High-Resolution Imaging and Analysis of Individual Nuclear Pore Complexes.

Field emission scanning electron microscopy (FESEM) is a well-established technique for acquiring three-dimensional surface images of nuclear pore complexes (NPCs). We present an optimized protocol for the exposure of mammalian cell nuclei and direct surface imaging of nuclear envelopes by FESEM, allowing for a detailed morphological comparison of individual NPCs, without the need for averaging techniques. This provides a unique high resolution tool for studying the effects of cellular stress, specific genetic manipulations and inherited diseases on the ultrastructure of human NPCs.

The perinuclear region concentrates disordered proteins with predicted phase separation distributed in a 3D network of cytoskeletal filaments and organelles.

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.

Nuclear pores dilate and constrict in cellulo.

In eukaryotic cells, nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins and form a cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter have been reported, but the physiological circumstances and the molecular details remain unknown. Here, we combined cryo­electron tomography with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. Although NPCs of exponentially growing cells adopted a dilated conformation, they reversibly constricted upon cellular energy depletion or conditions of hypertonic osmotic stress. Our data point to a model where the nuclear envelope membrane tension is linked to the conformation of the NPC.

Atg39 selectively captures inner nuclear membrane into lumenal vesicles for delivery to the autophagosome.

Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope-localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside-in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.

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.

LINC complex regulation of genome organization and function.

The regulation of genomic function is in part mediated through the physical organization and architecture of the nucleus. Disruption to nuclear organization and architecture is increasingly being recognized by its contribution to many diseases. The LINC complexes - protein structures traversing the nuclear envelope, that physically connect the nuclear interior, and hence the genome, to cytoplasmic cytoskeletal networks are an important component in the physical organization of the genome and its function. This connection, potentially allows for the constant detection of environmental mechanical stimuli, resulting in altered regulation of nuclear architecture and genome function, either directly or via the process of mechanotransduction. Here, we review the influences LINC complexes exert on genome functions and their impact on cellular/organismal health.

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.

The nuclear pore complex and the genome: organizing and regulatory principles.

The nuclear pore complex (NPC) is a massive nuclear envelope-embedded protein complex, the canonical function of which is to mediate selective nucleocytoplasmic transport. In addition to its transport function, the NPC has been shown to interact with the underlying chromatin and to influence both activating and repressive gene regulatory processes, contributing to the establishment and the epigenetic maintenance of cell identity. In this review, we discuss diverse gene regulatory functions of NPC components and emerging mechanisms underlying these functions, including roles in genome architecture, transcription complex assembly, chromatin remodeling, and coordination of transcription and mRNA export. These functional roles highlight the importance of the NPC as a nuclear scaffold directing genome organization and function.

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.

HIV-1 viral cores enter the nucleus collectively through the nuclear endocytosis-like pathway.

It is recognized that HIV-1 capsid cores are disassembled in the cytoplasm, releasing their genomes into the nucleus through nuclear pores, but there is also evidence showing the capsid (CA) exists in the nucleus. Whether HIV-1 enters the nucleus and how it enters the nucleus through the undersized nuclear pore remains mysterious. Based on multicolor labeling and real-time imaging of the viral and cellular components, our observations via light and electron microscopy suggest that HIV-1 selectively gathered at the microtubule organization center (MTOC), leading the nearby nuclear envelope (NE) to undergo deformation, invagination and restoration to form a nuclear vesicle in which the viral particles were wrapped; then, the inner membrane of the nuclear vesicle ruptured to release HIV-1 into the nucleus. This unexpected discovery expands our understanding of the complexity of HIV-1 nuclear entry, which may provide new insights to HIV-1 virology.

Nuclear lipid droplets form in the inner nuclear membrane in a seipin-independent manner.

Nuclear lipid droplets (LDs) in hepatocytes are derived from precursors of very-low-density lipoprotein in the ER lumen, but it is not known how cells lacking the lipoprotein secretory function form nuclear LDs. Here, we show that the inner nuclear membrane (INM) of U2OS cells harbors triglyceride synthesis enzymes, including ACSL3, AGPAT2, GPAT3/GPAT4, and DGAT1/DGAT2, and generates nuclear LDs in situ. mTOR inhibition increases nuclear LDs by inducing the nuclear translocation of lipin-1 phosphatidic acid (PA) phosphatase. Seipin, a protein essential for normal cytoplasmic LD formation in the ER, is absent in the INM. Knockdown of seipin increases nuclear LDs and PA in the nucleus, whereas seipin overexpression decreases these. Seipin knockdown also up-regulates lipin-1ß expression, and lipin-1 knockdown decreases the effect of seipin knockdown on nuclear LDs without affecting PA redistribution. These results indicate that seipin is not directly involved in nuclear LD formation but instead restrains it by affecting lipin-1 expression and intracellular PA distribution.

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.

Structural Analysis of Different LINC Complexes Reveals Distinct Binding Modes.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes are molecular tethers that span the nuclear envelope (NE) and physically connect the nucleus to the cytoskeleton. They transmit mechanical force across the NE in processes such as nuclear anchorage, nuclear migration, and homologous chromosome pairing during meiosis. LINC complexes are composed of KASH proteins traversing the outer nuclear membrane, and SUN proteins crossing the inner nuclear membrane. Humans have several SUN- and KASH-containing proteins, yet what governs their proper engagement is poorly understood. To investigate this question, we solved high resolution crystal structures of human SUN2 in complex with the KASH-peptides of Nesprin3, Nesprin4, and KASH5. In comparison to the published structures of SUN2-KASH1/2 we observe alternative binding modes for these KASH peptides. While the core interactions between SUN and the C-terminal residues of the KASH peptide are similar in all five complexes, the extended KASH-peptide adopts at least two different conformations. The much-improved resolution allows for a more detailed analysis of other elements critical for KASH interaction, including the KASH-lid and the cation loop, and a possible self-locked state for unbound SUN. In summary, we observe distinct differences between the examined SUN-KASH complexes. These differences may have an important role in regulating the SUN-KASH network.

Visualizing Nudivirus Assembly and Egress.

Enveloped viruses hijack cellular membranes in order to provide the necessary material for virion assembly. In particular, viruses that replicate and assemble inside the nucleus have developed special approaches to modify the nuclear landscape for their advantage. We used electron microscopy to investigate cellular changes occurring during nudivirus infection and we characterized a unique mechanism for assembly, packaging, and transport of new virions across the nuclear membrane and through the cytoplasm. Our three-dimensional reconstructions describe the complex remodeling of the nuclear membrane necessary to release vesicle-associated viruses into the cytoplasm. This is the first report of nuclear morphological reconfigurations that occur during nudiviral infection.IMPORTANCE The dynamics of nuclear envelope has a critical role in multiple cellular processes. However, little is known regarding the structural changes occurring inside the nucleus or at the inner and outer nuclear membranes. For viruses assembling inside the nucleus, remodeling of the intranuclear membrane plays an important role in the process of virion assembly. Here, we monitored the changes associated with viral infection in the case of nudiviruses. Our data revealed dramatic membrane remodeling inside the nuclear compartment during infection with Oryctes rhinoceros nudivirus, an important biocontrol agent against coconut rhinoceros beetle, a devastating pest for coconut and oil palm trees. Based on these findings, we propose a model for nudivirus assembly in which nuclear packaging occurs in fully enveloped virions.

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.

Super-Resolution Localisation of Nuclear PI(4)P and Identification of Its Interacting Proteome.

Phosphoinositides are glycerol-based phospholipids, and they play essential roles in cellular signalling, membrane and cytoskeletal dynamics, cell movement, and the modulation of ion channels and transporters. Phosphoinositides are also associated with fundamental nuclear processes through their nuclear protein-binding partners, even though membranes do not exist inside of the nucleus. Phosphatidylinositol 4-phosphate (PI(4)P) is one of the most abundant cellular phosphoinositides; however, its functions in the nucleus are still poorly understood. In this study, we describe PI(4)P localisation in the cell nucleus by super-resolution light and electron microscopy, and employ immunoprecipitation with a specific anti-PI(4)P antibody and subsequent mass spectrometry analysis to determine PI(4)P's interaction partners. We show that PI(4)P is present at the nuclear envelope, in nuclear lamina, in nuclear speckles and in nucleoli and also forms multiple small foci in the nucleoplasm. Nuclear PI(4)P undergoes re-localisation to the cytoplasm during cell division; it does not localise to chromosomes, nucleolar organising regions or mitotic interchromatin granules. When PI(4)P and PI(4,5)P2 are compared, they have different nuclear localisations during interphase and mitosis, pointing to their functional differences in the cell nucleus. Mass spectrometry identified hundreds of proteins, including 12 potentially novel PI(4)P interactors, most of them functioning in vital nuclear processes such as pre-mRNA splicing, transcription or nuclear transport, thus extending the current knowledge of PI(4)P's interaction partners. Based on these data, we propose that PI(4)P also plays a role in essential nuclear processes as a part of protein-lipid complexes. Altogether, these observations provide a novel insight into the role of PI(4)P in nuclear functions and provide a direction for further investigation.

Role of the nuclear membrane protein Emerin in front-rear polarity of the nucleus.

Cell polarity refers to the intrinsic asymmetry of cells, including the orientation of the cytoskeleton. It affects cell shape and structure as well as the distribution of proteins and organelles. In migratory cells, front-rear polarity is essential and dictates movement direction. While the link between the cytoskeleton and nucleus is well-studied, we aim to investigate if front-rear polarity can be transmitted to the nucleus. We show that the knock-down of emerin, an integral protein of the nuclear envelope, abolishes preferential localization of several nuclear proteins. We propose that the frontally biased localization of the endoplasmic reticulum, through which emerin reaches the nuclear envelope, is sufficient to generate its observed bias. In primary emerin-deficient myoblasts, its expression partially rescues the polarity of the nucleus. Our results demonstrate that front-rear cell polarity is transmitted to the nucleus and that emerin is an important determinant of nuclear polarity.

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.

Factors promoting nuclear envelope assembly independent of the canonical ESCRT pathway.

The nuclear envelope (NE) undergoes dynamic remodeling to maintain NE integrity, a process involving the inner nuclear membrane protein LEM2 recruiting CHMP7/Cmp7 and then ESCRT-III. However, prior work has hinted at CHMP7/ESCRT-independent mechanisms. To identify such mechanisms, we studied NE assembly in Schizosaccharomyces japonicus, a fission yeast that undergoes partial mitotic NE breakdown and reassembly. S. japonicus cells lacking Cmp7 have compromised NE sealing after mitosis but are viable. A genetic screen identified mutations that promote NE integrity in cmp7Δ cells. Unexpectedly, loss of Lem2 or its interacting partner Nur1 suppressed cmp7Δ defects. In the absence of Cmp7, Lem2 formed aggregates that appear to interfere with ESCRT-independent NE sealing. A gain-of-function mutation implicated a membrane and ESCRT-III regulator, Alx1, in this alternate pathway. Additional results suggest a potentially general role for unsaturated fatty acids in NE integrity. These findings establish the existence of mechanisms for NE sealing independent of the canonical ESCRT pathway.