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The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the membrane fission step that completes cytokinetic abscission and separates dividing cells. Filaments composed of ESCRT-III subunits constrict membranes of the intercellular bridge midbody to the abscission point. These filaments also bind and recruit cofactors whose activities help execute abscission and/or delay abscission timing in response to mitotic errors via the NoCut/Abscission checkpoint. We previously showed that the ESCRT-III subunit IST1 binds the cysteine protease CAPN7 (Calpain-7) and that CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance (Wenzelet al., 2022). Here, we report biochemical and crystallographic studies showing that the tandem MIT domains of CAPN7 bind simultaneously to two distinct IST1 MIT interaction motifs. Structure-guided point mutations in either CAPN7 MIT domain disrupted IST1 binding in vitro and in cells, and depletion/rescue experiments showed that the CAPN7-IST1 interaction is required for: 1) CAPN7 recruitment to midbodies, 2) efficient abscission, and 3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also required for abscission and checkpoint maintenance. Hence, IST1 recruits CAPN7 to midbodies, where its proteolytic activity is required to regulate and complete abscission.

Assembly of the nucleus following mitosis requires rapid and coordinate recruitment of diverse constituents to the inner nuclear membrane. We have identified an unexpected role for the nucleoporin Nup153 in promoting the continued addition of a subset of nuclear envelope proteins during initial expansion of nascent nuclei. Specifically, disrupting the function of Nup153 interferes with ongoing addition of B-type lamins, lamin B receptor (LBR), and SUN1 early in telophase, after the nuclear envelope (NE) has initially enclosed chromatin. In contrast, effects on lamin A and SUN2 were minimal, pointing to differential requirements for the ongoing targeting of nuclear envelope proteins. Further, distinct mis-targeting phenotypes arose among the proteins that require Nup153 for NE targeting. Thus, disrupting the function of Nup153 in nuclear formation reveals several previously undescribed features important for establishing nuclear architecture: 1) a role for a nuclear basket constituent in ongoing recruitment of nuclear envelope components, 2) two functionally separable phases of nuclear envelope formation in mammalian cells, and 3) distinct requirements of individual nuclear envelope residents for continued targeting during the expansion phase of NE reformation.

The 12 related human ESCRT-III proteins form filaments that constrict membranes and mediate fission, including during cytokinetic abscission. The C-terminal tails of polymerized ESCRT-III subunits also bind proteins that contain Microtubule-Interacting and Trafficking (MIT) domains. MIT domains can interact with ESCRT-III tails in many different ways to create a complex binding code that recruits essential cofactors to sites of ESCRT activity. Here, we have comprehensively and quantitatively mapped the interactions between all known ESCRT-III tails and 19 pure recombinant human MIT domains. We measured 228 pairwise interactions, quantified 58 positive interactions, and discovered 16 previously unreported interactions. We also report the crystal structure of the SPASTIN MIT domain in complex with the IST1 C-terminal tail. Three MIT enzymes were studied in detail and shown to: 1) localize to cytokinetic midbody membrane bridges through interactions with their specific ESCRT-III binding partners (SPASTIN-IST1, KATNA1-CHMP3, and CAPN7-IST1), 2) function in abscission (SPASTIN, KATNA1, and CAPN7), and 3) function in the “NoCut” abscission checkpoint (SPASTIN and CAPN7). Our studies define the human MIT-ESCRT-III interactome, identify new factors and activities required for cytokinetic abscission and its regulation, and provide a platform for analyzing ESCRT-III and MIT cofactor interactions in all ESCRT-mediated processes.

The abscission checkpoint regulates the ESCRT membrane fission machinery and thereby delays cytokinetic abscission to protect genomic integrity in response to residual mitotic errors. The checkpoint is maintained by Aurora B kinase, which phosphorylates multiple targets, including CHMP4C, a regulatory ESCRT-III subunit necessary for this checkpoint. We now report the discovery that cytoplasmic abscission checkpoint bodies (ACBs) containing phospho-Aurora B and tri-phospho-CHMP4C develop during an active checkpoint. ACBs are derived from mitotic interchromatin granules, transient mitotic structures whose components are housed in splicing-related nuclear speckles during interphase. ACB formation requires CHMP4C, and the ESCRT factor ALIX also contributes. ACB formation is conserved across cell types and under multiple circumstances that activate the checkpoint. Finally, ACBs retain a population of ALIX, and their presence correlates with delayed abscission and delayed recruitment of ALIX to the midbody where it would normally promote abscission. Thus, a cytoplasmic mechanism helps regulate midbody machinery to delay abscission., eLife digest When a cell divides, it must first carefully duplicate its genetic information and package these copies into compartments housed in the two new cells. Errors in this process lead to genetic mistakes that trigger cancer or other harmful biological events. Quality control checks exist to catch errors before it is too late. This includes a final ‘abscission’ checkpoint right before the end of division, when the two new cells are still connected by a thin membrane bridge. If cells fail to pass this ‘no cut’ checkpoint, they delay severing their connection until the mistake is fixed. A group of proteins called ESCRTs is responsible for splitting the two cells apart if nothing is amiss. The abscission checkpoint blocks this process by altering certain proteins in the ESCRT complex, but exactly how this works is not yet clear. To find out more, Williams et al. imaged ESCRT factors in a new experimental system in which the abscission checkpoint is active in many cells. This showed that, in this context, certain ESCRT components were rerouted from the thread of membrane between the daughter cells to previously unknown structures, which Williams et al. named abscission checkpoint bodies. These entities also sequestered other factors that participate in the abscission checkpoint and factors that contribute to gene expression. These results are key to better understand how cells regulate their division; in particular, they provide a new framework to explore when this process goes wrong and contributes to cancer.

The abscission checkpoint regulates the ESCRT membrane fission machinery and thereby delays cytokinetic abscission to protect genomic integrity in response to residual mitotic errors. The checkpoint is maintained by Aurora B kinase, which phosphorylates multiple targets, including CHMP4C, a regulatory ESCRT-III subunit necessary for this checkpoint. We now report the discovery that cytoplasmic abscission checkpoint bodies (ACBs) containing phospho-Aurora B and tri-phospho-CHMP4C develop in telophase under an active checkpoint. ACBs are derived from Mitotic Interchromatin Granules (MIGs), transient mitotic structures whose components are housed in splicing-related nuclear speckles during interphase. ACB formation requires CHMP4C, and the ESCRT factor ALIX also contributes. ACB formation is conserved across cell types and under multiple circumstances that activate the checkpoint. Finally, ACBs retain a population of ALIX, and their presence correlates with delayed recruitment of ALIX to the midbody where it would normally promote abscission. Thus, a cytoplasmic mechanism helps regulate midbody machinery to delay abscission.

SummaryShort linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins – structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

Thousands of nuclear pore complexes stud the nuclear envelope of a proliferating mammalian cell. At these sites, ∼30 proteins, each present in multiple copies, come together to provide a selective trafficking gateway. In addition to this vital canonical role in nucleocytoplasmic trafficking, a wide variety of other important biological functions are carried out by nuclear pore proteins. In this chapter, we survey the broad range of noncanonical roles that nuclear pore proteins play beyond the nuclear pore complex: during mitosis, in the regulation of gene expression, in response to DNA damage, at primary cilia, and as a signaling or tethering platform for other cellular processes. This chapter provides a snapshot of the scientific literature that emphasizes an ever-growing appreciation of the diverse contributions that nuclear pore proteins make to cell biology. This broader context is particularly important when considering experimental and disease-related phenotypes that arise when the role of a nuclear pore protein is impaired. An appreciation of non-canonical roles also underscores the potential for regulatory cross-talk between the status of nucleocytoplasmic trafficking and other cell functions.

Aurora B is a key regulator of cytokinetic abscission, yet the full range of cellular events and signaling factors that regulate Aurora B at this stage remains to be elucidated. Here it is shown that postmitotic genome surveillance is linked to the abscission checkpoint. This connection is dependent on ATR and a central Chk1-Aurora B signaling node., Aurora B regulates cytokinesis timing and plays a central role in the abscission checkpoint. Cellular events monitored by this checkpoint are beginning to be elucidated, yet signaling pathways upstream of Aurora B in this context remain poorly understood. Here we reveal a new connection between postmitotic genome surveillance and cytokinetic abscission. Underreplicated DNA lesions are known to be transmitted through mitosis and protected in newly formed nuclei by recruitment of 53BP1 and other proteins until repair takes place. We find that this genome surveillance initiates before completion of cytokinesis. Elevating replication stress increases this postmitotic process and delays cytokinetic abscission by keeping the abscission checkpoint active. We further find that ATR activity in midbody-stage cells links postmitotic genome surveillance to abscission timing and that Chk1 integrates this and other signals upstream of Aurora B to regulate when the final step in the physical separation of daughter cells occurs.

DNA double strand breaks are typically repaired through either the high-fidelity process of homologous recombination (HR), in which BRCA1 plays a key role, or the more error-prone process of non-homologous end joining (NHEJ), which relies on 53BP1. The balance between NHEJ and HR depends, in part, on whether 53BP1 predominates in binding to damage sites, where it protects the DNA ends from resection. The nucleoporin Nup153 has been implicated in the DNA damage response, attributed to a role in promoting nuclear import of 53BP1. Here, we define a distinct requirement for Nup153 in 53BP1 intranuclear targeting to damage foci and report that Nup153 likely facilitates the role of another nucleoporin, Nup50, in 53BP1 targeting. The requirement for Nup153 and Nup50 in promoting 53BP1 recruitment to damage foci induced by either etoposide or olaparib is abrogated in cells deficient for BRCA1 or its partner BARD1, but not in cells deficient for BRCA2. Together, our results further highlight the antagonistic relationship between 53BP1 and BRCA1 and place Nup153 and Nup50 in a molecular pathway that regulates 53BP1 function by counteracting BRCA1-mediated events.

Cells divide and accurately inherit genomic and cellular content through synchronized changes in cellular organization and chromosome dynamics. Although DNA segregation, nuclear reformation, and cytokinesis/abscission temporally overlap, little is known about how these distinct events are coordinated to ensure accurate cell division. Recently, we found that disruption of postmitotic nuclear pore complex assembly, an essential aspect of the newly forming nuclear envelope, triggers an Aurora B-dependent delay in abscission. This delay is further characterized by mislocalized, aberrantly active Aurora B in the cytoplasm of midbody-stage cells. These results support a model in which an Aurora B-mediated abscission checkpoint provides surveillance of nuclear pore complex formation to ensure that elements of nuclear architecture are fully formed before daughter cells are physically separated. Here we discuss the process of nuclear pore complex assembly, describe potential mechanisms that may explain how this process could be coordinated with abscission, and postulate why such a checkpoint mechanism may exist.

Accurate inheritance of genomic content during cell division is dependent on synchronized changes in cellular organization and chromosome dynamics. Elucidating how these events are coordinated is necessary for a complete understanding of cell proliferation. Previous in vitro studies have suggested that the nuclear pore protein Nup153 is a good candidate for participating in mitotic coordination. To decipher whether this is the case in mammalian somatic cells, we reduced the levels of Nup153 in HeLa cells and monitored consequences on cell growth. Reduction of Nup153 resulted in a delay during the late stages of mitosis accompanied by an increase in unresolved midbodies. Depletion of Nup153 to an even lower threshold led to a pronounced defect early in mitosis and an accumulation of cells with multilobed nuclei. Although global nucleocytoplasmic transport was not significantly altered under these depletion conditions, the FG-rich region of Nup153 was required to rescue defects in late mitosis. Thus, this motif may play a specialized role as cells exit mitosis. Rescue of the multilobed nuclei phenotype, in contrast, was independent of the FG-domain, revealing two separable roles for Nup153 in the execution of mitosis.

Previous studies have shown that a targeted deletion of Ovol1(previously known as movo1), encoding a member of the Ovo family of zinc-finger transcription factors, leads to germ cell degeneration and defective sperm production in adult mice. To explore the cellular and molecular mechanism of Ovol1 function, we have examined the mutant testis phenotype during the first wave of spermatogenesis in juvenile mice. Consistent with the detection of Ovol1 transcripts in pachytene spermatocytes of the meiotic prophase, Ovol1-deficient germ cells were defective in progressing through the pachytene stage. The pachytene arrest was accompanied by an inefficient exit from proliferation, increased apoptosis and an abnormal nuclear localization of the G2-M cell cycle regulator cyclin B1, but was not associated with apparent chromosomal or recombination defects. Transcriptional profiling and northern blot analysis revealed reduced expression of pachytene markers in the mutant, providing molecular evidence that pachytene differentiation was defective. In addition,the expression of Id2 (inhibitor of differentiation 2), a known regulator of spermatogenesis, was upregulated in Ovol1-deficient pachytene spermatocytes and repressed by Ovol1 in reporter assays. Taken together, our studies demonstrate a role for Ovol1 in regulating pachytene progression of male germ cells, and identify Id2 as a Ovol1target.

The ovo gene family consists of evolutionarily conserved genes including those cloned from Caenorhabditis elegans, Drosophila melanogaster, mouse, and human. Here we report the isolation and characterization of mouse Ovol2 (also known as movol2 or movo2) and provide evidence supporting the existence of multiple Ovol2 transcripts. These transcripts are produced by alternative promoter usage and alternative splicing and encode long and short OVOL2 protein isoforms, whose sequences differ from those previously reported. Mouse and human OVOL2 genes are expressed in overlapping tissues including testis, where Ovol2 expression is developmentally regulated and correlates with the meiotic/post-meiotic stages of spermatogenesis. Mouse Ovol2 maps to chromosome 2 in a region containing blind-sterile (bs), a spontaneous mutation that causes spermatogenic defects and germ cell loss. No mutation has been detected in the coding region of Ovol2 from bs mice, but Ovol2 transcription was dramatically reduced in testes from these mice, suggesting that Ovol2 is expressed in male germ cells.

Drosophila ovo / svb ( dovo ) is required for epidermal cuticle/denticle differentiation and is genetically downstream of the wg signaling pathway. Similarly, a mouse homolog of dovo , movo 1, is required for the proper formation of hair, a mammalian epidermal appendage. Here, we provide biochemical evidence that movo 1 encodes a nuclear DNA binding protein (mOvo1a) that binds to DNA sequences similar to those that dOvo binds to, further supporting the notion that mOvo1a and dOvo are genetically and biochemically homologous proteins. Additionally, we show that the movo 1 promoter is activated by the lymphoid enhancer factor 1 (LEF1)/β-catenin complex, a transducer of wnt signaling. Collectively, our findings suggest that movo 1 is a developmental target of wnt signaling during hair morphogenesis in mice, and that the wg / wnt-ovo link in epidermal appendage regulatory pathways has been conserved between mice and flies.

This study focuses on an unexpected role for particular nuclear pore proteins in the antagonism between the DNA repair factors BRCA1 and 53BP1, and the impact this has on the response to poly(A) ribose polymerase (PARP) inhibition. Repair of DNA double strand breaks, a cornerstone of genomic integrity, typically follows one of two distinct pathways: the high fidelity process of homologous recombination (HR) repair, in which BRCA1 plays a key role, and the more error-prone process of non-homologous end joining (NHEJ), which relies on 53BP1. The balance between NHEJ and HR depends, in part, on whether 53BP1 predominates in binding to damage sites, where it protects the DNA ends from resection and prevents HR from occurring. Conversely, BRCA1 binding counteracts 53BP1-mediated events to promote HR repair. While mutual antagonism between BRCA1 and 53BP1 has been appreciated for some time, factors that modulate this relationship are only beginning to be elucidated. The nuclear pore component Nup153 has previously been implicated in the DNA damage response, specifically in promoting nuclear import of 53BP1. To further explore the role of Nup153 in this context, we engineered an additional strong nuclear localization sequence (NLS) on 53BP1 and found that this overrides the requirement for Nup153 in nuclear import of 53BP1. While 53BP1-NLS is recruited normally to DNA damage foci in control-treated cells, its accumulation at these sites is still severely impaired in Nup153-depleted cells, indicating that Nup153 has an additional and distinct role in 53BP1 intranuclear targeting. These observations are further underscored by the additional finding that a second nuclear pore protein, Nup50, is also required for intranuclear targeting of 53BP1, yet makes no discernable contribution to 53BP1 nuclear import. Of note, depletion of a third nucleoporin (Tpr), which is part of the same structural element of the nuclear pore, did not alter this DNA damage response pathway. To gain insight into the pathway affected by Nup153 and Nup50 and to understand how their role fits into a clinically relevant context, we probed the consequences of reducing Nup153 and Nup50 levels in cells deficient in BRCA1 activity. We found that the requirement for Nup153 and Nup50 in promoting 53BP1 recruitment to damage foci is abrogated under these circumstances. In cells where HR repair is otherwise defective, however, robust recruitment of 53BP1 to DNA damage foci requires Nup153 and Nup50. Together, our results provide novel insight into 53BP1 regulation during the DNA damage response and are consistent with a model where Nup153 and Nup50 normally promote 53BP1 targeting by counteracting BRCA1-dependent events. These findings have important implications with regard to PARP inhibition, a therapeutic strategy thought to capitalize on defective HR repair by forcing inappropriate use of NHEJ, culminating in cellular toxicity. Accordingly, we found that decreased levels of Nup153 and Nup50 do not affect the 53BP1-dependent pathway in BRCA1-deficient cells exposed to PARP inhibitor treatment, but still impair this response in cells deficient for BRCA2 or when the ATM and ATR signaling pathways are inhibited. Our results indicate that Nup153 and Nup50, as factors that modulate 53BP1 activity, belong to a novel category of biomarkers that could be screened to make better predictions of response to PARP inhibitor treatment. Citation Format: Douglas R. Mackay, Katharine S. Ullman. Nup153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A46.

Real-time imaging coupled with a permeabilized cell system presents a very versatile platform to visualize the dynamic and intricate nature of nuclear envelope breakdown, one of the major morphological changes of mitosis. Here, we describe such a strategy in which the plasma membrane of cells expressing fluorescently tagged nucleoporin POM121 and Histone H2B is permeabilized with digitonin. These cells are then incubated with mitotic Xenopus egg extract to create conditions that recapitulate the major events of mitotic nuclear remodeling seen in live-cell imaging, providing the opportunity to probe mechanisms and pathways that coordinate nuclear disassembly.

Interactions between Nup50 and soluble transport factors underlie the efficiency of certain nucleocytoplasmic transport pathways. The platform on which these interactions take place is important to building a complete understanding of nucleocytoplasmic trafficking. Nup153 is the nucleoporin that provides this scaffold for Nup50. Here, we have delineated requirements for the interaction between Nup153 and Nup50, revealing a dual interface. An interaction between Nup50 and a region in the unique N-terminal region of Nup153 is critical for the nuclear pore localization of Nup50. A second site of interaction is at the distal tail of Nup153 and is dependent on importin α. Both of these interactions involve the N-terminal domain of Nup50. The configuration of the Nup153-Nup50 partnership suggests that the Nup153 scaffold provides not just a means of pore targeting for Nup50 but also serves to provide a local environment that facilitates bringing Nup50 and importin α together, as well as other soluble factors involved in transport. Consistent with this, disruption of the Nup153-Nup50 interface decreases efficiency of nuclear import.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Recent studies in Drosophila identified pygopus, which encodes a PHD finger protein, as an additional nuclear component of the canonical Wingless(Wg)/Wnt signaling pathway. In this study, we describe the molecular cloning and expression analysis of a mouse pygopus gene, mpygo2. mpygo2 transcripts were detected in almost all adult mouse tissues examined, whereas transcripts of another mouse pygopus gene, mpygo1, were detected only in heart tissue. Abundant mpygo2 transcripts were observed during embryogenesis in multiple developmental sites. Consistent with the demonstrated role of the Wnt-beta-catenin-LEF/TCF signaling pathway in mammalian skin development, mpygo2 expression was detected in the developing epidermis and hair follicles, which suggests that mpygo2 might mediate the effect of this signaling pathway in mouse skin.