Compromised nuclear envelope integrity drives TREX1-dependent DNA damage and tumor cell invasion.

Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.

Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.

Life-cycle-coupled evolution of mitosis in close relatives of animals.

Eukaryotes have evolved towards one of two extremes along a spectrum of strategies for remodelling the nuclear envelope during cell division: disassembling the nuclear envelope in an open mitosis or constructing an intranuclear spindle in a closed mitosis1,2. Both classes of mitotic remodelling involve key differences in the core division machinery but the evolutionary reasons for adopting a specific mechanism are unclear. Here we use an integrated comparative genomics and ultrastructural imaging approach to investigate mitotic strategies in Ichthyosporea, close relatives of animals and fungi. We show that species in this clade have diverged towards either a fungal-like closed mitosis or an animal-like open mitosis, probably to support distinct multinucleated or uninucleated states. Our results indicate that multinucleated life cycles favour the evolution of closed mitosis.

Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling.

Localized protein synthesis requires assembly and transport of translationally silenced ribonucleoprotein particles (RNPs), some of which are exceptionally large. Where in the cell such large RNP granules first assemble was heretofore unknown. We previously reported that during synapse development, a fragment of the Wnt-1 receptor, DFrizzled2, enters postsynaptic nuclei where it forms prominent foci. Here we show that these foci constitute large RNP granules harboring synaptic protein transcripts. These granules exit the nucleus by budding through the inner and the outer nuclear membranes in a nuclear egress mechanism akin to that of herpes viruses. This budding involves phosphorylation of A-type lamin, a protein linked to muscular dystrophies. Thus nuclear envelope budding is an endogenous nuclear export pathway for large RNP granules.

Spatial control of nucleoporin condensation by fragile X-related proteins.

Nucleoporins (Nups) build highly organized nuclear pore complexes (NPCs) at the nuclear envelope (NE). Several Nups assemble into a sieve-like hydrogel within the central channel of the NPCs. In the cytoplasm, the soluble Nups exist, but how their assembly is restricted to the NE is currently unknown. Here, we show that fragile X-related protein 1 (FXR1) can interact with several Nups and facilitate their localization to the NE during interphase through a microtubule-dependent mechanism. Downregulation of FXR1 or closely related orthologs FXR2 and fragile X mental retardation protein (FMRP) leads to the accumulation of cytoplasmic Nup condensates. Likewise, models of fragile X syndrome (FXS), characterized by a loss of FMRP, accumulate Nup granules. The Nup granule-containing cells show defects in protein export, nuclear morphology and cell cycle progression. Our results reveal an unexpected role for the FXR protein family in the spatial regulation of nucleoporin condensation.

Intracellular host cell membrane remodelling induced by SARS-CoV-2 infection in vitro.

BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.

Selective loss of a LAP1 isoform causes a muscle-specific nuclear envelopathy.

The nuclear envelope (NE) separates the nucleus from the cytoplasm in all eukaryotic cells. A disruption of the NE structure compromises normal gene regulation and leads to severe human disorders collectively classified as nuclear envelopathies and affecting skeletal muscle, heart, brain, skin, and bones. The ubiquitous NE component LAP1B is encoded by TOR1AIP1, and the use of an alternative start codon gives rise to the shorter LAP1C isoform. TOR1AIP1 mutations have been identified in patients with diverging clinical presentations such as muscular dystrophy, progressive dystonia with cerebellar atrophy, and a severe multi-systemic disorder, but the correlation between the mutational effect and the clinical spectrum remains to be determined. Here, we describe a novel TOR1AIP1 patient manifesting childhood-onset muscle weakness and contractures, and we provide clinical, histological, ultrastructural, and genetic data. We demonstrate that the identified TOR1AIP1 frameshift mutation leads to the selective loss of the LAP1B isoform, while the expression of LAP1C was preserved. Through comparative review of all previously reported TOR1AIP1 cases, we delineate a genotype/phenotype correlation and conclude that LAP1B-specific mutations cause a progressive skeletal muscle phenotype, while mutations involving a loss of both LAP1B and LAP1C isoforms induce a syndromic disorder affecting skeletal muscle, brain, eyes, ear, skin, and bones.

Reconstituting the reticular ER network - mechanistic implications and open questions.

The endoplasmic reticulum (ER) is a major membrane-bound organelle in all eukaryotic cells. This organelle comprises morphologically distinct domains, including the nuclear envelope and peripheral sheets and tubules. The tubules are connected by three-way junctions into a network. Several membrane proteins have been implicated in network formation; curvature-stabilizing proteins generate the tubules themselves, and membrane-anchored GTPases fuse tubules into a network. Recent experiments have shown that a tubular network can be formed with reconstituted proteoliposomes containing the yeast membrane-fusing GTPase Sey1 and a curvature-stabilizing protein of either the reticulon or REEP protein families. The network forms in the presence of GTP and is rapidly disassembled when GTP hydrolysis of Sey1 is inhibited, indicating that continuous membrane fusion is required for its maintenance. Atlastin, the ortholog of Sey1 in metazoans, forms a network on its own, serving both as a fusion and curvature-stabilizing protein. These results show that the reticular ER can be generated by a surprisingly small set of proteins, and represents an energy-dependent steady state between formation and disassembly. Models for the molecular mechanism by which curvature-stabilizing proteins cooperate with fusion GTPases to form a reticular network have been proposed, but many aspects remain speculative, including the function of additional proteins, such as the lunapark protein, and the mechanism by which the ER interacts with the cytoskeleton. How the nuclear envelope and peripheral ER sheets are formed remain major unresolved questions in the field. Here, we review reconstitution experiments with purified curvature-stabilizing proteins and fusion GTPases, discuss mechanistic implications and point out open questions.

Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays.

The linker of nucleoskeleton to cytoskeleton (LINC) complex is an essential multi-protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots, which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. By using bioinformatic, biochemical and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with Zea mays (Zm)SUN2 in vivo FRAP experiments using domain deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research.This article has an associated First Person interview with the first author of the paper.

Strong intracellular signal inactivation produces sharper and more robust signaling from cell membrane to nucleus.

For a chemical signal to propagate across a cell, it must navigate a tortuous environment involving a variety of organelle barriers. In this work we study mathematical models for a basic chemical signal, the arrival times at the nuclear membrane of proteins that are activated at the cell membrane and diffuse throughout the cytosol. Organelle surfaces within human B cells are reconstructed from soft X-ray tomographic images, and modeled as reflecting barriers to the molecules' diffusion. We show that signal inactivation sharpens signals, reducing variability in the arrival time at the nuclear membrane. Inactivation can also compensate for an observed slowdown in signal propagation induced by the presence of organelle barriers, leading to arrival times at the nuclear membrane that are comparable to models in which the cytosol is treated as an open, empty region. In the limit of strong signal inactivation this is achieved by filtering out molecules that traverse non-geodesic paths.

Fibroblasts lacking nuclear lamins do not have nuclear blebs or protrusions but nevertheless have frequent nuclear membrane ruptures.

The nuclear lamina, an intermediate filament meshwork lining the inner nuclear membrane, is formed by the nuclear lamins (lamins A, C, B1, and B2). Defects or deficiencies in individual nuclear lamin proteins have been reported to elicit nuclear blebs (protrusions or outpouchings of the nuclear envelope) and increase susceptibility for nuclear membrane ruptures. It is unclear, however, how a complete absence of nuclear lamins would affect nuclear envelope morphology and nuclear membrane integrity (i.e., whether nuclear membrane blebs or protrusions would occur and, if not, whether cells would be susceptible to nuclear membrane ruptures). To address these issues, we generated mouse embryonic fibroblasts (MEFs) lacking all nuclear lamins. The nuclear lamin-deficient MEFs had irregular nuclear shapes but no nuclear blebs or protrusions. Despite a virtual absence of nuclear blebs, MEFs lacking nuclear lamins had frequent, prolonged, and occasionally nonhealing nuclear membrane ruptures. By transmission electron microscopy, the inner nuclear membrane in nuclear lamin-deficient MEFs have a "wavy" appearance, and there were discrete discontinuities in the inner and outer nuclear membranes. Nuclear membrane ruptures were accompanied by a large increase in DNA damage, as judged by γ-H2AX foci. Mechanical stress increased both nuclear membrane ruptures and DNA damage, whereas minimizing transmission of cytoskeletal forces to the nucleus had the opposite effects.

Aspergillus nidulans in the post-genomic era: a top-model filamentous fungus for the study of signaling and homeostasis mechanisms.

The accessibility to next-generation sequencing (NGS) techniques has enabled the sequencing of hundreds of genomes of species representing all kingdoms. In the case of fungi, genomes of more than a thousand of species are publicly available. This is far from covering the number of 2.2-3.8 million fungal species estimated to populate the world but has significantly improved the resolution of the fungal tree of life. Furthermore, it has boosted systematic evolutionary analyses, the development of faster and more accurate diagnostic analyses of pathogenic strains or the improvement of several biotechnological processes. Nevertheless, the diversification of the nature of fungal species used as model has also weakened research in other species that were traditionally used as reference in the pre-genomic era. In this context, and after more than 65 years since the first works published by Pontecorvo, Aspergillus nidulans remains as one of the most referential model filamentous fungus in research fields such as hyphal morphogenesis, intracellular transport, developmental programs, secondary metabolism, or stress response. This mini-review summarizes how A. nidulans has contributed to the progress in these fields during the last years, and discusses how it could contribute in the future, assisted by NGS and new-generation molecular, microscopy, or cellular tools.

Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor.

Analysis of fluorescence fluctuation experiments by the mean-segmented Q (MSQ) method was recently used to successfully characterize the oligomeric state and mobility of proteins within the nuclear envelope (NE) of living cells. However, two significant shortcomings of MSQ were recognized. Non-ideal detector behavior due to dead-time and afterpulsing as well as the lack of error analysis currently limit the potential of MSQ. This paper presents time-shifted MSQ (tsMSQ), a new formulation of MSQ that is robust with respect to dead-time and afterpulsing. In addition, a protocol for performing error analysis on tsMSQ data is introduced to assess the quality of fit models and estimate the uncertainties of fit parameters. Together, these developments significantly simplify and improve the analysis of fluorescence fluctuation data taken within the NE. To demonstrate these new developments, tsMSQ was used to characterize the oligomeric state and mobility of the luminal domains of two inner nuclear membrane SUN proteins. The results for the luminal domain of SUN2 obtained through tsMSQ without correction for non-ideal detector effects agree with a recent study that was conducted using the original MSQ formulation. Finally, tsMSQ was applied to characterize the oligomeric state and mobility of the luminal domain of the germline-restricted SUN3.

Nuclear pore protein TPR associates with lamin B1 and affects nuclear lamina organization and nuclear pore distribution.

The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.

A fluorescence method to visualize the nuclear boundary by the lipophilic dye DiI.

Here, we describe a procedure to fluorescently contrast the nuclear boundary using the lipophilic carbocyanine dye DiI in cultured human cells. Our procedure is simple and is applicable to detect nuclear boundary defects, which may be relevant to studies on nuclear envelope dynamics, micronuclei formation and cancer biology. ABBREVIATIONS: DiI: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiO: 3,3'-dioctadecyloxacarbocyanine perchlorate; NE: nuclear envelope; RanBP2: Ran-binding protein 2/Nucleoporin 358.

Loss of MT1-MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid.

MT1-MMP (MMP14) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14(-/-) mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1-MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1-MMP induces a senescent phenotype characterized by up-regulation of p16(INK4a) and p21(CIP1/WAF) (1), increased activity of senescence-associated ß-galactosidase, generation of a senescence-associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14(-/-) mice with all-trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.

Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture.

The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.

Unr defines a novel class of nucleoplasmic reticulum involved in mRNA translation.

Unr (officially known as CSDE1) is a cytoplasmic RNA-binding protein with roles in the regulation of mRNA stability and translation. In this study, we identified a novel function for Unr, which acts as a positive regulator of placental development. Unr expression studies in the developing placenta revealed the presence of Unr-rich foci that are apparently located in the nuclei of trophoblast giant cells (TGCs). We determined that what we initially thought to be foci, were actually cross sections of a network of double-wall nuclear membrane invaginations that contain a cytoplasmic core related to the nucleoplasmic reticulum (NR). We named them, accordingly, Unr-NRs. Unr-NRs constitute a novel type of NR because they contain high levels of poly(A) RNA and translation factors, and are sites of active translation. In murine tissues, Unr-NRs are only found in two polyploid cell types, in TGCs and hepatocytes. In vitro, their formation is linked to stress and polyploidy because, in three cancer cell lines, cytotoxic drugs that are known to promote polyploidization induce their formation. Finally, we show that Unr is required in vivo for the formation of Unr-containing NRs because these structures are absent in Unr-null TGCs.

Equine Myxovirus Resistance Protein 2 Restricts Lentiviral Replication by Blocking Nuclear Uptake of Capsid Protein.

Human myxovirus resistance protein 2 (huMxB) has been shown to be a determinant type I interferon (IFN)-induced host factor involved in the inhibition of human immunodeficiency virus type 1 (HIV-1) as well as many other primate lentiviruses. This blocking occurs after the reverse transcription of viral RNA and ahead of integration into the host DNA, which is closely connected to the ability of the protein to bind the viral capsid. To date, Mx2s derived from nonprimate animals have shown no capacity for HIV-1 suppression. In this study, we examined the restrictive effect of equine Mx2 (eqMx2) on both equine infectious anemia virus (EIAV) and HIV-1 and investigated possible mechanisms for its specific function. We demonstrated that IFN-α/ß upregulates the expression of eqMx2 in equine monocyte-derived macrophages (eMDMs). The overexpression of eqMx2 significantly suppresses the replication of EIAV, HIV-1, and simian immunodeficiency viruses (SIVs) but not that of murine leukemia virus (MLV). The knockdown of eqMx2 transcription weakens the inhibition of EIAV replication by type I interferon. Interestingly, data from immunofluorescence assays suggest that the subcellular localization of eqMx2 changes following virus infection, from being dispersed in the cytoplasm to being accumulated at the nuclear envelope. Furthermore, eqMx2 blocks the nuclear uptake of the proviral genome by binding to the viral capsid. The N-terminally truncated mutant of eqMx2 lost the ability to bind the viral capsid as well as the restriction effect for lentiviruses. These results improve our understanding of the Mx2 protein in nonprimate animals.IMPORTANCE Previous research has shown that the antiviral ability of Mx2s is confined to primates, particularly humans. EIAV has been shown to be insensitive to restriction by human MxB. Here, we describe the function of equine Mx2. This protein plays an important role in the suppression of EIAV, HIV-1, and SIVs. The antiviral activity of eqMx2 depends on its subcellular location as well as its capsid binding capacity. Our results showed that following viral infection, eqMx2 changes its original cytoplasmic location and accumulates at the nuclear envelope, where it binds to the viral capsid and blocks the nuclear entry of reverse-transcribed proviral DNAs. In contrast, huMxB does not bind to the EIAV capsid and shows no EIAV restriction effect. These studies expand our understanding of the function of the equine Mx2 protein.

The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling.

Over the past decade, there has been growing interest in developing biosensors and devices with nanoscale and vertical topography. Vertical nanostructures induce spontaneous cell engulfment, which enhances the cell-probe coupling efficiency and the sensitivity of biosensors. Although local membranes in contact with the nanostructures are found to be fully fluidic for lipid and membrane protein diffusions, cells appear to actively sense and respond to the surface topography presented by vertical nanostructures. For future development of biodevices, it is important to understand how cells interact with these nanostructures and how their presence modulates cellular function and activities. How cells recognize nanoscale surface topography has been an area of active research for two decades before the recent biosensor works. Extensive studies show that surface topographies in the range of tens to hundreds of nanometers can significantly affect cell functions, behaviors, and ultimately the cell fate. For example, titanium implants having rough surfaces are better for osteoblast attachment and host-implant integration than those with smooth surfaces. At the cellular level, nanoscale surface topography has been shown by a large number of studies to modulate cell attachment, activity, and differentiation. However, a mechanistic understanding of how cells interact and respond to nanoscale topographic features is still lacking. In this Account, we focus on some recent studies that support a new mechanism that local membrane curvature induced by nanoscale topography directly acts as a biochemical signal to induce intracellular signaling, which we refer to as the curvature hypothesis. The curvature hypothesis proposes that some intracellular proteins can recognize membrane curvatures of a certain range at the cell-to-material interface. These proteins then recruit and activate downstream components to modulate cell signaling and behavior. We discuss current technologies allowing the visualization of membrane deformation at the cell membrane-to-substrate interface with nanometer precision and demonstrate that vertical nanostructures induce local curvatures on the plasma membrane. These local curvatures enhance the process of clathrin-mediated endocytosis and affect actin dynamics. We also present evidence that vertical nanostructures can induce significant deformation of the nuclear membrane, which can affect chromatin distribution and gene expression. Finally, we provide a brief perspective on the curvature hypothesis and the challenges and opportunities for the design of nanotopography for manipulating cell behavior.