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Background: Triple-negative breast cancer (TNBC) is a sub-classification of breast carcinomas, which leads to poor survival outcomes for patients. TNBCs do not possess the hormone receptors that are frequently targeted as a therapeutic in other cancer subtypes and, therefore, chemotherapy remains the standard treatment for TNBC. Nuclear envelope proteins are frequently dysregulated in cancer cells, supporting their potential as novel cancer therapy targets. The Lem-domain (Lem-D) (LAP2, Emerin, MAN1 domain, and Lem-D) proteins are a family of inner nuclear membrane proteins, which share a ~45-residue Lem-D. The Lem-D proteins, including Ankle2, Lemd2, TMPO, and Emerin, have been shown to be associated with many of the hallmarks of cancer. This study aimed to define the association between the Lem-D proteins and TNBC and determine whether these proteins could be promising therapeutic targets. Methods: GENT2, TCGA, and KM plotter were utilized to investigate the expression and prognostic implications of several Lem-D proteins: Ankle2, TMPO, Emerin, and Lemd2 in publicly available breast cancer patient data. Immunoblotting and immunofluorescent analysis of immortalized non-cancerous breast cells and a panel of TNBC cells were utilized to establish whether protein expression of the Lem-D proteins was significantly altered in TNBC. SiRNA was used to decrease individual Lem-D protein expression, and functional assays, including proliferation assays and apoptosis assays, were conducted. Results: The Lem-D proteins were generally overexpressed in TNBC patient samples at the mRNA level and showed variable expression at the protein level in TNBC cell lysates. Similarly, protein levels were generally negatively correlated with patient survival outcomes. siRNA-mediated depletion of the individual Lem-D proteins in TNBC cells induced aberrant nuclear morphology, decreased proliferation, and induced cell death. However, minimal effects on nuclear morphology or cell viability were observed following Lem-D depletion in non-cancerous MCF10A cells. Conclusion: There is evidence to suggest that Ankle2, TMPO, Emerin, and Lemd2 expressions are correlated with breast cancer patient outcomes, but larger patient sample numbers are required to confirm this. siRNA-mediated depletion of these proteins was shown to specifically impair TNBC cell growth, suggesting that the Lem-D proteins may be a specific anti-cancer target.

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The nuclear lamina (NL) changes composition for regulation of nuclear events. We investigated changes that occur in Drosophila oogenesis, revealing switches in NL composition during germ cell differentiation. Germline stem cells (GSCs) express only LamB and predominantly emerin, whereas differentiating nurse cells predominantly express LamC and emerin2. A change in LamC-specific localization also occurs, wherein phosphorylated LamC redistributes to the nuclear interior only in the oocyte, prior to transcriptional reactivation of the meiotic genome. These changes support existing concepts that LamC promotes differentiation, a premise that was tested. Remarkably ectopic LamC production in GSCs did not promote premature differentiation. Increased LamC levels in differentiating germ cells altered internal nuclear structure, increased RNA production, and reduced female fertility due to defects in eggshell formation. These studies suggest differences between Drosophila lamins are regulatory, not functional, and reveal an unexpected robustness to level changes of a major scaffolding component of the NL.

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Barrier-to-autointegration factor (BAF) protein is a DNA-binding protein that crosslinks chromatin to allow mitotic nuclear envelope (NE) assembly. The LAP2-emerin-MAN1 (LEM)-domain protein LEMD2 and ESCRT-II/III hybrid protein CHMP7 close NE holes surrounding spindle microtubules (MTs). BAF binds LEM-domain family proteins to repair NE ruptures in interphase, but whether BAF-LEM binding participates in NE hole closure around spindle MTs is not known. Here, we took advantage of the stereotypical event of NE formation in fertilized Caenorhabditis elegans oocytes to show that BAF-LEM binding and LEM-2-CHMP-7 have distinct roles in NE closure around spindle MTs. LEM-2 and EMR-1 (homologs of LEMD2 and emerin) function redundantly with BAF-1 (the C. elegans BAF protein) in NE closure. Compromising BAF-LEM binding revealed an additional role for EMR-1 in the maintenance of the NE permeability barrier. In the absence of BAF-LEM binding, LEM-2-CHMP-7 was required for NE assembly and embryo survival. The winged helix domain of LEM-2 recruits CHMP-7 to the NE in C. elegans and a LEM-2-independent nucleoplasmic pool of CHMP-7 also contributes to NE stability. Thus, NE hole closure surrounding spindle MTs requires redundant mechanisms that safeguard against failure in NE assembly to support embryogenesis.

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The Drosophila ovary represents an outstanding model for investigating tissue homeostasis. Females continuously produce oocytes throughout their lifetime. However, as females age, fecundity declines, in part, due to changes in ovarian niche function and germline stem cell (GSC) homeostasis. Understanding the dynamics of GSC maintenance will provide needed insights into how coordinated tissue homeostasis is lost during aging. Critical regulators of GSC maintenance are proteins that reside in the nuclear lamina (NL), including the NL proteins emerin and Barrier-to-Autointegration Factor (BAF). Continued investigation of how emerin, BAF, and other NL proteins contribute to GSC function depends upon the availability of antibodies for NL proteins, a limiting resource. In this chapter, we discuss strategies for using clustered regularly interspaced short palindromic repeats (CRISPR) genomic editing to produce endogenously tagged NL genes to circumvent this obstacle, using the generation of the gfp-baf allele as an example. We describe strategies for validation of tagged alleles. Finally, we outline methods for immunohistochemical analysis of resulting tagged-NL proteins.

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The nuclear envelope (NE) creates a barrier between the cytosol and nucleus during interphase that is key for cellular compartmentalization and protecting genomic DNA. NE rupture can expose genomic DNA to the cytosol and allow admixture of the nuclear and cytosolic constituents, a proposed mechanism of cancer and NE-associated diseases. Barrier-to-autointegration factor (BAF) is a DNA-binding protein that localizes to NE ruptures where it recruits LEM-domain proteins, A-type lamins, and participates in rupture repair. To further reveal the mechanisms by which BAF responds to and aids in repairing NE ruptures, we investigated known properties of BAF including LEM domain binding, lamin binding, compartmentalization, phosphoregulation of DNA binding, and BAF dimerization. We demonstrate that it is the cytosolic population of BAF that functionally repairs NE ruptures, and phosphoregulation of BAF's DNA-binding that enables its ability to facilitate that repair. Interestingly, BAF's LEM or lamin binding activity appears dispensable for its role in functional repair. Furthermore, we demonstrate that BAF functions to reduce the extent of leakage though NE ruptures, suggesting that BAF effectively forms a diffusion barrier prior to NE repair. Collectively, these results enhances our knowledge of the mechanisms by which BAF responds to NE ruptures and facilitates their repair.

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Despite significant advances in our understanding of tumourigenesis and cancer therapeutics, cancer continues to account for 30% of worldwide deaths. Therefore, there remains an unmet need for the development of cancer therapies to improve patient quality of life and survival outcomes. The inner nuclear membrane has an essential role in cell division, cell signalling, transcription, cell cycle progression, chromosome tethering, cell migration and mitosis. Furthermore, expression of several inner nuclear membrane proteins has been shown to be frequently altered in tumour cells, resulting in the dysregulation of cellular pathways to promote tumourigenesis. However, to date, minimal research has been conducted to investigate how targeting these dysregulated and variably expressed proteins may provide a novel avenue for cancer therapies. In this review, we present an overview of the involvement of the inner nuclear membrane proteins within the hallmarks of cancer and how they may be exploited as potent anti-cancer therapeutics.

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The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.

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BACKGROUND: Mammalian LEM-domain proteins (LEMs) are encoded by seven genes, including LAP2, EMD, LEMD1, LEMD2, LEMD3, ANKLE1, and ANKLE2. Though some LEMs were involved in various tumor progression, the expression and prognostic values of LEMs in prostate adenocarcinoma (PRAD) have yet to be analyzed. METHODS: Herein, we investigated the expression, survival data, and immune infiltration levels of LEMs in PRAD patients from ATCG, TIMER, LinkedOmics, and TISIDB databases. We also further validated the mRNA and protein expression levels of ANKLE1, EMD, and LEMD2 in human prostate tumor specimens by qPCR, WB, and IHC. RESULTS: We found that all LEM expressions, except for that of LAP2, were markedly altered in PRAD compared to the normal samples. Among all LEMs, only the expressions of ANKLE1, EMD, and LEMD2 were correlated with advanced tumor stage and survival prognosis in PRAD. Consistent with the predicted computational results, the mRNA and protein expression levels of these genes were markedly increased in the PRAD group. We then found that ANKLE1, EMD, and LEMD2 expressions were markedly correlated with immune cell infiltration levels. High ANKLE1, EMD, and LEMD2 expressions predicted a worse prognosis in PRAD based on immune cells. DNA methylation or/and copy number variations may contribute to the abnormal upregulation of ANKLE1, EMD, and LEMD2 in PRAD. CONCLUSIONS: Taken together, this study implied that ANKLE1, EMD, and LEMD2 were promising prognosis predictors and potential immunotherapy targets for PRAD patients.

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Dictyostelium amoebae perform a semi-closed mitosis, in which the nuclear envelope is fenestrated at the insertion sites of the mitotic centrosomes and around the central spindle during karyokinesis. During late telophase the centrosome relocates to the cytoplasmic side of the nucleus, the central spindle disassembles and the nuclear fenestrae become closed. Our data indicate that Dictyostelium spastin (DdSpastin) is a microtubule-binding and severing type I membrane protein that plays a role in this process. Its mitotic localization is in agreement with a requirement for the removal of microtubules that would hinder closure of the fenestrae. Furthermore, DdSpastin interacts with the HeH/ LEM-family protein Src1 in BioID analyses as well as the inner nuclear membrane protein Sun1, and shows subcellular co-localizations with Src1, Sun1, the ESCRT component CHMP7 and the IST1-like protein filactin, suggesting that the principal pathway of mitotic nuclear envelope remodeling is conserved between animals and Dictyostelium amoebae.

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The nuclear envelope (NE) separates genomic DNA from the cytoplasm in eukaryotes. The structure of the NE is dynamically altered not only in mitotic disassembly and reassembly but also during interphase. Recent studies have shown that the NE is frequently damaged by various cellular stresses that degenerate NE components and/or disrupt their functional interactions. These stresses are referred to as 'NE stress'. Accumulating evidence has demonstrated that NE stress potentially causes severe cellular dysfunctions, such as cell death and genome instability. In this review, the concept of NE stress, the processes repairing damage of the NE caused by NE stress, and the molecular mechanisms by which NE stress contributes to disease pathogenesis are introduced.

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The late-acting endosomal sorting complex required for transport (ESCRT) machinery has been implicated in facilitating the resealing of the nuclear envelope (NE) after mitosis, enabling compartmentalization of the genome away from the cytoplasm. Here, we leverage the stereotypic first division of the C. elegans embryo to identify additional functions of the ESCRT machinery in maintaining the structure of the inner nuclear membrane. Specifically, impaired ESCRT function results in a defect in the pruning of inner nuclear membrane invaginations, which arise normally during NE reformation and expansion. Additionally, in combination with a hypomorphic mutation that interferes with assembly of the underlying nuclear lamina, inhibition of ESCRT function significantly perturbs NE architecture and increases chromosome segregation defects, resulting in penetrant embryonic lethality. Our findings highlight links between ESCRT-mediated inner nuclear membrane remodeling, maintenance of nuclear envelope morphology, and the preservation of the genome during early development.

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The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication, and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin-binding protein Barrier-to-Autointegration Factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to the deformation of NL structure, and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate the contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf misregulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored the expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained misexpressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in the repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global misexpression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found upregulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.

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The eukaryotic genome inside the nucleus is enveloped by two membranes, the Outer Nuclear Membrane (ONM) and the Inner Nuclear Membrane (INM). Tethered to the INM is the nuclear lamina, a fibrillar network composed of lamins-the nuclear intermediate filaments, and membrane associated proteins. The nuclear lamina interacts with several nuclear structures, including chromatin. As most nuclear functions, including regulation of gene expression, chromosome segregation and duplication as well as nuclear structure, are highly conserved in metazoans, the Caenorhabditis elegans nematode serves as a powerful model organism to study nuclear processes and architecture. This translucent organism can easily be observed under a microscope as a live embryo, larvae and even adult. Here we will review the data on nuclear lamina composition and functions gathered from studies using C. elegans model organisms: We will discuss genome spatial organization and its contribution to gene expression. We will review both the interaction between the cytoplasm and the nucleus and mechanotransduction mechanism. Finally, we will discuss disease causing mutation in nuclear lamins, including the use of this animal model in diseases research.

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Thyroid cancer (TC) is the most common type of endocrine malignancy in humans, and its relative incidence has increased continuously in recent years. However, the primary molecular mechanisms of thyroid tumorigenesis and progression remain unclear. Papillary TC (PTC) is the most common subtype of TC. Recent studies have reported that one of the tumorigenesis and progression mechanisms is driven by genetic alterations that regulate the TC cell signaling pathway. In the present study, RNA sequencing (RNA-seq) was performed on 79 paired PTC and adjacent normal thyroid tissues to further study the molecular mechanisms of TC. Reverse transcription-quantitative PCR was used to detect the expression levels of LEM domain containing 1 (LEMD1) in 47 paired PTC and adjacent normal thyroid tissue samples. Initial analysis revealed that LEMD1 expression was significantly upregulated in TC tissues compared with that in normal tissues. The results of the thyroid RNA-seq datasets from The Cancer Genome Atlas were consistent with the RNA-seq analysis results of the present study. High LEMD1 expression increased the risk of lymph node metastasis in patients with TC. The biological function of LEMD1 on cell proliferation, migration, invasion and apoptosis was investigated in vitro via small interfering RNA and overexpression vector. Gene set enrichment analysis indicated that high LEMD1 expression was associated with epithelial-mesenchymal transition (EMT) and the Wnt/ß-catenin signaling pathway. Western blotting revealed that LEMD1 modulated the protein expression levels of E-cadherin, N-cadherin, vimentin, ß-catenin and cleaved-caspase 3. In conclusion, the present results indicated that LEMD1 may drive TC cell tumorigenesis and progression by activating the Wnt/ß-catenin signaling pathway and EMT.

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The nuclear envelope (NE) is a critical barrier between the cytosol and nucleus that is key for compartmentalization within the cell and serves an essential role in organizing and protecting genomic DNA. Rupturing of the NE through loss of constitutive NE proteins and/or mechanical force applied to the nucleus results in the unregulated mixing of cytosolic and nuclear compartments, leading to DNA damage and genomic instability. Nuclear rupture has recently gained interest as a mechanism that may participate in various NE-associated diseases as well as cancer. Remarkably, these rupturing events are often transient, with cells being capable of rapidly repairing nuclear ruptures. Recently, we identified Barrier-to-Autointegration Factor (BAF), a DNA-binding protein involved in post-mitotic NE reformation and cytosolic viral regulation, as an essential protein for nuclear rupture repair. During interphase, the highly mobile cytosolic BAF is primed to monitor for a compromised NE by rapidly binding to newly exposed nuclear DNA and subsequently recruiting the factors necessary for NE repair. This review highlights the recent findings of BAF's roles in rupture repair, and offers perspectives on how regulatory factors that control BAF activity may potentially alter the cellular response to nuclear ruptures and how BAF may participate in human disease.

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Stem cell homeostasis requires nuclear lamina (NL) integrity. In Drosophila germ cells, compromised NL integrity activates the ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 2 (Chk2) checkpoint kinases, blocking germ cell differentiation and causing germline stem cell (GSC) loss. Checkpoint activation occurs upon loss of either the NL protein emerin or its partner barrier-to-autointegration factor, two proteins required for nuclear reassembly at the end of mitosis. Here, we examined how mitosis contributes to NL structural defects linked to checkpoint activation. These analyses led to the unexpected discovery that wild-type female GSCs utilize a non-canonical mode of mitosis, one that retains a permeable but intact nuclear envelope and NL. We show that the interphase NL is remodeled during mitosis for insertion of centrosomes that nucleate the mitotic spindle within the confines of the nucleus. We show that depletion or loss of NL components causes mitotic defects, including compromised chromosome segregation associated with altered centrosome positioning and structure. Further, in emerin mutant GSCs, centrosomes remain embedded in the interphase NL. Notably, these embedded centrosomes carry large amounts of pericentriolar material and nucleate astral microtubules, revealing a role for emerin in the regulation of centrosome structure. Epistasis studies demonstrate that defects in centrosome structure are upstream of checkpoint activation, suggesting that these centrosome defects might trigger checkpoint activation and GSC loss. Connections between NL proteins and centrosome function have implications for mechanisms associated with NL dysfunction in other stem cell populations, including NL-associated diseases, such as laminopathies.

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Cell aging is the result of deteriorating competence in maintaining cellular homeostasis and quality control. Certain cell types are able to rejuvenate through asymmetric cell division by excluding aging factors, including damaged cellular compartments and extrachromosomal rDNA circles, from entering the daughter cell. Recent findings from the budding yeast S. cerevisiae have shown that gametogenesis represents another type of cellular rejuvenation. Gametes, whether produced by an old or a young mother cell, are granted a renewed replicative lifespan through the formation of a fifth nuclear compartment that sequesters the harmful senescence factors accumulated by the mother. Here, we describe the importance and mechanism of cellular remodeling at the nuclear envelope mediated by ESCRT-III and the LEM-domain proteins, with a focus on nuclear pore biogenesis and chromatin interaction during gamete rejuvenation.

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Misassembled nuclear pore complexes (NPCs) are removed by sealing off the surrounding nuclear envelope (NE), which is conducted by the endosomal sorting complexes required for transport (ESCRT) machinery. Recruitment of ESCRT proteins to the NE is mediated by the interaction between the ESCRT member Chm7 and the inner nuclear membrane protein Heh1, which belongs to the conserved LEM family. Increased ESCRT recruitment results in excessive membrane scission at damage sites but its regulation remains poorly understood. Here, we show that Hub1-mediated alternative splicing of HEH1 pre-mRNA, resulting in production of its shorter form Heh1-S, is critical for the integrity of the NE in Saccharomyces cerevisiae ESCRT-III mutants lacking Hub1 or Heh1-S display severe growth defects and accumulate improperly assembled NPCs. This depends on the interaction of Chm7 with the conserved MSC domain, which is only present in the longer variant Heh1-L. Heh1 variants assemble into heterodimers, and we demonstrate that a unique splice segment in Heh1-S suppresses growth defects associated with the uncontrolled interaction between Heh1-L and Chm7. Together, our findings reveal that Hub1-mediated splicing generates Heh1-S to regulate ESCRT recruitment to the NE.This article has an associated First Person interview with the first author of the paper.

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Cellular aging occurs as a cell loses its ability to maintain homeostasis. Aging cells eliminate damaged cellular compartments and other senescence factors via self-renewal. The mechanism that regulates cellular rejuvenation remains to be further elucidated. Using budding yeast gametogenesis as a model, we show here that the endosomal sorting complex required for transport (ESCRT) III regulates nuclear envelope organization. During gametogenesis, the nuclear pore complex (NPC) and other senescence factors are sequestered away from the prospore nuclei. We show that the LEM-domain protein Heh1 (Src1) facilitates the nuclear recruitment of ESCRT-III, which is required for meiotic NPC sequestration and nuclear envelope remodeling. Furthermore, ESCRT-III-mediated nuclear reorganization appears to be critical for gamete rejuvenation, as hindering this process curtails either directly or indirectly the replicative lifespan in gametes. Our findings demonstrate the importance of ESCRT-III in nuclear envelope remodeling and its potential role in eliminating senescence factors during gametogenesis.

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The nuclear envelope (NE) consists of the inner and outer nuclear membranes (INM and ONM), and the nuclear pore complex (NPC), which penetrates the double membrane. ONM continues with the endoplasmic reticulum (ER). INM and NPC can interact with chromatin to regulate the genetic activities of the chromosome. Studies in the fission yeast Schizosaccharomyces pombe have contributed to understanding the molecular mechanisms underlying heterochromatin formation by the RNAi-mediated and histone deacetylase machineries. Recent studies have demonstrated that NE proteins modulate heterochromatin formation and functions through interactions with heterochromatic regions, including the pericentromeric and the sub-telomeric regions. In this review, we first introduce the molecular mechanisms underlying the heterochromatin formation and functions in fission yeast, and then summarize the NE proteins that play a role in anchoring heterochromatic regions and in modulating heterochromatin formation and functions, highlighting roles for a conserved INM protein, Lem2.

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