Seminars in Cell and Developmental Biology 123 (2022) 124–130 Contents lists available at ScienceDirect Seminars in Cell and Developmental Biology journal homepage: www.elsevier.com/locate/semcdb A matter of wrapper: Defects in the nuclear envelope of lagging and bridging chromatin threatens genome integrity Marina Rodriguez-Muñoz, Teresa Anglada *, Anna Genescà * Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain A R T I C L E I N F O A B S T R A C T Keywords: The nuclear envelope surrounds the eukaryotic genome and, through the nuclear pore complexes, regulates Nuclear envelope transport in and out of the nucleus. Correct nucleo-cytoplasm compartmentations are essential for nuclear DNA bridge functions such as DNA replication or repair. During metazoan mitosis, the nuclear envelope disintegrates to allow Micronucleus the segregation of the two copies of DNA between daughter cells. At the end of mitosis, it reforms on each group DNA damage Chromothripsis of chromosomes in the daughter cells. However, nuclear envelope reformation is delayed on lagging chromo­ somes and DNA bridges. Defects in the coordination between nuclear envelope reformation and chromosome segregation impair the nuclear functions. Mechanical stress to which micronuclei and DNA bridges are subjected to combined with their particular architecture and the altered nuclear functions result in DNA damage. While micronuclei and DNA bridges were considered for more than 100 years as mere indicators of chromosomal instability, rapid technological advances are helping to better understand the biological consequences of these aberrant nuclear morphologies. Recent studies provide interesting evidence that micronuclei and chromatin bridges act as a key platforms for a catastrophic mutational process observed in cancers called chromothripsis and a trigger for the innate immune response. Therefore, they could affect cellular functions by both genetic and non-genetic means. 1. Introduction dramatically in the last 10 years with recent technological advances for visualizing and recording living cells and massive DNA sequencing. Nuclear morphology is not a trivial matter. The nucleus in most These new approaches have led to relevant progress in our under­ normal (non-transformed) cells has a regular and ellipsoid shape, but it standing of the biological consequences of MN and have created wide­ is often irregular in cancer cells. Alterations of nuclear structure in spread interest in these nuclear structures. We now know that tumor cells include changes in nuclear size and shape as well as in the micronuclei lack structural integrity and they are not fully functional as organization of heterochromatin. All these morphological changes are they cannot properly perform basic nuclear functions such as DNA characteristic of certain types of tumors and stages and they have been replication, transcription or DNA damage repair. These new in­ used in the diagnosis of cancer for many years. For example, the Pap vestigations provide evidence that micronucleation can set in motion a smear developed in the early 20th century by George Papanicolau to deep genome remodelling of cells, thus taking MN to a new dimension: simultaneously detect specific nuclear and cytoplasmic features of can­ from passive indicators of chromosome instability to sources of DNA cer cells in cervical samples. Changes in the nuclear architecture reveal a damage and karyotypic diversity in cell populations. characteristic that is transversal to cancer cells: their inability to main­ Unlike micronuclei, DNA bridges between daughter cell nuclei have tain the integrity of their genome. long been recognized as a source of DNA damage and intratumoral ge­ Among abnormal nuclear morphologies, micronuclei (MN) have netic heterogeneity [14,32]. According to the Breakage-Fusion-Bridge been used for many decades as biomarkers of chromosomal damage, (BFB) model [32], DNA bridges can break and initiate a cycle capable genomic instability and exposure to mutagenic agents [11,21]. Their of self-feeding when the broken ends fuse with another broken end. The scoring was considered as the gold standard for detecting exposure to variety of genome changes caused by the presence of bridges in cells has mutagenic agents. However, our perspective on MN has changed suddenly spread to unforeseen limits. Classical studies showed that, * Corresponding authors. E-mail addresses: Marina.Rodriguez.Munoz@uab.cat (M. Rodriguez-Muñoz), Teresa.Anglada@uab.cat (T. Anglada), Anna.Genesca@uab.cat (A. Genescà). https://doi.org/10.1016/j.semcdb.2021.03.004 Received 29 December 2020; Accepted 8 March 2021 Available online 21 March 2021 1084-9521/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). M. Rodriguez-Muñoz et al. Seminars in Cell and Developmental Biology 123 (2022) 124–130 through BFB cycles, chromosomal bridges could generate mutations, 3. Defective nuclear envelope on bridging and lagging chromosome rupture, amplifications and highly rearranged chromo­ chromosomes somes. But it was not until recently that we learned that the repertoire of genomic alterations that can be attributed to DNA bridges is much The nuclear envelope (NE) consists of two lipid bilayer membranes broader, including phenomena capable of dramatically changing the supported by the nuclear lamina and perforated by the nuclear pores. On genome of cells such as duplication of the entire genome and the nuclear side, the nuclear lamina –a network of intermediate fila­ chromothripsis. ments composed of two types of lamin proteins, the B type lamin and the The evolution in our knowledge of the consequences of micronuclei A type (Lmn A/C), and integral membrane proteins– provides me­ and DNA bridges derive, to a great extent, from our understanding that chanical support to the NE and contribute to chromatin organization these structures are surrounded by a defective nuclear envelope. Defects [16]. Scattered in the envelope, nuclear pores constitute large protein­ in the wrapping of micronuclei and chromatin bridges make not only aceous channels for the selective transport of macromolecules and their nuclear functions to be altered, but also their DNA to be inappro­ diffusion of ions and metabolites between the nucleus and the cyto­ priately exposed to the cytoplasmic environment during the interphase, plasm. In eukaryotic cells, the NE has evolved to form a stable physical triggering the innate immune response. Taken together, it suggests that barrier between the nucleus and the cytoplasm, normally breaking down MN and DNA bridges could affect cellular functions by both genetic and only during mitosis. NE reassembly is facilitated by the non-genetic means. microtubule-mediated accumulation of the DNA cross-bridging bar­ rier-to-autointegration factor (BAF) at the surface of anaphase chro­ 2. Molecular origin of DNA bridges and micronuclei mosomes. Relying on its ability to bridge distant DNA sites, BAF shapes a single nucleus from a set of individualized chromosomes at the end of There are different molecular mechanisms by which DNA bridges mitosis [38]. Following an initial phase during which BAF localizes on and micronuclei can be formed, but for most of them, mitosis is a the entire surface of the compact mass of late anaphase chromatin, BAF common key process. This stage of the cell cycle is inherently stressful continues to enrich in central regions of the assembling nuclear rim close for cells. During mitosis, actin and tubulin cytoskeleton suffer dramatic to the spindle microtubules. This region of the nuclear envelope near to changes to reshape the cell. The nuclear envelope is torn apart and the the pole-to-pole axis of the mitotic spindle is named the “core” domain replicated chromosomes condense and move inside the cell; first in a [17]. congressional movement towards the equatorial cell plane and then, To form a functional interphase nucleus, segregating chromosomes towards the cell poles for segregating the two copies of the replicated have to recruit not only membranes and lamins but also nucleoporins to DNA between daughter cells. In this stage of the cell cycle, in which constitute nuclear pore complexes (NPCs). Therefore nuclear membrane changes in the cellular architecture are of such magnitude, the mal­ recruitment and NPC assembly must be coordinated to avoid the for­ function of the molecular machinery involved has serious consequences mation of a closed NE that lacks NPCs [3]. However, the presence of for the cell. lagging and bridging chromatin in dividing cells compromises the ar­ Lagging chromosomes during anaphase are prone to be subsequently chitecture and the function of the NE on missegregated chromatin. The incorporated into MN. Laggards can originate from different mecha­ NE of micronuclei typically possess a lower density of nuclear pores nisms. They frequently arise because of erroneous attachments to mi­ relative to primary nuclei and exhibit inefficient transport of nuclear crotubules, such as those of merotelic chromosomes in which single proteins [19,42,7]. Similar to MN, the NE on chromatin bridges is kinetochores attach to microtubules emanating from different poles [6]. frequently altered. Although positive for the chromatin binding BAF, it Laggards also result from unattached acentric fragments, which can be is typically depleted for many important proteins, including A/C- and derived from unrepaired DNA double-strand breaks caused by DNA B-type Lamins and NPCs [29]. Alterations in NE composition affect the replication stress or as a result of misrepair of DNA breaks [4,11]. transport of proteins that need to be shuttled through and as a conse­ Lagging chromosomes can also appear due to the untimely loss of sister quence of it, the nuclear functions become defective. For instance, the chromatid cohesion because of the defective removal of cohesins that DNA replication and DNA repair functions in micronuclei are strikingly tether sister chromatids [39] or from abnormal congress of chromo­ impaired [7,42]. Therefore, the nucleo-cytoplasmic compartmentation somes before their segregation due to malfunction of microtubule and transport defects of the NE on missegregated bridging and lagging plus-end directed kinetochore motor proteins [12]. Whatever their chromosomes are due to an abnormal assembly of NPCs and other NE origin, lagging chromosomes become MN after their nuclear envelope components. reassemble independently from the primary nucleus, being spatially What causes the NE on lagging and bridging chromosomes to be separated. defective? Why are MN and DNA bridges depleted of NPCs and other NE As opposed to laggards, bridging chromatin completely spans the components? Studies published in the last decade in the fruit fly segregating masses of chromosomes during anaphase. Bridges are most Drosophila melanogaster pointed to a biochemical model to explain a often the manifestation of dicentric chromatids in which each centro­ spatial regulation of NE postmitotic reassembly. According to these mere of a continuous DNA fibre is pulled towards opposite spindle poles. studies, NE reassembles around segregating chromosomes only once These rearranged chromosomes with two centromeres in a single DNA they have left the mitotic midzone where the concentration of active molecule are most frequently formed because of misjoined DNA breaks Aurora B enzyme is high [1,2] (Fig. 1). These observations suggested or after fusion of chromosomes with dysfunctional telomeres [13]. that a surveillance mechanism would be mediated by localized con­ Chromatin bridges are also formed during mitosis when the success of centrations of active Aurora B kinase: by keeping key substrates phos­ complete replication is compromised and it generates chromosomes phorylated, chromosome decondensation and NE reassembly on with non-replicated segments interspersed with other replicated seg­ late-segregating chromosomes that occupy the spindle midzone would ments [23]. Although to a lesser extent, DNA bridges are also caused by be delayed to facilitate their inclusion in the primary nucleus [46]. defects in chromatin condensation and sister chromatid cohesion. However, if the missegregated chromosome fails to incorporate in the Chromatin bridges can persist beyond anaphase and form stabilized primary nucleus, delayed NE reformation will finally have impact on the nucleoplasmic connections between the two daughter cells. At the end of composition and function of the NE that wraps the missegregated mitosis, the nuclear envelope must reassemble around each group of chromatin. chromosomes. However, the formation of this envelope on mis­ Other recent studies point to mechanical causes for the deficient NE segregated bridging chromatin, as well as on lagging chromosomes, is assembly on late segregating chromosomes in human cells (Fig. 1). Ac­ usually defective due to the spatial location of these structures in the cording to these studies, altered NE assembly on lagging chromosomes is dividing cell. not the consequence of a beneficial checkpoint delay, but rather a 125 M. Rodriguez-Muñoz et al. Seminars in Cell and Developmental Biology 123 (2022) 124–130 interphase. In agreement with this, Liu and colleagues [25] showed that loosening of spindle microtubule bundling by siRNA-mediated depletion of the kinesin KIF4A partially reversed the defect in NPC and other non-core NE proteins on lagging chromosomes. Whatever the causes of poor NE assembly on missegregated chro­ matin are, whether it is due to mechanical or biochemical causes, NE defects in micronuclei and DNA bridges seem to be linked to the position that the chromatin occupies within the dividing cell. Alteration of the NE composition affects not only basic biological processes, such as DNA replication or repair that requires timely transport of proteins but also affects proper nucleo-cytoplasmic compartmentalization. 4. Nuclear envelope rupture of micronuclei and chromatin bridges An intriguing phenomenon was reported in 2012 in human cancer cell lines. Using live-cell imaging, Hetzer’s laboratory characterized singular episodes wherein the primary nuclei of proliferating cells from different cancer cell lines become temporarily ruptured during inter­ phase [44]. The interphase loss of the nuclear permeability barrier allowed nucleoplasmic proteins to leak out and cytoplasmic proteins to leak in. Still images in U2OS cells revealed that nuclear ruptures initi­ Fig. 1. Two models for nuclear envelope reassembly in telophase. Upper panel: ated from localized deformations of the NE that expanded forming NE According to the chromosome separation checkpoint model, segregated chro­ herniations, which are eventually ruptured. mosome masses (light blue) recruit membranes (dark gray) and lamin filaments Analysis of NE rupture has identified two significant contributors to (red), but NE and NPCs (purple) assembly is locally delayed on chromosome NE instability: nuclear lamina organization and mechanical stress regions close to Aurora B gradient (orange). Under this model, Aurora B activity (Fig. 2). Experiments in cancer cell lines showing that lamin B1 over­ mediates a surveillance mechanism that prevents chromosome decondensation expression is sufficient to prevent NE rupture and that lamin depletion and NE reassembly until the effective separation of sister chromatids is ach­ promotes envelope rupture revealed the important role of the nuclear ieved. Localized delays in nuclear envelope formation have been proposed to lamina in NE integrity [44]. However, external physical forces can also facilitate inclusion of late-segregating chromosome fragments. Based on this induce NE rupture. Transient rupture at the leading end of the nucleus model, the reassembly of the NE is delayed on lagging chromosomes and DNA was visualized in dendritic cells, fibroblasts, and cancer cells forced to bridges. Lower panel: According to the spindle inhibition model, the non-core migrate through small pores [8,37]. Significantly, NE rupture in cells NE (including NPCs) assembly is inhibited by the mitotic spindle (green) in telophase cells. It is proposed that the chromosome regions in contact with the subjected to mechanical stress relies on the assembly of contractile actin spindle assemble the core NE (pale gray), whereas the chromosome peripheral bundles in the cytoplasm that interact with the nucleus via the linker of regions assemble the non-core NE (dark gray) including NPCs (purple). On the nucleoskeleton and cytoskeleton (LINC) complex. Experiments showing basis of this model, the NE around lagging chromosomes and DNA bridges re­ that inhibition of cytosolic myosin II activity or loss of the LINC complex sembles the core domain of the NE and fails to recruit NPCs and nuclear lamina reduced the occurrence of NE rupture sustained the notion that NE (red) despite assembling the core membrane. (For interpretation of the refer­ rupture depends on an increase in intranuclear pressure from ences to color in this figure legend, the reader is referred to the web version of actin-based nucleus confinement [20]. According to the emerging this article.) model, weak membrane areas caused by defects in lamina organization, rupture when mechanical forces on the nucleus, such as actin-based pathological outcome. NPCs are assembled in two stages, during telo­ nucleus compression and stretching, increases the internal pressure on phase concomitantly with membrane recruitment on chromatin (hereon the nuclear membrane. referred to as postmitotic assembly) and, later on, during interphase Recent studies have anticipated that nuclear lamina organization when the nuclear envelope is already sealed [45]. The mechanisms for defects and mechanical stress may converge in generating NE rupture in postmitotic and interphase NPC assembly are fundamentally different relevant pathophysiological processes such as the migration of tumor and have different kinetics, with the first one being much faster than the cells during the process of invasion. Consistent with this concept, a second one [10,35]. By using a combination of live-cell imaging and localized detachment of the nuclear membranes from the nuclear lamina high-resolution 3D electron microscopy, Otsuka and colleagues [36] in the expanding nuclear blebs of cancer cells migrating through small reported that postmitotic NPC reassembly proceeds by a radial dilation pores was reported by Denais et al. [8]. Moreover, staining of NE com­ of small membrane openings pre-existing in the endoplasmic reticulum. ponents showed that nuclear pores and lamin B1 were excluded from the In contrast to the postmitotic process, the interphase NPC assembly re­ ruptured region at the tip of the nucleus [37]. Yet another example of the lies on de novo membrane fusion as it proceeds by an inside-out evagi­ convergent contribution of mechanical stress and nuclear envelope de­ nation of the inner nuclear membrane that grows depth until it fuses fects on NE rupture is found in cells born with chromatin bridges. By with the outer nuclear membrane [35]. Similar to the assembly of other physically connecting the two nuclei, chromatin bridges exert pulling NE components, NPC formation in telophase cells is locally delayed in forces on the nucleus and induce abnormal teardrop morphologies in the so-called “core” NE domains located in the pole-to-pole axis of the both daughter nuclei. As a consequence of this, nuclear lamins are lost mitotic spindle due to dense spindle microtubules on the DNA surface around the chromatin bridges, which are then prone to rupture [29]. [5] (Fig. 1). In the subsequent interphase, the core domains of the fully Micronuclei also have a high probability of NE rupture. However, in sealed interphasic NE progressively assemble new NPCs, but this new contrast to chromatin bridges, MN are not subjected to heavy mechan­ wave of NPC assembly during interphase relies on pre-existing NPCs in ical stress. Research suggests strongly that compositional defects, adjacent regions of the nucleus. An important consequence inferred including nuclear lamina defects that manifest with the appearance of from this model is that reduced numbers of functional NPC on the en­ gaps in the lamina, are responsible for MN disruption [19,48]. As dis­ velope of abnormal nuclear structures that emerge at the end of mitosis, cussed in the previous section, the defects in the NE composition of MN such as MN and chromatin bridges, hinder NPC formation throughout are probably linked to the position that the missegregated chromatin 126 M. Rodriguez-Muñoz et al. Seminars in Cell and Developmental Biology 123 (2022) 124–130 Fig. 2. Mechanisms of nuclear envelope rupture. NE rupture occurs frequently as a result of defects in the organization of the nu­ clear lamina (red) that give rise to gaps in the lamina meshwork. NE rupture can also occur as a result of increased membrane stress associ­ ated with mechanical forces or architectural restrictions. Upper panel: Confined migration of cells leads to the formation of nuclear mem­ brane protrusions at the leading edge of the nucleus as the cell moves through space con­ strictions. Nuclear blebs form when nuclear membranes (dark gray) detach from the nuclear lamina (red), bulge into the cytoplasm and eventually rupture. DNA double-strand breaks might result from NE openings. Rupture of the primary nucleus during confined cell migration is transient as cells can restore the nuclear membrane integrity during interphase. Middle panel: Micronuclear envelope rupture requires nuclear lamina defects, but the role of me­ chanical stress is unknown. Disruption of the micronucleus results in DNA damage as a consequence of defects in nuclear functions (deficient DNA replication and DNA repair). Unlike the primary nucleus, micronuclear en­ velope rupture is irreversible. This is probably a consequence of the high curvature of the micronuclear envelope. Lower panel: nuclear lamina defects and increased mechanical stress is associated with NE rupture of DNA bridges. DNA damage can result from NE rupture of DNA bridges, although mechanical stress on the DNA bridge due to migration of the connected daughter cells also play an important role. Like micronuclei, NE rupture on bridging DNA is also irreversible. (For interpretation of the ref­ erences to color in this figure legend, the reader is referred to the web version of this article.) occupies within the dividing cell. fatal for cells, DNA DSBs might arise from it. Transient rupture of the primary nucleus envelope is a source of DNA Cells are protected against the deleterious consequences of NE damage. Intense γH2AX staining, a marker of DNA double-strand breaks rupture by a rapid reestablishment of NE integrity. The envelope of the (DSBs), was observed at chromatin protrusions of cells that were forced primary nucleus reseals rapidly during interphase assisted by compo­ to pass through small pores [8]. The confirmation that DNA damage was nents of the ESCRT III (endosomal sorting complexes required for caused by migration-induced nuclear deformation and subsequent NE transport III) membrane-remodelling machinery [8,37]. However, NE rupture was obtained by live-cell imaging on cells expressing fluo­ rupture in MN is irreparable as demonstrated with nuclear and cyto­ rescently labeled 53BP1, another marker of DNA damage [8,22]. Alto­ plasmic fluorescent reporters [19]. The irreversibility in the loss of gether, this suggested that although a transient loss of NE integrity is not compartmentalization of MN potentially arise from its extreme 127 M. Rodriguez-Muñoz et al. Seminars in Cell and Developmental Biology 123 (2022) 124–130 membrane curvature [28]. Recent evidence revealed that high nuclear DNA synthesis due to leakage of essential nuclear components [19]. curvature imposed by a physical external probe on the primary nucleus DNA damage associated with replication stress might be exacerbated by promotes NE rupture and favors sustained DNA damage [47]. Once deficient DNA damage response signalling. Consistent with this, launched, loss of NE integrity is probably also permanent in chromatin although MN present significant labeling of the DNA damage marker bridges. In this case, it is not as a result of the curvature of its NE but of γH2AX, they show poor recruitment of DNA repair factors such as 53BP1 the distance between the nuclei to which the bridge is anchored. In a [41,42]. However, the acquisition of massive DNA damage does not scenario such as that posed in MNs and DNA bridges, in which the cell is always require NE rupture. Defects in the unruptured NE cause micro­ unable to repair collapsed NE, damage to the DNA of these structures is nuclear DNA replication to be slower, and as a consequence of it exacerbated. proliferating cells enter mitosis when replication of micronuclear DNA is not yet complete. The persistence of a large number of unrepaired DNA 5. DNA damage associated with micronuclei breaks and unresolved replication intermediates could be catastrophic upon mitotic breakdown of the micronuclear envelope and condensa­ Micronuclei have been associated with DNA damage from the late of tion of chromosomes [7,25]. According to these models, the micro­ 1960s when a pioneering study using cytogenetic approaches reported nucleated chromosomes undergo an asynchronous DNA synthesis with in human cells the presence of pulverized chromosomes presumably the primary nucleus thus suffering from a disturbance in its performance derived from MN [33]. Direct evidence for the presence of massive DNA of condensation of not yet fully replicated chromosomes, leading damage in MN was obtained using γH2AX immunolabeling in irradiated eventually to a phenotype of localized shattering in mitosis. human fibroblasts. Uniform intense γH2AX labeling of MN revealed Yet a second mechanism, which is not associated with DNA repli­ massive fragmentation of the micronuclear chromatin [41]. Although cation, might potentially link micronucleation with massive DNA dam­ processes involving a selective degradation of the micronuclear DNA age. Upon disruption of the micronuclear envelope, not only leakage of could not be excluded, chromothriptic shattering of micronuclear nuclear proteins, but also an influx of cytosolic proteins into the chromatin would also lead to the reported uniform γH2AX labeling micronucleus has been observed. NE collapse leads the MN content pattern. exposed to the cytoplasmic environment as evidenced by the rapid Chromothripsis, a mutagenic phenomenon relevant in cancer accumulation of a fluorescent form of the cytosolic DNA sensor cyclic development, consists of a catastrophic shattering of one or more GMP-AMP synthase (cGAS) [31]. Although the downstream conse­ chromosome regions in a single event, followed by a seemingly random quences of cGAS pathway activation on the micronuclear chromosomes repair of the DNA fragments that lead to the generation of highly rear­ are unknown, upon rupture the micronuclear chromosomes become ranged chromosomes [40]. Considering that chromothripsis is most exposed to harmful cytoplasmic components such as endo- and exo­ frequently restricted to a single chromosome, the physical isolation of nucleases. Taken together, several non-mutually exclusive mechanisms missegregated chromosomes within micronuclei offers a mechanistic associated with defects in nuclear functions arising from defective nu­ explanation for this phenomenon. Indeed, Crasta et al. [7] found that clear envelope could act to promote massive DNA damage in acquisition of DNA damage in MN was often associated with DNA micronuclei. replication. MN undergo defective and asynchronous DNA replication — a defect that was associated with “pulverized” appearance of single 6. DNA damage associated with chromatin bridges chromosomes in micronucleated mitotic cells. By using live-cell imag­ ing, the authors tracked the fate of newly generated MN and reported Several studies have reported an association between the BFB cycles that chromosome aberrations acquired in MN could be incorporated into and chromothripsis [24,34]. While the original BFB model places the the genome as the micronuclear chromatin either reincorporated with resolution of chromatin bridges during the last stages of the cell division, the primary nucleus after anaphase or persisted as a micronucleus. more recent studies indicate that they persist through mitosis and Confirmation that genomic rearrangements specifically involved the cytokinesis to form stabilized nucleoplasmic connections between the missegregated chromosomes and occurred within one cell cycle came two daughter cells. NE rupture of chromatin bridges during interphase is from the elegant combination of live-cell imaging with single-cell DNA highly correlated with chromothripsis and it was suggested to be a sequencing. DNA sequencing of pairs of daughter cells derived from a critical initiating event for the localized shattering of DNA in these micronucleated cell progenitor identified a concentration of rearrange­ abnormal nuclear structures [29]. These authors examined the behavior ments associated with the gained haplotype in one of the daughters [48]. of dicentric chromosomes using spinning-disk confocal imaging and More recently, an experimental approach to induce the formation of MN reported that, after persisting for many hours, resolution of chromatin from a specific chromosome corroborated the association between bridges is facilitated by the cytoplasmic 3′ repair exonuclease 1 micronucleation and chromothriptic genome rearrangements. By (TREX1). It was proposed that this enzyme gains access to the bridging selectively inactivating the Y centromere, Ly and colleagues showed chromatin through NE rupture and it contributes to the resolution of the both at the cytogenetic and the DNA sequencing level that the mis­ bridge because the amount of ssDNA in the bridge — a likely key step in segregated Y chromosome exhibit 120-fold higher probability of this process — was shown to be decreased after inhibiting NE rupture. developing structural rearrangements [26,27]. According to these au­ Whole-genome sequencing of clones derived from cells born with DNA thors, the cascade of events is initiated by centromere inactivation, bridges showed rearrangements with spatial clustering of breakpoints, a followed by missegregation and fragmentation of micronucleated hallmark of chromothripsis [29,30]. However, even though NE rupture chromatin. In the following interphase, canonical non-homologous end frequently associates with DNA breaks, it is unclear whether this is the joining, but not homology-dependent repair, facilitates the religation of primary cause of damage on the bridging DNA as it is not known which chromosome fragments if the content of the micronucleus was included is the source of the nicks in the double-stranded DNA that allow the in the primary nucleus, but not if it was kept separate after the second exonuclease TREX1 to initiate resection. mitosis. In a recent study, Umbreit et al. [43] reported that deformation of While the association between MN and chromothripsis is clear, one bridging chromatin in migrating cells is sufficient to generate simple question remains to be addressed: why might chromosomes sequestered breaks and local DNA fragmentation. The authors observed that motile in MN experience massive fragmentation? Micronucleation of a chro­ cell lines broke DNA bridges during interphase, whereas less motile cell mosome can lead to DNA cleavage through different mechanisms. A first lines rarely underwent bridge breakage. They concluded that mechani­ type of mechanisms depends on the loss of nuclear functions due to NE cal forces generated by the interphase actomyosin cytoskeleton stretch defects and NE disruption during interphase. The collapse of the and break chromosome bridges, promoting local chromosome frag­ micronuclear envelope upon DNA replication initiation slows or stalls mentation. By using a combination of live-cell imaging with single-cell 128 M. Rodriguez-Muñoz et al. Seminars in Cell and Developmental Biology 123 (2022) 124–130 whole-genome sequencing, the authors observed that, in a fraction of consequences of micronuclei and DNA bridges do not limit to the cells the cells born with DNA bridges, the immediate genomic consequences wearing these abnormal structures but they expand to the whole of bridge breakage are relatively simple patterns of copy number alter­ organism. ations localized near the sites of breakage. However, highly complex rearrangements suggestive of errors in the replication of bridging DNA Funding were also observed in some cells suggesting that defective DNA repli­ cation generates additional DNA damage and chromothripsis [43]. A The Genome Integrity Group at the Universitat Autònoma de Bar­ possibility is that DNA bridge breakage involves both mechanical and celona is funded by La Marató de TV3 Foundation (201918-30), the biochemical mechanisms as access to DNA of enzymes, such as cyto­ Consejo de Seguridad Nuclear and the Generalitat de Catalunya (2017- plasmic nucleases, might be enhanced by mechanical tension through SGR-503). MRM is beneficiary of the Ph.D. program fellowships of the actomyosin-mediated disruption of the NE. Universitat Autònoma de Barcelona. 7. Non-genetic effects of the nuclear envelope rupture of Conflicts of Interest micronuclei and chromatin bridges The authors declare that they have no conflicts of interest. Two papers published in 2017 established a connection between aberrantly shaped nuclei and activation of the proinflammatory References response [18,31]. The papers described a new way of activating the innate immune system through MN surveillance by cGAS, a cytosolic [1] O. Afonso, I. Matos, A.J. Pereira, P. Aguiar, M.A. Lampson, H. Maiato, Feedback control of chromosome separation by a midzone Aurora B gradient, Science 345 DNA sensor that induces the production of cytokines through the (6194) (2014) 332–336, https://doi.org/10.1126/science.1251121. cGAS-STING pathway, thus extending the effects of nuclear atypia on [2] O. Afonso, C.M. Castellani, L.P. Cheeseman, J.G. Ferreira, B. Orr, L.T. Ferreira, J. the non-genetic field. Harding et al. [18] observed that inhibiting the J. Chambers, E. Morais-de-Sá, T.J. Maresca, H. Maiato, Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk, eLife 8 (2019) 1–31, c-NHEJ after high doses of radiation decreased the innate immune https://doi.org/10.7554/eLife.47646. response instead of activating it. Because there was a strong G2 cell cycle [3] W. Antonin, J. Ellenberg, E. Dultz, Nuclear pore complex assembly through the cell arrest when c-NHEJ was inhibited in irradiated cells, the authors spec­ cycle: regulation and membrane organization, FEBS Lett. 582 (14) (2008) 2004–2016, https://doi.org/10.1016/j.febslet.2008.02.067. ulated that the activation of the innate immune response required [4] R. a Burrell, S.E. McClelland, D. Endesfelder, P. Groth, M.C. Weller, N. Shaikh, passing through mitosis and subsequent formation of MN. The authors E. Domingo, N. Kanu, S.M. Dewhurst, E. Gronroos, S.K. Chew, A.J. Rowan, observed that cGAS is concentrated on disrupted MN and reported that A. Schenk, M. Sheffer, M. Howell, M. Kschischo, A. Behrens, T. Helleday, J. Bartek, inflammatory cytokine production was inhibited by genetic ablation of I.P. Tomlinson, C. Swanton, Replication stress links structural and numerical cancer chromosomal instability, Nature 494 (7438) (2013) 492–496, https://doi. cGAS. MacKenzie et al. [31] arrived at the same conclusion using a org/10.1038/nature11935. different experimental system. They reported an accumulation of cGAS [5] I.J. De Castro, R.S. Gil, L. Ligammari, M.L. Di Giacinto, P. Vagnarelli, CDK1 and in MN formed in mouse embryonic fibroblasts lacking RNase H2, an PLK1 coordinate the disassembly and reassembly of the nuclear envelope in vertebrate mitosis, Oncotarget 9 (8) (2018) 7763–7773. 〈www.impactjournals.co enzyme that has a role in ribonucleotide excision repair. Consistent with m/oncotarget/〉. the results reported by Harding et al. [18], they also reported a higher [6] D. Cimini, F. Degrassi, Aneuploidy: a matter of bad connections, Trends Cell Biol. degree of proinflammatory gene expression in cells with 15 (8) (2005) 442–451, https://doi.org/10.1016/j.tcb.2005.06.008. [7] K. Crasta, N.J. Ganem, R. Dagher, A.B. Lantermann, E.V. Ivanova, Y. Pan, L. Nezi, radiation-induced MN as compared with non-micronucleated cells. The A. Protopopov, D. Chowdhury, D. Pellman, DNA breaks and chromosome preceding studies support a model whereby loss of micronuclear enve­ pulverization from errors in mitosis, Nature 482 (7383) (2012) 53–58, https://doi. lope integrity can provide access of cytoplasmic factors to micronuclear org/10.1038/nature10802. [8] C.M. Denais, R.M. Gilbert, P. Isermann, A.L. McGregor, M. te Lindert, B. Weigelin, DNA and by extension, to other nuclear shape defects such as DNA P.M. Davidson, P. Friedl, K. Wolf, J. Lammerding, Nuclear envelope rupture and bridges. According to this model, NE rupture leads to cGAS accumula­ repair during cancer cell migration, Science 352 (6283) (2016) 353–358, https:// tion on the micronuclear and bridging chromatin, which activates cGAS doi.org/10.1126/science.aad7297. [9] Z. Dou, K. Ghosh, M.G. Vizioli, J. Zhu, P. Sen, K.J. Wangensteen, J. Simithy, Y. Lan, to produce cGAMP and signal to the STING adaptor protein, leading to Y. Lin, Z. Zhou, B.C. Capell, C. Xu, M. Xu, J.E. Kieckhaefer, T. Jiang, M. Shoshkes- expression of interferons and pro-inflammatory cytokines [9,15]. Carmel, K.M.A.A. Tanim, G.N. Barber, J.T. Seykora, S.E. Millar, K.H. Kaestner, B. While the cGAS-STING pathway evolved in eukaryotic cells as a A. Garcia, P.D. Adams, S.L. Berger, Cytoplasmic chromatin triggers inflammation in major mechanism for the detection of bacterial and viral infection, senescence and cancer, Nature 550 (7676) (2017) 402–406, https://doi.org/ 10.1038/nature24050. activation of cGAS by self DNA has dual opposite consequences. [10] E. Dultz, J. Ellenberg, Live imaging of single nuclear pores reveals unique assembly Whereas it represents a threat to the organism as it triggers autoim­ kinetics and mechanism in interphase, J. Cell Biol. 191 (1) (2010) 15–22, https:// munity, it may also act as an immune surveillance mechanism that de­ doi.org/10.1083/jcb.201007076. [11] M. Fenech, Cytokinesis-block micronucleus cytome assay, Nat. Protoc. 2 (5) (2007) tects and potentially suppresses a range of neoplasia-inducing processes. 1084–1104, https://doi.org/10.1038/nprot.2007.77. Importantly, these processes have a common denominator: they are [12] C.L. Fonseca, H.L.H. Malaby, L.A. Sepaniac, W. Martin, C. Byers, A. Czechanski, based on the formation of aberrant nuclear structures that suffer D. Messinger, M. Tang, R. Ohi, L.G. Reinholdt, J. Stumpff, Mitotic chromosome alignment ensures mitotic fidelity by promoting interchromosomal compaction frequent and irreversible rupture of their wrapping. during anaphase, J. Cell Biol. 218 (4) (2019) 1148–1163, https://doi.org/ 10.1083/jcb.201807228. 8. Conclusions [13] A. Genescà, J. Pampalona, C. Frías, D. Domínguez, L. Tusell, Role of telomere dysfunction in genetic intratumor diversity, Adv. Cancer Res. (2011) 11–41, https://doi.org/10.1016/B978-0-12-387688-1.00002-8. Nuclear architecture defects are now appreciated as sources of phe­ [14] D. Gisselsson, L. Pettersson, M. Hoglund, M. Heidenblad, L. Gorunova, J. Wiegant, nomena such as chromothripsis, capable of radically changing the F. Mertens, P. Dal Cin, F. Mitelman, N. Mandahl, Chromosomal breakage-fusion- bridge events cause genetic intratumor heterogeneity, Proc. Natl. Acad. Sci. USA genome of cells. Massive DNA damage has been associated with defects 97 (10) (2000) 5357–5362, https://doi.org/10.1073/pnas.090013497. in nuclear functions arising from defective nuclear envelope and ectopic [15] S. Glück, B. Guey, M.F. Gulen, K. Wolter, T.W. Kang, N.A. Schmacke, access of cytoplasmic nucleases to missegregated chromatin. Also, me­ A. Bridgeman, J. Rehwinkel, L. Zender, A. Ablasser, Designed experiments and analysed the data, Nat. Cell Biol. 19 (9) (2017) 1061–1070, https://doi.org/ chanical stress has been identified as an initiating event for chromo­ 10.1038/ncb3586. thripsis. However, not only the repertoire of genetic consequences has [16] Y. Gruenbaum, R. Foisner, Lamins: nuclear intermediate filament proteins with expanded but also the type of consequences. We now understand that fundamental functions in nuclear mechanics and genome regulation, Annu. Rev. micronuclei and DNA bridges can also lead to important non-genetic Biochem. 84 (2015) 131–164, https://doi.org/10.1146/annurev-biochem-060614- 034115. effects through the cGAS-STING pathway, which activates the innate [17] T. Haraguchi, T. Kojidani, T. Koujin, T. Shimi, H. Osakada, C. Mori, A. Yamamoto, immune system through secretion of cytokines. Therefore, the direct Y. Hiraoka, Live cell imaging and electron microscopy reveal dynamic processes of 129 M. Rodriguez-Muñoz et al. Seminars in Cell and Developmental Biology 123 (2022) 124–130 BAF-directed nuclear envelope assembly, J. Cell Sci. 121 (15) (2008) 2540–2554, [34] K. Nones, N. Waddell, N. Wayte, A.M. Patch, P. Bailey, F. Newell, O. Holmes, J. https://doi.org/10.1242/jcs.033597. L. Fink, M.C.J. Quinn, Y.H. Tang, G. Lampe, K. Quek, K.A. Loffler, S. Manning, [18] S.M. Harding, J.L. Benci, J. Irianto, D.E. Discher, A.J. Minn, R.A. Greenberg, S. Idrisoglu, D. Miller, Q. Xu, N. Waddell, P.J. Wilson, T.J.C. Bruxner, A.N. Christ, Mitotic progression following DNA damage enables pattern recognition within I. Harliwong, C. Nourse, E. Nourbakhsh, M. Anderson, S. Kazakoff, C. Leonard, micronuclei, Nature 548 (7668) (2017) 466–470, https://doi.org/10.1038/ S. Wood, P.T. Simpson, L.E. Reid, L. Krause, D.J. Hussey, D.I. Watson, R.V. Lord, nature23470. D. Nancarrow, W.A. Phillips, D. Gotley, B.M. Smithers, D.C. Whiteman, N. [19] E.M. Hatch, A.H. Fischer, T.J. Deerinck, M.W. Hetzer, Catastrophic nuclear K. Hayward, P.J. Campbell, J.V. Pearson, S.M. Grimmond, A.P. Barbour, Genomic envelope collapse in cancer cell micronuclei, Cell 154 (1) (2013) 47–60, https:// catastrophes frequently arise in esophageal adenocarcinoma and drive doi.org/10.1016/j.cell.2013.06.007. tumorigenesis, Nat. Commun. 5 (2014) 5224, https://doi.org/10.1038/ [20] E.M. Hatch, M.W. Hetzer, Nuclear envelope rupture is induced by actin-based ncomms6224. nucleus confinement, J. Cell Biol. 215 (1) (2016) 27–36, https://doi.org/10.1083/ [35] S. Otsuka, K.H. Bui, M. Schorb, M.J. Hossain, A.Z. Politi, B. Koch, M. Eltsov, jcb.201603053. M. Beck, J. Ellenberg, Nuclear pore assembly proceeds by an inside-out extrusion [21] N. Holland, C. Bolognesi, M. Kirschvolders, S. Bonassi, E. Zeiger, S. Knasmueller, of the nuclear envelope, eLife 5 (2016) 1–23, https://doi.org/10.7554/ M. Fenech, The micronucleus assay in human buccal cells as a tool for eLife.19071. biomonitoring DNA damage: the HUMN project perspective on current status and [36] S. Otsuka, A.M. Steyer, M. Schorb, J.K. Hériché, M.J. Hossain, S. Sethi, knowledge gaps, Mutat. Res. Rev. Mutat. Res. 659 (1–2) (2008) 93–108, https:// M. Kueblbeck, Y. Schwab, M. Beck, J. Ellenberg, Postmitotic nuclear pore assembly doi.org/10.1016/j.mrrev.2008.03.007. proceeds by radial dilation of small membrane openings, Nat. Struct. Mol. Biol. 25 [22] J. Irianto, Y. Xia, C.R. Pfeifer, A. Athirasala, J. Ji, C. Alvey, M. Tewari, R. (1) (2018) 21–28, https://doi.org/10.1038/s41594-017-0001-9. R. Bennett, S.M. Harding, A.J. Liu, R.A. Greenberg, D.E. Discher, DNA damage [37] M. Raab, M. Gentili, H. de Belly, H.R. Thiam, P. Vargas, A.J. Jimenez, follows repair factor depletion and portends genome variation in cancer cells after F. Lautenschlaeger, R. Voituriez, A.M. Lennon-Duménil, N. Manel, M. Piel, ESCRT pore migration, Curr. Biol. 27 (2) (2017) 210–223, https://doi.org/10.1016/j. III repairs nuclear envelope ruptures during cell migration to limit DNA damage cub.2016.11.049. and cell death, Science 352 (6283) (2016) 359–362, https://doi.org/10.1126/ [23] E. Lecona, O. Fernández-Capetillo, Replication stress and cancer: it takes two to science.aad7611. tango, Exp. Cell Res. 329 (1) (2014) 26–34, https://doi.org/10.1016/j. [38] M. Samwer, M.W.G. Schneider, R. Hoefler, P.S. Schmalhorst, J.G. Jude, J. Zuber, D. yexcr.2014.09.019. W. Gerlich, DNA cross-bridging shapes a single nucleus from a set of mitotic [24] Y. Li, C. Schwab, S.L. Ryan, E. Papaemmanuil, H.M. Robinson, P. Jacobs, A. chromosomes, Cell 170 (5) (2017) 956–972.e23, https://doi.org/10.1016/j. V. Moorman, S. Dyer, J. Borrow, M. Griffiths, N.A. Heerema, A.J. Carroll, P. Talley, cell.2017.07.038. N. Bown, N. Telford, F.M. Ross, L. Gaunt, R.J.Q. McNally, B.D. Young, P. Sinclair, [39] H. Sapkota, E. Wasiak, J.R. Daum, G.J. Gorbsky, Multiple determinants and V. Rand, M.R. Teixeira, O. Joseph, B. Robinson, M. Maddison, N. Dastugue, consequences of cohesion fatigue in mammalian cells, Mol. Biol. Cell 29 (15) P. Vandenberghe, C. Haferlach, P.J. Stephens, J. Cheng, P. Van Loo, M.R. Stratton, (2018) 1811–1824, https://doi.org/10.1091/mbc.E18-05-0315. P.J. Campbell, C.J. Harrison, Constitutional and somatic rearrangement of [40] P.J. Stephens, C.D. Greenman, B. Fu, F. Yang, G.R. Bignell, L.J. Mudie, E. chromosome 21 in acute lymphoblastic leukaemia, Nature 508 (7494) (2014) D. Pleasance, K.W. Lau, D. Beare, L.A. Stebbings, S. McLaren, M.L. Lin, D. 98–102, https://doi.org/10.1038/nature13115. J. McBride, I. Varela, S. Nik-Zainal, C. Leroy, M. Jia, A. Menzies, A.P. Butler, J. [25] S. Liu, M. Kwon, M. Mannino, N. Yang, F. Renda, A. Khodjakov, D. Pellman, W. Teague, M.A. Quail, J. Burton, H. Swerdlow, N.P. Carter, L.A. Morsberger, Nuclear envelope assembly defects link mitotic errors to chromothripsis, Nature C. Iacobuzio-Donahue, G.A. Follows, A.R. Green, A.M. Flanagan, M.R. Stratton, P. 561 (7724) (2018) 551–555, https://doi.org/10.1038/s41586-018-0534-z. A. Futreal, P.J. Campbell, Massive genomic rearrangement acquired in a single [26] P. Ly, L.S. Teitz, D.H. Kim, O. Shoshani, H. Skaletsky, D. Fachinetti, D.C. Page, D. catastrophic event during cancer development, Cell 144 (1) (2011) 27–40, https:// W. Cleveland, Selective Y centromere inactivation triggers chromosome shattering doi.org/10.1016/j.cell.2010.11.055. in micronuclei and repair by non-homologous end joining, Nat. Cell Biol. 19 (1) [41] M. Terradas, M. Martín, L. Tusell, A. Genescà, DNA lesions sequestered in (2017) 68–75, https://doi.org/10.1038/ncb3450. micronuclei induce a local defective-damage response, DNA Repair 8 (10) (2009) [27] P. Ly, S.F. Brunner, O. Shoshani, D.H. Kim, W. Lan, T. Pyntikova, A.M. Flanagan, 1225–1234, https://doi.org/10.1016/j.dnarep.2009.07.004. S. Behjati, D.C. Page, P.J. Campbell, D.W. Cleveland, Chromosome segregation [42] M. Terradas, M. Martín, L. Hernández, L. Tusell, A. Genescà, Nuclear envelope errors generate a diverse spectrum of simple and complex genomic defects impede a proper response to micronuclear DNA lesions, Mutat. Res. 729 rearrangements, Nat. Genet. 51 (4) (2019) 705–715, https://doi.org/10.1038/ (1–2) (2012) 35–40, https://doi.org/10.1016/j.mrfmmm.2011.09.003. s41588-019-0360-8. [43] N.T. Umbreit, C.Z. Zhang, L.D. Lynch, L.J. Blaine, A.M. Cheng, R. Tourdot, L. Sun, [28] P. Ly, D.W. Cleveland, Rebuilding chromosomes after catastrophe: emerging H.F. Almubarak, K. Judge, T.J. Mitchell, A. Spektor, D. Pellman, Mechanisms mechanisms of chromothripsis, Trends Cell Biol. 27 (12) (2017) 917–930, https:// generating cancer genome complexity from a single cell division error, Science 368 doi.org/10.1016/j.tcb.2017.08.005. (6488) (2020) eaba0712, https://doi.org/10.1126/science.aba0712. [29] J. Maciejowski, Y. Li, N. Bosco, P.J. Campbell, T. de Lange, Chromothripsis and [44] J.D. Vargas, E.M. Hatch, D.J. Anderson, M.W. Hetzer, Transient nuclear envelope kataegis induced by telomere crisis, Cell 163 (7) (2015) 1641–1654, https://doi. rupturing during interphase in human cancer cells, Nucleus 3 (1) (2012) 88–100, org/10.1016/j.cell.2015.11.054. https://doi.org/10.4161/nucl.18954. [30] J. Maciejowski, A. Chatzipli, A. Dananberg, K. Chu, E. Toufektchan, L.J. Klimczak, [45] C. Wandke, U. Kutay, Enclosing chromatin: reassembly of the nucleus after open D.A. Gordenin, P.J. Campbell, T. de Lange, APOBEC3-dependent kataegis and mitosis, Cell 152 (6) (2013) 1222–1225, https://doi.org/10.1016/j. TREX1-driven chromothripsis during telomere crisis, Nat. Genet. 52 (9) (2020) cell.2013.02.046. 884–890, https://doi.org/10.1038/s41588-020-0667-5. [46] B. Warecki, W. Sullivan, Micronuclei Formation Is Prevented by Aurora, 210 [31] K.J. MacKenzie, P. Carroll, C.A. Martin, O. Murina, A. Fluteau, D.J. Simpson, (September) (2018), 171–187. doi: 10.1534/genetics.118.301031. N. Olova, H. Sutcliffe, J.K. Rainger, A. Leitch, R.T. Osborn, A.P. Wheeler, [47] Y. Xia, I.L. Ivanovska, K. Zhu, L. Smith, J. Irianto, C.R. Pfeifer, C.M. Alvey, J. Ji, M. Nowotny, N. Gilbert, T. Chandra, M.A.M. Reijns, A.P. Jackson, cGAS D. Liu, S. Cho, R.R. Bennett, A.J. Liu, R.A. Greenberg, D.E. Discher, Nuclear rupture surveillance of micronuclei links genome instability to innate immunity, Nature at sites of high curvature compromises retention of DNA repair factors, J. Cell Biol. 548 (7668) (2017) 461–465, https://doi.org/10.1038/nature23449. 217 (11) (2018) 3796–3808, https://doi.org/10.1083/jcb.201711161. [32] B. McClintock, The stability of broken ends of chromosomes in Zea mays, Genetics [48] C.-Z. Zhang, A. Spektor, H. Cornils, J.M. Francis, E.K. Jackson, S. Liu, M. Meyerson, 26 (2) (1941) 234–282. D. Pellman, Chromothripsis from DNA damage in micronuclei, Nature 522 (7555) [33] H. Kato, A.A. Sandberg, Chromosome pulverization in human binucleate cells (2015) 179–184, https://doi.org/10.1038/nature14493. following colcemid treatment, J Cell Biol. 34 (1) (1967) 35–45, https://doi.org/ 10.1083/jcb.34.1.35. 130