from mitosis to promote the fidelity of the process. At the moment, we have just an initial view of how a chromosome separation checkpoint might work. It remains unclear how this spatial control feeds back information to the molecular clocks that control the anaphase-telophase transition. Moreover, it remains to be determined how a potential chromosome separation checkpoint is coordinated with the completion of cytokinesis, while expanding the repertoire of potential Aurora B targets on both chromatin and nuclear envelope. It will also be essential to uncover to what extent other errors invisible to the SAC, such as telomere fusions or catenated DNA [114, 115], are sensitive to a midzone Aurora B gradient and how they impact the anaphase-telophase transition and mi-

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totic fidelity. Finally, it will be important to determine whether the proposed chromosome separation checkpoint is impaired in cancer cells; if so, this could explain, at least in part, the abnormally high rate of micronuclei formation and the propensity for numerical and structural aberrations of chromosomes in cancer cells.

Acknowledgements We thank all members of the CID lab, Cristina Ferra´s in particular, for insightful discussions, as well as Anto´nio Pereira and Jorge Ferreira for the critical reading of the manuscript. We also thank Aaron F. Mertz for careful proofreading. H.M. is funded by PRECISE grant from the European Research Council. The authors declare no conflict of interest.

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