Twenty years of merotelic kinetochore attachments: a historical perspective

Abstract

Micronuclei, small DNA-containing structures separate from the main nucleus, were used for decades as an indicator of genotoxic damage. Micronuclei containing whole chromosomes were considered a biomarker of aneuploidy and were believed to form, upon mitotic exit, from chromosomes that lagged behind in anaphase as all other chromosomes segregated to the pole of the mitotic spindle. However, the mechanism responsible for inducing anaphase lagging chromosomes remained unknown until just over twenty years ago. Here, I summarize what preceded and what followed this discovery, highlighting some of the open questions and opportunities for future investigation.

Introduction

During mitosis, the sister chromatids of the replicated chromosomes must separate and segregate to opposite poles, and this process must be executed accurately to ensure that each daughter inherits a complete copy of the genome. A variety of mitotic defects can lead to chromosome missegregation and result in genetic imbalances in the daughter cells [reviewed in (Baudoin and Cimini 2018b)]. Many of these errors are prevented by a biochemical signaling pathway, known as the spindle assembly checkpoint (or SAC), that monitors chromosome attachment to the mitotic spindle (Lara-Gonzalez, et al. 2021). Anaphase lagging chromosomes, however, are caused by a defect that can escape SAC detection and therefore represent a major threat to mitotic fidelity. Upon mitotic exit, anaphase lagging chromosomes often form micronuclei in the nascent daughter cell (Cimini, et al. 2002, He, et al. 2019) and micronuclei were shown to cause a variety of adverse effects (Krupina, et al. 2021). Although other chromosome segregation defects can be observed in cancer cells and tumor tissues (Gisselsson, et al. 2004, Stewenius, et al. 2007, Tucker, et al. 2023), anaphase lagging chromosomes were shown to be prevalent in chromosomally unstable cancer cells, mouse tumor models, and certain human tumors (Bakhoum, et al. 2011, Bakhoum, et al. 2015, Bakhoum, et al. 2014, Thompson and Compton 2008, Venkatesan, et al. 2021, Zaki, et al. 2014). Similarly, micronuclei are often found in cancer cells and tumor tissues (Gisselsson, et al. 2001, Streffer, et al. 1985), and have been shown to be an important biomarker associated with increased risk of disease, including cancer (Bonassi, et al. 2011a, Bonassi, et al. 2011b). The mechanism responsible for the formation of lagging chromosomes was discovered just over twenty years ago (Cimini, et al. 2001). That discovery became the seed for a series of new research questions, ranging from the correction mechanisms, to the mechanisms that increase these errors in cancer cells, to the biology of micronuclei, and more. In this review, I summarize questions, knowledge, and ideas that preceded the identification of the mechanism underlying anaphase lagging chromosomes. Then, I briefly summarize all the findings that followed that discovery and are still a fertile ground of investigation for researchers in the chromosome segregation, aneuploidy, and cancer biology communities.

Anaphase lagging chromosomes and kinetochore-positive micronuclei as biomarkers of aneuploidy

An anaphase lagging chromosome is defined as a chromosome that lags behind during anaphase, when all other chromosomes segregate to the spindle poles, and it can form a micronucleus upon mitotic exit (Figure 1B). A micronucleus, as the name suggests, is a small structure consisting of DNA encapsulated by a nuclear envelope, which is separated from the main nucleus. By the early 1990s, the cellular toxicology community viewed anaphase lagging chromosomes as precursors of certain micronuclei, whose occurrence was considered to be a biomarker of aneuploidy (Antoccia, et al. 1991, Bonatti, et al. 1992, Degrassi and Tanzarella 1988, Thomson and Perry 1988). This was partly based on detailed cytology studies indicating that missegregation of chromosomes or chromatids and subsequent micronucleus formation could lead to aneuploidy in the cell progeny (Gustavino, et al. 1994, Rizzoni, et al. 1989). Detecting and quantifying micronuclei is simple and fast and therefore they have been used as a measure of genotoxic damage for decades (Pincu, et al. 1985) in a standardized assay named the in vitro micronucleus assay (Fenech 2000). However, micronuclei can form as a result of either missegregation of whole chromosomes (Figure 1B) or acentric chromosome fragments (Figure 1A) that cannot interact with the mitotic spindle, and the simple visualization of micronuclei does not provide information on their origin. In the late 1980s, the genetic toxicology community recognized the need to identify genotoxic agents that affected cells and organisms in ways other than by damaging their DNA or inducing gene mutations. Specifically, it had become apparent that certain chemical agents do not cause DNA damage (i.e., are not clastogenic), but can cause aneuploidy (i.e., are aneugenic), which can severely impair cell and organismal physiology. At that time, the method of choice for assessing aneuploidy was counting chromosomes in chromosome spreads. However, such approach is time consuming and has limited statistical power, given that typically only ~50 chromosome spreads are analyzed per sample. Therefore, there was a need to develop an assay that allowed to quantify aneuploidy quickly and easily. The in vitro micronucleus assay appeared to have the right requisites, but only if strategies could be developed to distinguish micronuclei that contained entire chromosomes (from aneugenic effects) from those containing chromosome fragments (from clastogenic effects). To this end, attempts were made to discriminate micronuclei based on their size and/or their DNA content, assuming that micronuclei that were larger and/or contained more DNA, were more likely to contain entire chromosomes compared to smaller micronuclei (Hogstedt and Karlsson 1985, Pincu, et al. 1985, Yamamoto and Kikuchi 1980). This idea was justified, given that micronuclei containing larger chromosomes were recently reported to have a larger area (Mammel, et al. 2022). However, these approaches are laborious and difficult to standardize and therefore were never adopted as routine methods in the genetic toxicology field. The discovery of antibodies from scleroderma patients (CREST) that localized to the kinetochore (Moroi, et al. 1980) enabled the discrimination between micronuclei containing entire chromosomes (CREST-positive micronuclei) and micronuclei containing chromosome fragments (CREST-negative micronuclei) (Figure 1A–B). A number of studies focused on assessing the ability of such approach to predict the aneugenic potential of physical and chemical agents (Antoccia, et al. 1991, Fenech and Morley 1989, Hennig, et al. 1988, Schuler, et al. 1997). As opposed to the detection of anaphase lagging chromosomes or counting chromosomes in chromosome spreads, this approach could be used to quantify micronuclei in thousands of interphase cells in tens of minutes (Fenech 2000). A variation of this approach was subsequently developed to selectively identify micronuclei in cells that were proliferative and underwent cell division during or after exposure. This method was based on the idea that aneuploidy arising in proliferative cells would affect the cell population, and hence organismal health; moreover, this approach would prevent micronuclei already present in the cell population from confounding the data. In this approach, named the cytokinesis-block assay, cells were treated, during or after exposure, with cytochalasin (an inhibitor of actin polymerization) for a time window corresponding approximately to cell cycle duration prior to fixation and analysis. All the cells undergoing cell division during the cytochalasin treatment would be unable to complete cytokinesis and would therefore appear as binucleate cells; analysis of micronuclei could then focus specifically on the binucleate cell population (Figure 1C). The combination of the cytochalasin treatment with kinetochore staining was viewed as a powerful method in the genetic toxicology community (Fenech 2000), although comparison of rates of anaphase lagging chromosomes vs. CREST-positive micronuclei in binucleate cells revealed that this method underestimated the number of anaphase lagging chromosomes (Cimini, et al. 1999).

Figure 1.

Diagram showing formation of micronuclei upon mitotic exit. Chromosomes/DNA are depicted in blue, kinetochores in orange. A. Formation of a kinetochore-negative micronucleus from a lagging chromosome fragment. B. Formation of a kinetochore-positive micronucleus from an anaphase lagging chromosome. C. Formation of a kinetochore-positive micronucleus in a binucleate cell in the cytokinesis-block assay.

Although the genetic toxicology community broadly used centromere/kinetochore-positive micronuclei as a readout of aneuploidy and overall agreed that such micronuclei originated from anaphase lagging chromosomes, the mechanism causing lagging chromosomes remained unknown.

Merotelic kinetochore attachment: the underlying cause of anaphase lagging chromosomes

Because the genetic toxicology community was not accustomed to thinking about cellular mechanisms and the cell biology community was not focused on aneuploidy and its consequence, the cellular mechanism causing chromosomes to lag behind during anaphase was unknown until just over 20 years ago. Nevertheless, some ideas were shared in informal discussions and included overriding of the SAC in the presence of unattached chromosomes/kinetochores, accidental detachment of microtubules from kinetochores during anaphase, and failure of sister chromatids to separate (which would lead to the formation of micronuclei with two kinetochores) as the potential causes of anaphase lagging chromosomes. The idea that anaphase lagging chromosomes may result from a “leaky” SAC was perhaps influenced by the term used for chromosomes in prometaphase cells that were positioned close the spindle poles. These chromosomes were often referred to as “lagging chromosomes” and perhaps this terminology led to the idea that these unaligned prometaphase chromosomes failed to align at the metaphase plate prior to anaphase onset and were the precursors of anaphase lagging chromosomes. We now prefer to refer to such prometaphase chromosomes as “unaligned” chromosomes to differentiate them from the anaphase lagging chromosomes (Baudoin and Cimini 2018b). It was also shown that unaligned chromosomes are not seen at anaphase onset in cells with anaphase lagging chromosomes and that cells with anaphase lagging chromosomes do not experience a pre-anaphase mitotic delay (Cimini, et al. 2002), which typically occurs when unaligned chromosomes are present (Rieder, et al. 1995, Rieder, et al. 1994). These observations definitely dismissed the idea of anaphase onset in the presence of unattached kinetochores as the underlying cause of anaphase lagging chromosomes. The accidental detachment of microtubules from the kinetochore was also not plausible, given that the stability of microtubules had been shown to dramatically increase in anaphase compared to metaphase (Zhai, et al. 1995) and we now know that microtubule pulling forces stabilize kinetochore-microtubule attachments (Akiyoshi, et al. 2010), thus making detachment in anaphase highly unlikely. Finally, failure of sister chromatids to separate was also very unlikely, given that staining with individual centromeric probes showed micronuclei contained individual centromeres and anaphase lagging chromosomes consisted of individual daughter chromosomes and not paired chromatids (Catalan, et al. 2000, Cimini, et al. 1997, Cimini, et al. 1999, Falck, et al. 2002).

Detailed analysis of chromosomes/kinetochores and microtubules revealed that the underlying cause of anaphase lagging chromosomes was merotelic kinetochore attachment (Figure 2), a type of kinetochore-microtubule attachment in which a single kinetochore binds microtubules from both spindle poles instead of just one (Cimini, et al. 2001). This type of attachment had previously been reported in cold- or drug-treated crane-fly spermatocytes, where the term merotelic was first used (Janicke and LaFountain 1984, Ladrach and LaFountain 1986), and in cells undergoing cell division with individual chromatids or chromosome fragments containing single kinetochores (Khodjakov, et al. 1997, Wise and Brinkley 1997, Yu and Dawe 2000). The observation that merotelic kinetochore attachment could explain the origin of anaphase lagging chromosomes in mitotic cells provided a possible mechanism for the aneuploidy and chromosomal instability (CIN) frequently observed in cancer cells (Bolhaqueiro, et al. 2019, Lengauer, et al. 1997, Nicholson and Cimini 2013, Rajagopalan and Lengauer 2004, Weaver and Cleveland 2006). In support of this idea, subsequent studies showed that most cancer cells display high rates of anaphase lagging chromosomes (Bakhoum, et al. 2014, Ganem, et al. 2009, Silkworth, et al. 2009, Thompson and Compton 2008) and lagging chromosomes were also observed in tumor tissue samples (Bakhoum, et al. 2011, Zaki, et al. 2014), although this may not be the case for all tumor types (Gisselsson, et al. 2004, Stewenius, et al. 2007, Tucker, et al. 2023). Merotelic kinetochore attachment was also shown to be a key mechanism of aneuploidy in human pluripotent stem cells (Deng, et al. 2023) and in mammalian oocytes, where it can promote chromosome missegregation in both meiosis I and II (Cheng, et al. 2017, Holubcova, et al. 2015, Kouznetsova, et al. 2014, Shomper, et al. 2014, Zielinska, et al. 2015), although other mechanisms of chromosome missegregation have also been described in meiotic cells. For instance, one study identified lack of kinetochore-microtubule attachment as a major cause of lagging chromosome formation and aneuploidy in aged mouse oocytes (Mihajlovic, et al. 2021).

Figure 2.

Fate of anaphase lagging chromosomes depends on the relative size of the microtubule bundles connecting the merotelic kinetochore to the two spindle poles. A. Example of live cell (top) and diagram (middle) of a “balanced” merotelically attached anaphase lagging chromosome. In the images (taken two minutes apart), the lagging kinetochore can be seen persisting at the cell equator as all the other chromosomes move poleward. As a result of this, a whole chromosome-containing micronucleus is formed in one of the daughter cells (bottom). B. Example of live cell (top) and diagram (middle) of an “unbalanced” merotelically attached anaphase lagging chromosome. In the images (taken three minutes apart), two merotelically attached kinetochores can be seen. However, because of the different size of the two microtubule bundles connecting the merotelic kinetochores to the two spindle poles, the chromosomes’ position is shifted close to the group of normally segregating chromosomes on the right. As a result of this, the chromosome(s) will be included in the main nucleus in one of the daughter cells (bottom). In the images, kinetochores are in green and microtubules are red. In the diagrams, chromosomes/DNA are depicted in blue, kinetochores in orange. Live cell images are modified from Cimini et al., 2004 (A) and Cimini et al., 2006 (B).

Overall, there is currently strong agreement that merotelically attached lagging chromosomes represent a major source of aneuploidy and chromosomal instability in normal and cancer cells.

How do merotelic attachments arise?

Establishment of kinetochore-microtubule attachments is a stochastic process and therefore different types of attachments can be established in early mitosis [reviewed in (Gregan, et al. 2011, Sarangapani and Asbury 2014)]. Merotelic kinetochore attachments form often during the early stages of mitosis in normal bipolar cells, but they decrease as mitosis progresses (Cimini, et al. 2003). Ultimately, chromosome mis-segregation due to merotelic kinetochore attachment will depend on the overall balance between rates of formation of such mis-attachments and the rates at which they are corrected. The rates of merotelic attachment formation were shown to increase in several contexts, mostly having to do with either kinetochore structure or spindle geometry. For instance, certain fission yeast mutants are believed to form high rates of merotelic attachments because of disorganized kinetochore structure (Gregan, et al. 2011, Gregan, et al. 2007, Pidoux, et al. 2000, Rumpf, et al. 2010). Similarly, a C. elegans mutant in which the kinetochore is twisted around the chromatin forms high rates of merotelic attachments (Stear and Roth 2002). Finally, in Indian muntjac cells, whose chromosomes possess kinetochores of varying sizes, the probability of merotelic attachment increases with kinetochore size (Baudoin and Cimini 2018a, Drpic, et al. 2018).

Spindle multipolarity is one spindle geometry defect that was shown to increase the rates of merotelic kinetochore attachments. This happens because within a multipolar spindle it is easier for a single kinetochore to face more than one spindle pole than it would be within a bipolar spindle (Ganem, et al. 2009, Paul, et al. 2009, Silkworth and Cimini 2012, Silkworth, et al. 2009) and indeed it was shown that increasing numbers of spindle poles correlate with increasing numbers of merotelic attachments (Paul, et al. 2009). This can explain earlier observations that lagging chromosomes are very commonly observed in multipolar anaphases (Sluder, et al. 1997) and in cells recovering from a nocodazole-induced mitotic arrest (Cimini, et al. 2001, Cimini, et al. 2003, Cimini, et al. 1999, Ladrach and LaFountain 1986). Indeed, in cells recovering from a nocodazole arrest, microtubules assemble from the two centrosomes as well as from multiple ectopic microtubule organizing centers (MTOCs) (Cimini, et al. 2003). However, these ectopic MTOCs eventually cluster with the two centrosomes to form a bipolar spindle (Cimini, et al. 2003). The transient multipolarity state is sufficient to enhance the rates of merotelic kinetochore attachment and hence the rates of anaphase lagging chromosomes (Ganem, et al. 2009, Silkworth and Cimini 2012, Silkworth, et al. 2009). Notably, this transient multipolarity followed by centrosome clustering is believed to play an important role in promoting chromosome missegregation in cancer cells with supernumerary centrosomes (Brinkley 2001, Ganem, et al. 2009, Silkworth, et al. 2009). Increased rates of merotelic attachment formation are also observed when the centrosomes/spindle poles in mitotic cells do not separate prior to nuclear envelope breakdown (Kaseda, et al. 2012, Nam and van Deursen 2014, Silkworth and Cimini 2012, Silkworth, et al. 2012). This happens because, if the spindle poles are in close proximity while kinetochore-microtubule attachments are established, the chance of a single kinetochore to bind microtubules from both spindle poles is higher than it would be if the two spindle poles were at diametrically opposed positions around the chromosomes (Silkworth and Cimini 2012, Silkworth, et al. 2012).

Because merotelic kinetochores are attached to microtubules, the SAC is silenced and therefore higher frequencies of merotelic attachments invariably translate into higher frequencies of anaphase lagging chromosomes. Nevertheless, correction mechanisms exist in both prometaphase/metaphase and anaphase to reduce the rates of chromosome missegregation from merotelic attachment. The next section will focus on such mechanisms.

Correction of merotelic kinetochore attachments throughout mitosis

Merotelic kinetochore attachments are very common in early mitosis, but their numbers decrease as mitosis progresses (Cimini, et al. 2004, Cimini, et al. 2003). The Aurora B kinase, which was already known to be essential for chromosome bi-orientation in budding yeast (Biggins, et al. 1999, Tanaka, et al. 2002), has been described as a major player in the merotelic error correction that occurs in prometaphase and metaphase (Cimini, et al. 2006) thanks to its ability to phosphorylate kinetochore substrates, specifically the Hec1 subunit of the Ndc80 complex, and cause microtubule detachment (Cheeseman, et al. 2006, Ciferri, et al. 2008, DeLuca, et al. 2006). Detachment of microtubules from the kinetochore provides the opportunity for loss of attachments from the incorrect pole and formation of new attachments to microtubules from the correct pole. Importantly, measurements of kinetochore-microtubule turnover showed that cells with high rates of anaphase lagging chromosomes have more stable kinetochore-microtubule attachments in either prometaphase, metaphase, or both (Bakhoum, et al. 2009a). The exact mechanism by which Aurora B kinase destabilizes kinetochore-microtubule attachments is still debated, with large part of the debate revolving around whether the pool of Aurora B localized at the centromere or a pool localized at the kinetochore is responsible for the phosphorylation of Hec1 required to detach microtubules [for a review on this topic see (Broad and DeLuca 2020)]. The centromere-localized kinesin-13s MCAK and Kif2b were also shown to play a key role in the correction of kinetochore-microtubule mis-attachments, as their suppression results in reduced kinetochore-microtubule turnover and increased rates of anaphase lagging chromosomes (Bakhoum, et al. 2009b, Kline-Smith, et al. 2004, Maney, et al. 1998). Interestingly, Auora B-dependent phosphorylation was also shown to modulate MCAK activity (Andrews, et al. 2004, Lan, et al. 2004, Zhang, et al. 2007). Importantly, potentiating MCAK using a small molecule was shown to reduce the rates of anaphase lagging chromosomes in cancer cells, suggesting that this missegregation errors could be a druggable target, although the cancer cells quickly adapted and reestablished their pre-treatment rates of lagging chromosomes (Orr, et al. 2016). A recent study has also suggested that an interplay between Aurora B kinase activity and mechanical tension specifically contributes to correction of merotelic kinetochore attachments as opposed to kinetochore-microtubule mis-attachments lacking tension (Chen, et al. 2021). This model is somewhat inconsistent with in vitro data showing that tension can suppress Aurora B-mediated detachment of kinetochore-bound microtubules (de Regt, et al. 2022), but this may be because the interplay between tension and Aurora B influences amphitelic and merotelic attachments differently. Indeed, the Aurora B-mediated correction of merotelic attachments may specifically depend on the deformation of merotelic kinetochores in metaphase and on the enrichment of Aurora B at those sites (Cimini, et al. 2004, Cimini, et al. 2006, Knowlton, et al. 2006, Trivedi, et al. 2019). Finally, there is evidence that, although a defective SAC may not directly cause anaphase lagging chromosomes, premature SAC silencing reduces the time window for the pre-anaphase correction of merotelic attachments, leading to increased rates of anaphase lagging chromosomes (Cimini, et al. 2003).

Merotelic kinetochore attachments do not sustain SAC signaling the way unattached kinetochores do [for a review on the SAC see (Lara-Gonzalez, et al. 2021)]. Therefore, although many merotelic attachments can be corrected via the mechanisms mentioned above, some can persist into anaphase. Early studies showed that merotelic kinetochore attachments that persist into anaphase can lead to different outcomes. In some cases, merotelically attached lagging chromosomes remain positioned at the spindle equator, whereas in other cases they are shifted toward one of the two groups of segregating chromosomes (Cimini, et al. 2004, Thompson and Compton 2011). Anaphase lagging chromosomes that remain close to the spindle equator, invariably form micronuclei (Cimini, et al. 2002), whereas those that are shifted towards one of the two spindle poles can be incorporated into the main nucleus (Thompson and Compton 2011). The differential positioning of lagging chromosomes during anaphase was shown to depend on the relative number of microtubules emanating from the kinetochore of the lagging chromosome to the two opposite poles (Cimini, et al. 2004, Thompson and Compton 2011) (Figure 2). Specifically, if the lagging chromosome is bound to microtubule bundles of similar thickness (i.e., containing similar numbers of microtubules; Figure 2A), then the chromosome will remain close to the cell equator (Cimini, et al. 2004), whereas if the two microtubule bundles are of different thickness (as measured by fluorescence intensity; Figure 2B), then the chromosome will be shifted to one side (Cimini, et al. 2004), typically sufficiently close to one of the two groups of segregating chromosomes to avoid missegregation. Initially, this behavior was attributed to the effect tension can have on kinetochore-bound microtubules, with high tension promoting polymerization (Maddox, et al. 2003). In the case of differentially sized microtubule bundles attached to the kinetochore, the smaller bundle will experience higher tension and therefore will lengthen during anaphase, resulting in a shift of the lagging chromosome away from the equator and close to the pole connected to the thicker bundle (Cimini, et al. 2004) (Figure 2B). In recent years, two groups have highlighted a contribution of Aurora B kinase, which localizes at the spindle midzone in anaphase and can directly phosphorylate substrates at the kinetochore of lagging chromosomes (Papini, et al. 2021), in minimizing the number of anaphase lagging chromosomes resulting in chromosome missegregation (Orr, et al. 2021, Sen, et al. 2021), although there is still some debate on the exact mechanism by which this happens [reviewed in (Maiato and Silva 2023)]. An “anaphase correction mechanism” was also invoked in an earlier study in mouse oocytes showing a ten-fold ratio between the number of lagging chromosomes in anaphase II and the number of chromosomes that eventually missegregated (Kouznetsova, et al. 2019). Similar observations were also reported for anaphase II in insect spermatocytes (Janicke, et al. 2007). In at least one cell line, it was shown that in cases in which a lagging chromosome is incorporated into the main nucleus, this is often the correct nucleus (Thompson and Compton 2011).

Despite all the safety mechanisms described in this section, some anaphase lagging chromosomes will remain in proximity of the cell equator through anaphase and beyond and this can have dire consequences [reviewed in (Krupina, et al. 2021)], as discussed in the next section.

Micronucleus formation and downstream effects

When an anaphase lagging chromosome persists in proximity of the spindle equator through the end of cell division, it invariably forms a micronucleus upon mitotic exit (Cimini, et al. 2002, Thompson and Compton 2011). It should be noted that anaphase lagging chromosomes are not the only source of micronuclei, which can also form from “polar chromosomes” that do not align prior to anaphase onset (Ferrandiz, et al. 2022, Gomes, et al. 2022) or fragments of DNA resulting from pre-mitotic DNA damage or chromosome bridges that break during cell division (Hennig, et al. 1988, Hoffelder, et al. 2004, Pampalona, et al. 2016, Schuler, et al. 1997, Umbreit, et al. 2020). Over the past decade, many studies have focused on the biology of micronuclei and on the fate of cells that enter mitosis with a micronucleus (Crasta, et al. 2012, Hatch, et al. 2013, He, et al. 2019, Liu, et al. 2018, Soto, et al. 2018, Vazquez-Diez, et al. 2016). Most of these studies used experimental protocols that resulted in increased rates of anaphase lagging chromosomes as a way to increase the number of analyzable micronuclei (Crasta, et al. 2012, Hatch, et al. 2013, He, et al. 2019, Liu, et al. 2018, Soto, et al. 2018). Thus, a wealth of knowledge has been accumulated on the biology, fate, and impact of lagging chromosome-derived micronuclei. An early study showed that micronuclei accumulated DNA damage due to defective transport of DNA repair enzymes and other proteins inside the micronucleus (Crasta, et al. 2012). Similar findings were also reported in mouse embryos, where micronuclei displayed nuclear envelope defects, accumulation of DNA damage, and impaired kinetochore assembly, which in turn led to persistent missegregation of the micronucleated chromosome (Vazquez-Diez, et al. 2016). These findings are consistent with earlier studies that analyzed micronuclei containing chromosome fragments instead of whole chromosomes. For instance, micronuclei emerging from chromosome bridge breakage were shown to display defective nuclear pore complex assembly, nuclear import defects, reduced transcriptional activity, and chromatin fragmentation (Hoffelder, et al. 2004). Similarly, radiation-induced micronuclei (which likely contain DNA fragments) displayed defective recruiting of DNA repair factors (Terradas, et al. 2012, Terradas, et al. 2009), likely due to nuclear envelope assembly defects arising from the inability of lagging DNA to recruit non-core nuclear envelope proteins, including nuclear pore complexes, during nuclear envelope reassembly upon mitotic exit (Liu, et al. 2018). Consequently, micronuclei fail to properly import key proteins that are necessary for the integrity of their nuclear envelope and DNA.

Micronuclei were also shown to frequently experience nuclear envelope rupture (Hatch, et al. 2013, Maciejowski and Hatch 2020). A recent study showed that micronuclei containing larger and more gene-dense chromosomes are less likely to rupture (Mammel, et al. 2022), but the chance of membrane rupture is never zero. When the micronucleus envelope ruptures, the micronuclear DNA can become exposed to cytoplasmic content, including nucleases that can digest and fragment the DNA, similarly to what was observed for persistent chromatin bridges (Maciejowski, et al. 2015). Defective DNA metabolism (e.g., defective DNA repair and replication) within micronuclei and micronucleus membrane rupture have been proposed as the underlying causes of the “chromosome pulverization” reported in micronucleated cells that reenter mitosis (Crasta, et al. 2012, Kato and Sandberg 1968). Importantly, this chromosome pulverization is believed to be a precursor of chromothripsis, a catastrophic event in which a single chromosome or chromosome region undergoes shattering and re-stitching to generate a highly rearranged genomic region and that is detected in a substantial number of tumors (Cortes-Ciriano, et al. 2020, Stephens, et al. 2011). Several studies have shown clear evidence of chromothripsis being triggered by encapsulation of chromosomes into micronuclei (Ly, et al. 2019, Ly, et al. 2017, Zhang, et al. 2015). Ruptured micronuclei were also shown to activate the cGAS-STING pathway responsible for cellular immune response to cytosolic DNA (Harding, et al. 2017, Maciejowski and Hatch 2020, Mackenzie, et al. 2017), and this was in turn shown to promote cancer progression by driving metastasis (Bakhoum, et al. 2018, Krupina, et al. 2021, Maciejowski and Hatch 2020).

Finally, even when they remain intact and transport of DNA repair factors is not impaired, micronuclei were shown to display abnormal mitotic behavior when the micronucleated cell reentered mitosis (He, et al. 2019, Soto, et al. 2018). This was due to a number of mechanisms, including defective or delayed chromosome condensation and defective recruitment of kinetochore proteins (He, et al. 2019, Soto, et al. 2018). Due to defective chromosome condensation, micronuclear DNA could also become trapped within the cleavage furrow and cause it to regress (He, et al. 2019), resulting in the formation of a binucleate, tetraploid cell. This is relevant because tetraploidy has been shown to promote tumorigenesis (Fujiwara, et al. 2005, Nguyen, et al. 2009) and whole genome doubling is a common event during tumor evolution (Carter, et al. 2012, Zack, et al. 2013).

In conclusion, micronuclei arising from anaphase lagging chromosomes represent a serious threat to genomic stability. This is true even if the lagging chromosome segregates to and forms a micronucleus in the correct daughter cell.

Concluding remarks

The study that identified merotelic kinetochore attachment as the underlying cause of anaphase lagging chromosomes represented an important juncture. The mitosis community recognized the importance of aneuploidy and chromosomal instability (CIN) and embarked on investigations aimed at determining the contribution of anaphase lagging chromosomes to CIN in cancer cells (Bakhoum, et al. 2014, Thompson and Compton 2008), examining what mechanisms may enhance the formation of merotelic attachments (Ganem, et al. 2009, Kline-Smith, et al. 2004, Silkworth, et al. 2012, Silkworth, et al. 2009) or promote their correction (Bakhoum, et al. 2009b, Cimini, et al. 2006, DeLuca, et al. 2006, Knowlton, et al. 2006), and explore the long-term consequences of lagging chromosomes (Crasta, et al. 2012, He, et al. 2019, Ly, et al. 2019, Ly, et al. 2017, Soto, et al. 2018, Vazquez-Diez, et al. 2016, Zhang, et al. 2015).

What we have learned in recent years about the fate of lagging chromosomes and the micronuclei derived from them reinforces the traditional view that these structures are good biomarkers of genotoxic damage (Fenech, et al. 2020), highlighting that this story has come full circle. We have learned a tremendous amount of detail along the way and this will serve the scientific community well as we try to fill the remaining gaps and answer new questions.

As described earlier, there is still substantial debate on the exact mechanisms that contribute to correction of kinetochore mis-attachments, including merotely, and this area of investigation will likely continue to be prolific. A specific question that remains unanswered is whether a correlation exists between chromosome identity and efficiency of mis-attachment correction. Such a bias could explain recent reports that certain human chromosomes are more prone to missegregate than others (Bochtler, et al. 2019, Klaasen, et al. 2022, Worrall, et al. 2018). It will be interesting to investigate whether this bias in chromosome missegregation depends on a bias in formation and/or correction of merotelic attachments.

It will also be important to gain a deeper understanding of how merotelic kinetochore attachment contributes to cancer. It is clear that lagging chromosomes are only one type of chromosome segregation errors found in cancer cells. It will be important to focus on examining tumor patient tissues and other experimental systems to better understand the contribution of different types of mitotic defects and their inter-connections. For instance, chromosome bridges, which are often seen in tumor cells (Bakhoum, et al. 2011, Bolhaqueiro, et al. 2019, Zaki, et al. 2014), may arise as a result of DNA damage in a micronucleus that originally formed from a lagging chromosome. This type of temporal information cannot be obtained from analyzing fixed tissue samples and new strategies may be needed to study the evolution of cell populations in order to understand these interdependencies. Such studies would untangle the many persistent questions regarding the role of aneuploidy in cancer. Indeed, although it is generally acknowledged that aneuploidy is a cancer hallmark, the exact role of aneuploidy has not been fully elucidated and how aneuploidy accumulates in cancer cells is still unclear.

Notably, the recently described link between micronuclei and activation of the cGAS-STING pathway and the evidence that activation of this pathway can promote tumorigenic phenotypes (Bakhoum, et al. 2018, Harding, et al. 2017, Krupina, et al. 2021, Maciejowski and Hatch 2020, Mackenzie, et al. 2017) opens up new areas of investigation and opportunities to exploit the phenotypic traits of CIN cancers and their immune signature for therapeutic purposes.

Finally, there have been, over the years, reports on a potential interplay between canonical tumor suppressor genes or oncogenes, including P53, P21, MYC, K-RAS, RAF/MEK/ERK, and fidelity of mitotic chromosome segregation, tolerance to aneuploidy, and CIN (Annibali, et al. 2014, Giaretti 1997, Gronroos and Lopez-Garcia 2018, Kreis, et al. 2014, Littler, et al. 2019, Perera and Venkitaraman 2016, Rizzotto, et al. 2021, Zerbib, et al. 2023). However, while there is substantial evidence that P53 protects cells from aneuploidy, tetraploidy, and CIN (Rizzotto, et al. 2021), the relation between other tumor suppressor genes or oncogenes and chromosome missegregation, ploidy alterations, and CIN remains largely unexplored. Future studies in this area promise to uncover new molecular details on the mechanisms of accurate vs. defective chromosome segregation, bring to light pathways to CIN and CIN tolerance, and possibly inform new therapeutic strategies for cancer and other diseases.

Availability of data and material

No original data or material are reported in this review article.