The dynamic nuclear envelope: resilience in health and dysfunction in disease.

The canonical appearance of the nucleus depends on constant adaptation and remodeling of the nuclear envelope in response to changing biomechanical forces and metabolic demands. Dynamic events at the nuclear envelope play a vital role in supporting key nuclear functions as well as conferring plasticity to this organelle. Moreover, imbalance of these dynamic processes is emerging as a central feature of disease etiology. This review focuses on recent advances that shed light on the myriad events at the nuclear envelope that contribute to resilience and flexibility in nuclear architecture.

The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission.

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 Calpain-7 (CAPN7) and that CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance (Wenzel et al., 2022). Here, we report biochemical and crystallographic studies showing that the tandem microtubule-interacting and trafficking (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.

Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission.

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 is used to recruit essential cofactors to sites of ESCRT activity. Here, we have comprehensively and quantitatively mapped the interactions between all known ESCRT-III tails and 19 recombinant human MIT domains. We measured 228 pairwise interactions, quantified 60 positive interactions, and discovered 18 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.

A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents.

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 (NE) proteins during initial expansion of nascent nuclei. Specifically, disrupting the function of Nup153 interferes with ongoing addition of B-type lamins, lamin B receptor, and SUN1 early in telophase, after the NE has initially enclosed chromatin. In contrast, effects on lamin A and SUN2 were minimal, pointing to differential requirements for the ongoing targeting of NE proteins. Further, distinct mistargeting 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 NE formation in mammalian cells, and 3) distinct requirements of individual NE residents for continued targeting during the expansion phase of NE reformation.

Nuclear pore complexes concentrate on Actin/LINC/Lamin nuclear lines in response to mechanical stress in a SUN1 dependent manner.

Formation of robust actomyosin stress fibers (SF) in response to cell stretch plays a key role in the transfer of information from the cytoplasm into the nucleus. Actin/LINC/Lamin (ALL) nuclear lines provide mechanical linkage between the actin cytoskeleton and the lamin nucleoskeleton across the nuclear envelope. To understand the establishment of ALL lines, we used live cell imaging of cells exposed to cyclic stretch. We discovered that nuclear pore complexes (NPCs) concentrate along ALL lines that are generated in response to uniaxial cyclic stretch. The ALL-associated NPCs display increased fluorescence intensity of nucleoporins Pom121, TPR and Nup153 relative to nucleoporins that are distal to the ALL lines. Here we test the hypothesis that a LINC complex component of ALL lines, SUN1 is involved in the integration of NPCs with ALL lines. We generated CRISPR SUN1 knockdown and knockout cell lines and show that SUN1 is essential for normal integration of NPCs to ALL lines. Loss or elimination of SUN1 significantly diminishes NPC/ALL line integration, demonstrating a key role for SUN1 in the recruitment or stabilization of NPCs to a discrete subdomain of the nuclear envelope at ALL lines. This work provides new insight into the mechanism by which cells respond to mechanical force through nuclear envelope remodeling.

Identification of abscission checkpoint bodies as structures that regulate ESCRT factors to control abscission timing.

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.

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.

Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short 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.

LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformation.

During cell division, remodelling of the nuclear envelope enables chromosome segregation by the mitotic spindle1. The reformation of sealed nuclei requires ESCRTs (endosomal sorting complexes required for transport) and LEM2, a transmembrane ESCRT adaptor2-4. Here we show how the ability of LEM2 to condense on microtubules governs the activation of ESCRTs and coordinated spindle disassembly. The LEM motif of LEM2 binds BAF, conferring on LEM2 an affinity for chromatin5,6, while an adjacent low-complexity domain (LCD) promotes LEM2 phase separation. A proline-arginine-rich sequence within the LCD binds to microtubules and targets condensation of LEM2 to spindle microtubules that traverse the nascent nuclear envelope. Furthermore, the winged-helix domain of LEM2 activates the ESCRT-II/ESCRT-III hybrid protein CHMP7 to form co-oligomeric rings. Disruption of these events in human cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to defects in nuclear integrity and DNA damage. We propose that during nuclear reassembly LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins7,8, suggest that phase separation may contribute to other critical envelope functions, including interphase repair8-13 and chromatin organization14-17.

Mechanical stress triggers nuclear remodeling and the formation of transmembrane actin nuclear lines with associated nuclear pore complexes.

Mechanical stimulation of fibroblasts induces changes in the actin cytoskeleton including stress fiber (SF) reinforcement and realignment. Here we characterize the nuclear response to mechanical stimulation (uniaxial cyclic stretch). Using fluorescence microscopy and quantitative image analysis we find that stretch-induced nuclear elongation and alignment perpendicular to the stretch vector are dependent on formin-regulated actin polymerization. The mechanosensitive transcription factors Yes-associated protein/Transcriptional coactivator with PDZ domain (YAP/TAZ) and myocardin-related transcription factor (MRTF-A, also known as MKL1 and MAL1) accumulate in the nucleus and activate their target genes in response to uniaxial cyclic stretch. We show that transmembrane actin nuclear (TAN) lines are induced by stretch stimulation and nuclear envelope (NE) proteins including nesprins, SUN2, and lamins form Linkers of the Nucleoskeleton and Cytoskeleton (LINC) complexes aligned with actin SFs. These NE structures are altered by pharmacological treatments (Cytochalasin D and Jasplakinolide) or genetic disruption (zyxin gene deletion) that alter actin, and their persistence requires maintenance of stretch stimulation. Nuclear pore complexes (NPCs) accumulate at TAN lines providing a potential mechanism for linking mechanical cues to NPC function.

A cancer-associated polymorphism in ESCRT-III disrupts the abscission checkpoint and promotes genome instability.

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the endosomal-sorting complexes required for transport (ESCRT) machinery and is tightly regulated by charged multivesicular body protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4C (rs35094336, CHMP4CT232) increases cancer susceptibility. Here, we explain the structural and functional basis for this cancer association: The CHMP4CT232 allele unwinds the C-terminal helix of CHMP4C, impairs binding to the early-acting ESCRT factor ALIX, and disrupts the abscission checkpoint. Cells expressing CHMP4CT232 exhibit increased levels of DNA damage and are sensitized to several conditions that increase chromosome missegregation, including DNA replication stress, inhibition of the mitotic checkpoint, and loss of p53. Our data demonstrate the biological importance of the abscission checkpoint and suggest that dysregulation of abscission by CHMP4CT232 may synergize with oncogene-induced mitotic stress to promote genomic instability and tumorigenesis.

Nup153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events.

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.

LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells.

Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In Schizosaccharomyces pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope.

Coordinated events of nuclear assembly.

Each time a metazoan cell undergoes open mitosis, the nucleus is dismantled in order to partition DNA content to the daughter cells. After chromosomes separate, changes at the chromatin surface usher in reestablishment of nuclear architecture. Proteins destined for the nuclear envelope are attracted to chromatin and concomitantly recruit membrane. As nuclear envelope and protein constituents spread to coat chromatin, distinct regions emerge-some rich in rapid pore formation, others occupied by microtubules that remain attached to kinetochores. Microtubule connections present physical barriers that must be remodeled in order for the nuclear envelope to seal. Regions of the nascent nuclear envelope that are initially characterized by contrasting repertoires of nuclear envelope proteins rapidly coalesce as nuclei expand and enter interphase.

Dual colorimetric and fluorogenic probes for visualizing tyrosine phosphatase activity.

Two resorufin-based substrates for protein tyrosine phosphatase (PTP) activity have been synthesized. These substrates provide sensitive fluorogenic readouts of PTP activity in vitro and in living cells at both acidic and neutral pH. In addition, the presence of the pathogenic bacteria Staphylococcus aureus was detected visually using a colorimetric readout.

Repo-Man/PP1 regulates heterochromatin formation in interphase.

Repo-Man is a protein phosphatase 1 (PP1) targeting subunit that regulates mitotic progression and chromatin remodelling. After mitosis, Repo-Man/PP1 remains associated with chromatin but its function in interphase is not known. Here we show that Repo-Man, via Nup153, is enriched on condensed chromatin at the nuclear periphery and at the edge of the nucleopore basket. Repo-Man/PP1 regulates the formation of heterochromatin, dephosphorylates H3S28 and it is necessary and sufficient for heterochromatin protein 1 binding and H3K27me3 recruitment. Using a novel proteogenomic approach, we show that Repo-Man is enriched at subtelomeric regions together with H2AZ and H3.3 and that depletion of Repo-Man alters the peripheral localization of a subset of these regions and alleviates repression of some polycomb telomeric genes. This study shows a role for a mitotic phosphatase in the regulation of the epigenetic landscape and gene expression in interphase.

ATR and a Chk1-Aurora B pathway coordinate postmitotic genome surveillance with cytokinetic abscission.

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.

shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance.

The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.

Enhanced arginine methylation of programmed cell death 4 protein during nutrient deprivation promotes tumor cell viability.

The role of programmed cell death 4 (PDCD4) in tumor biology is context-dependent. PDCD4 is described as a tumor suppressor, but its coexpression with protein arginine methyltransferase 5 (PRMT5) promotes accelerated tumor growth. Here, we report that PDCD4 is methylated during nutrient deprivation. Methylation occurs because of increased stability of PDCD4 protein as well as increased activity of PRMT5 toward PDCD4. During nutrient deprivation, levels of methylated PDCD4 promote cell viability, which is dependent on an enhanced interaction with eIF4A. Upon recovery from nutrient deprivation, levels of methylated PDCD4 are regulated by phosphorylation, which controls both the localization and stability of methylated PDCD4. This study reveals that, in response to particular environmental cues, the role of PDCD4 is up-regulated and is advantageous for cell viability. These findings suggest that the methylated form of PDCD4 promotes tumor viability during nutrient deprivation, ultimately allowing the tumor to grow more aggressively.