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Lamins interact with a host of nuclear membrane proteins, transcription factors, chromatin regulators, signaling molecules, splicing factors, and even chromatin itself to form a nuclear subcompartment, the nuclear lamina, that is involved in a variety of cellular processes such as the governance of nuclear integrity, nuclear positioning, mitosis, DNA repair, DNA replication, splicing, signaling, mechanotransduction and -sensation, transcriptional regulation, and genome organization. Lamins are the primary scaffold for this nuclear subcompartment, but interactions with lamin-associated peptides in the inner nuclear membrane are self-reinforcing and mutually required. Lamins also interact, directly and indirectly, with peripheral heterochromatin domains called lamina-associated domains (LADs) and help to regulate dynamic 3D genome organization and expression of developmentally regulated genes.

The nuclear compartment is delimited by a specialized expanded sheet of the endoplasmic reticulum (ER) known as the nuclear envelope (NE). Compared to the outer nuclear membrane and the contiguous peripheral ER, the inner nuclear membrane (INM) houses a unique set of transmembrane proteins that serve a staggering range of functions. Many of these functions reflect the exceptional position of INM proteins at the membrane-chromatin interface. Recent research revealed that numerous INM proteins perform crucial roles in chromatin organization, regulation of gene expression, genome stability, and mediation of signaling pathways into the nucleus. Other INM proteins establish mechanical links between chromatin and the cytoskeleton, help NE remodeling, or contribute to the surveillance of NE integrity and homeostasis. As INM proteins continue to gain prominence, we review these advancements and give an overview on the functional versatility of the INM proteome. © 2021, Cold Spring Harbor Perspectives in Biology by Cold Spring Harbor Laboratory Press ISSN:1943-0264

The nucleus displays a wide range of sizes and shapes in different species and cell types, yet its size scaling and many of the key structural constituents that determine its shape are highly conserved. In this review, we discuss the cellular properties and processes that contribute to nuclear size and shape control, drawing examples from across eukaryotes and highlighting conserved themes and pathways. We then outline physiological roles that have been uncovered for specific nuclear morphologies and disease pathologies associated with aberrant nuclear morphology. We argue that a comparative approach, assessing and integrating observations from different systems, will be a powerful way to help us address the open questions surrounding functional roles of nuclear size and shape in cell physiology.

In eukaryotic cells, the genetic material is segregated inside the nucleus. This compartmentalization of the genome requires a transport system that allows cells to move molecules across the nuclear envelope, the membrane-based barrier that surrounds the chromosomes. Nuclear pore complexes (NPCs) are the central component of the nuclear transport machinery. These large protein channels penetrate the nuclear envelope, creating a passage between the nucleus and the cytoplasm through which nucleocytoplasmic molecule exchange occurs. NPCs are one of the largest protein assemblies of eukaryotic cells and, in addition to their critical function in nuclear transport, these structures also play key roles in many cellular processes in a transport-independent manner. Here we will review the current knowledge of the NPC structure, the cellular mechanisms that regulate their formation and maintenance, and we will provide a brief description of a variety of processes that NPCs regulate.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that the nucleoplasmic pool of lamin C rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The accumulation of lamin C at the rupture sites required both the immunoglobulin-like fold domain that binds to barrier-to-autointegration factor (BAF) and a nuclear localization signal. The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF, and cGAS concertedly accumulate at sites of NE rupture for rapid repair.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a protein complex across the nuclear envelope and has maintained its general assembly mode throughout evolution. SUN and KASH proteins, which are the major components of LINC complex, interact with each other in the nuclear lumen to transmit forces across the nuclear envelope and have diverse functions. However, research of LINC complex in budding yeast has been limited due to the lack of identification of a canonical KASH protein and a cytoskeleton factor. Here, we review recent findings that addressed these puzzles in budding yeast. We highlight the distinct assembly model of the telomere-associated LINC complex in budding yeast, which could be beneficial for identifying LINC variants in other eukaryotes.

Background The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. Results Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. Conclusions Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

Nucleophagy, the selective subtype of autophagy that predominantly targets only a selected and (nonessential) portion of the nucleus, and rarely the nucleus in its entirety, for degradation, reinforces the paradigm that nucleophagy recycling is a meticulous and highly delicate process guarded by fail-safe mechanisms. Our goal in this commentary is to encourage autophagy researchers and other scientists to explore nucleophagy blind spots and gain advanced insights into the diverse roles of this process and its selective modality as they pertain to intranuclear quality control and cellular homeostasis. Identifying and deciphering nucleophagic signaling, regulation, molecular mechanism(s) and its mediators, cargo composition and nuclear membrane dynamics under numerous physiological and/or pathological settings will provide important advances in our understanding of this critical type of organelle-selective autophagy. Abbreviations: INM, inner nuclear membrane; LN, late nucleophagy; mRNA, messenger RNA; NE, nuclear envelope; NL, nuclear lamina; NPC(s), nuclear pore complex(es); NVJ(s), nucleus-vacuole junction(s); ONM, outer nuclear membrane; PMN, piecemeal microautophagy of the nucleus; PND, programmed nuclear death; PNuD, programmed nuclear destruction; rDNA/rRNA, ribosomal DNA/RNA

During cell growth and motility in crowded tissues or interstitial spaces, cells must integrate multiple physical and biochemical environmental inputs. After a number of recent studies, the view of the nucleus as a passive object that cells have to drag along has become obsolete, placing the nucleus as a central player in sensing some of these inputs. In the present review, we will focus on changes in nuclear shape caused by external and internal forces. Depending on their magnitude, nuclear deformations can generate signaling events that modulate cell behavior and fate, or be a source of perturbations or even damage, having detrimental effects on cellular functions. On very large deformations, nuclear envelope rupture events become frequent, leading to uncontrolled nucleocytoplasmic mixing and DNA damage. We will also discuss the consequences of repeated compromised nuclear integrity, which can trigger DNA surveillance mechanisms, with critical consequences to cell fate and tissue homeostasis.

This article comments on:Masuda K, Hikida R, Fujino K. 2021. The plant nuclear lamina proteins NMCP1 and NMCP2 form a filamentous network with lateral filament associations. Journal of Experimental Botany 72, 6190–6204.

Background The microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes. Results As starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 μm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions. Conclusions Tau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

Proper mitotic progression inSchizosaccharomyces pomberequires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB – yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high- resolution structured illumination microscopy (SIM), we show that the conserved SUN- domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.

"Interphase epichromatin" describes the surface of chromatin located adjacent to the interphase nuclear envelope. It was discovered in 2011 using a bivalent anti-nucleosome antibody (mAb PL2-6), now known to be directed against the nucleosome acidic patch. The molecular structure of interphase epichromatin is unknown, but is thought to be heterochromatic with a high density of "exposed" acidic patches. In the 1960s, transmission electron microscopy of fixed, dehydrated, sectioned, and stained inactive chromatin revealed "unit threads," frequently organized into parallel arrays at the nuclear envelope, which were interpreted as regular helices with similar to 30-nm center-to-center distance. Also observed in certain cell types, the nuclear envelope forms a "sandwich" around a layer of closely packed unit threads (ELCS, envelope-limited chromatin sheets). Discovery of the nucleosome in 1974 led to revised helical models of chromatin. But these models became very controversial and the existence of in situ 30-nm chromatin fibers has been challenged. Development of cryo-electron microscopy (Cryo-EM) gave hope that in situ chromatin fibers, devoid of artifacts, could be structurally defined. Combining a contrast-enhancing phase plate and cryo-electron tomography (Cryo-ET), it is now possible to visualize chromatin in a "close-to-native" situation. ELCS are particularly interesting to study by Cryo-ET. The chromatin sheet appears to have two layers of similar to 30-nm chromatin fibers arranged in a criss-crossed pattern. The chromatin in ELCS is continuous with adjacent interphase epichromatin. It appears that hydrated similar to 30-nm chromatin fibers are quite rare in most cells, possibly confined to interphase epichromatin at the nuclear envelope.

A large proportion of the metazoan genome is spatially segregated at the nuclear periphery through genomic contacts with the nuclear lamina, a thin meshwork of lamin filaments that lines the inner-nuclear membrane. Lamina-associated domains are believed to contribute to the regulation of gene transcription and to provide structural three-dimensional support to the organization of the genome in A and B compartments and topologically associating domains. In this review, we will evaluate recent work addressing the role of lamina-associated domains in three-dimensional genome organization and propose experimental frameworks that may expand our understanding of their interdependence.

Micronuclei are small membrane-bounded compartments with a DNA content encapsulated by a nuclear envelope and spatially separated from the primary nucleus. Micronuclei have long been linked to chromosome instability, genome rearrangements, and mutagenesis. They are frequently found in cancers, during senescence, and after genotoxic stress. Compromised integrity of the micronuclear envelope delays or disrupts DNA replication, inhibits DNA repair, and exposes micronuclear DNA directly to cytoplasm. Micronuclei play a central role in tumorigenesis, with micronuclear DNA being a source of complex genome rearrangements (including chromothripsis) and promoting a cyclic GMP–AMP synthase (cGAS)-mediated cellular immune response that may contribute to cancer metastasis. Here, we discuss recent findings on how micronuclei are generated, what the consequences are, and what cellular mechanisms can be applied to protect against micronucleation.

The defining feature of the eukaryotic cell, the nucleus, is bounded by a double envelope. This envelope and the nuclear pores within it play a critical role in separating the genome from the cytoplasm. It also presents cells with a challenge. How are cells to remodel the nuclear compartment boundary during mitosis without compromising nuclear function? In the two billion years since the emergence of the first cells with a nucleus, eukaryotes have evolved a range of strategies to do this. At one extreme, the nucleus is disassembled upon entry into mitosis and then reassembled anew in the two daughter cells. At the other, cells maintain an intact nuclear compartment boundary throughout the division process. In this review, we discuss common features of the division process that underpin remodelling mechanisms, the topological challenges involved and speculate on the selective pressures that may drive the evolution of distinct modes of division.

As one of the core framework proteins of nuclear pore complex (NPC), nucleoporin Nupl70 acts as a structural adapter between the nucleolus and nuclear pore membrane and maintains the stability of NPC structure through interaction with other proteins. In this study, we identified a Nup170 protein in the microsporidian Nosema bombycis for the first time and named it as NbNup170. Secondary structure prediction showed that the NbNup170 contains α-helices and random coils. The three-dimensional structure of NbNup170 is elliptical in shape. Phylogenetic analysis based on the Nup170 and homologous sequences showed that N. bombycis clustered together with Vairimorpha ceranae and Vairimorpha apis. The immunofluorescence localization results showed that the NbNup170 was located on the plasma membrane of the dormant spore and transferred to the surface of sporoplasm in a punctate pattern when the dormant spore has finished germination, and that NbNup170 was distributed on the nuclear membrane and both sides of the nuclei of early proliferative phase, and only on the nuclear membrane during sporogonic phase in the N. bombycis. qPCR analysis showed that the relative expression level of NbNup170 maintained at a low level from 30 to 78 h post-infection with N. bombycis, then reached the highest at 102 h, while that of NbNup170 was repressed at a very low level throughout its life cycle by RNA interference. These results suggested that NbNup170 protein is involved in the proliferative phase and active during the sporogonic phase of N. bombycis.

Background Macrophages are important cells of the innate immune system and contribute to a variety of physiological and pathophysiological responses. Monovalent and divalent ion channels have been studied in macrophage function, and while much research is still required, a role for these channels is beginning to emerge in macrophages. In addition to the plasma membrane, ion channels are also found in intracellular membranes including mitochondrial, lysosomal and nuclear membranes. While studying the function of plasma membrane located large conductance voltage- and calcium-activated potassium channels (BK channels) in a macrophage cell line RAW264.7, we became aware of the expression of these ion channels in other cellular locations. Methods Immunofluorescence and Western blot analysis were used to identify the expression of BK channels. To demonstrate a functional role for the nuclear located channel, we investigated the effect of the lipid soluble BK channel inhibitor paxilline on CREB phosphorylation. Results Treatment of resting macrophages with paxilline resulted in increased CREB phosphorylation. To confirm a role for nuclear BK channels, these experiments were repeated in isolated nuclei and similar results were found. Ca2+ and calmodulin-dependent kinases (CaMK) have been demonstrated to regulate CREB phosphorylation. Inhibition of CaMKII and CaMKIV resulted in the reversal of paxilline-induced CREB phosphorylation. Conclusions These results suggest that nuclear BK channels regulate CREB phosphorylation in macrophages. Nuclear located ion channels may therefore be part of novel signalling pathways in macrophages and should be taken into account when studying the role of ion channels in these and other cells. Graphic abstract

Sirtuin 3 (SIRT3) is an NAD+-dependent deacetylase linked to a broad range of physiological and pathological processes, including aging and aging-related diseases. However, the role of SIRT3 in regulating human stem cell homeostasis remains unclear. Here we found that SIRT3 expression was downregulated in senescent human mesenchymal stem cells (hMSCs). CRISPR/Cas9-mediated depletion of SIRT3 led to compromised nuclear integrity, loss of heterochromatin and accelerated senescence in hMSCs. Further analysis indicated that SIRT3 interacted with nuclear envelope proteins and heterochromatin-associated proteins. SIRT3 deficiency resulted in the detachment of genomic lamina-associated domains (LADs) from the nuclear lamina, increased chromatin accessibility and aberrant repetitive sequence transcription. The re-introduction of SIRT3 rescued the disorganized heterochromatin and the senescence phenotypes. Taken together, our study reveals a novel role for SIRT3 in stabilizing heterochromatin and counteracting hMSC senescence, providing new potential therapeutic targets to ameliorate aging-related diseases.

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., Research article: Schematic view of the route of nascent severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) through the compartments of the viral factory. SARS‐CoV‐2 infection induces an intense intracellular membrane remodelling in Vero cells. This alteration protects and delimits the locus of viral replication, morphogenesis and maturation. The intermediate steps of SARS‐CoV‐2 morphogenesis were approached by transmission electron microscopy and electron tomography, revealing and describing different membrane structures and the assembly of viral particles by budding through the endoplasmic reticulum‐Golgi intermediate compartment.

A total of 10-20% of plasma membrane proteins are anchored by glycosylphosphatidylinositol (GPI). GPI is attached to proteins by GPI transamidase (GPI-T), which contains five subunits named PIGK, PIGS, PIGT, PIGU, and GPAA1. We previously reported that PIGT localizes near the nucleus in Drosophila. However, localizations of the other four subunits remain unknown. Here, we show that a catalytic subunit of GPI-T, PIGK, mainly localizes to the endoplasmic reticulum (ER), while the other four subunits localize to the nuclear envelope (NE) and ER. The NE/ER localization ratio of PIGS differs between cell types and developmental stages. Our results suggest that GPI-T catalyzes GPI attachment in the ER and the other four subunits may have other unknown functions in the NE.

Adherent cells utilize local environmental cues to make decisions on their growth and movement. We have previously shown that HEK293 cells grown on the fibronectin stripe patterns were elongated. Here we show that Piezo1 function is involved in cell spreading. Piezo1 expressing HEK cells plated on fibronectin stripes elongated, while a knockout of Piezo1 eliminated elongation. Inhibiting Piezo1 conductance using GsMTx4 or Gd3+ blocked cell spreading, but the cells grew thin tail-like extensions along the patterns. Images of GFP-tagged Piezo1 showed plaques of Piezo1 moving to the extrusion edges, co-localized with focal adhesions. Surprisingly, in non-spreading cells Piezo1 was located primarily on the nuclear envelope. Inhibiting the Rho-ROCK pathway also reversibly inhibited cell extension indicating that myosin contractility is involved. The growth of thin extrusion tails did not occur in Piezo1 knockout cells suggesting that Piezo1 may have functions besides acting as a cation channel.

Although nuclei are the defining features of eukaryotes, we still do not fully understand how the nuclear compartment is duplicated and partitioned during division. This is especially the case for organisms that do not completely disassemble their nuclear envelope upon entry into mitosis. In studying this process in Drosophila neural stem cells, which undergo asymmetric divisions, we find that the nuclear compartment boundary persists during mitosis thanks to the maintenance of a supporting nuclear lamina. This mitotic nuclear envelope is then asymmetrically remodeled and partitioned to give rise to two daughter nuclei that differ in envelope composition and exhibit a30-fold difference in volume. The striking difference in nuclear size was found to depend on two consecutive processes: asymmetric nuclear envelope resealing at mitotic exit at sites defined by the central spindle, and differential nuclear growth that appears to depend on the available local reservoir of ER/nuclear membranes, which is asymmetrically partitioned between the two daughter cells. Importantly, these asymmetries in size and composition of the daughter nuclei, and the associated asymmetries in chromatin organization, all become apparent long before the cortical release and the nuclear import of cell fates determinants. Thus, asymmetric nuclear remodeling during stem cell divisions may contribute to the generation of cellular diversity by initiating distinct transcriptional programs in sibling nuclei that contribute to later changes in daughter cell identity and fate.

The fungal kingdom is large and diverse, representing extremes of ecology, life cycles and morphology. At a cellular level, the diversity among fungi is particularly apparent at the spindle pole body (SPB). This nuclear envelope embedded structure, which is essential for microtubule nucleation, shows dramatically different morphologies between different fungi. However, despite phenotypic diversity, many SPB components are conserved, suggesting commonalities in structure, function and duplication. Here, I review the organization of the most well-studied SPBs and describe how advances in genomics, genetics and cell biology have accelerated knowledge of SPB architecture in other fungi, providing insights into microtubule nucleation and other processes conserved across eukaryotes.

The eukaryotic nucleus shows organized structures of chromosomes, transcriptional components and their associated proteins. It has been believed that such a dense nuclear environment prevents the formation of a cytoskeleton-like network of protein filaments. However, accumulating evidence suggests that the cell nucleus also possesses structural filamentous components to support nuclear organization and compartments, which are referred to as nucleoskeleton proteins. Nucleoskeleton proteins including lamins and actin influence nuclear dynamics including transcriptional regulation, chromatin organization and DNA damage responses. Furthermore, these nucleoskeleton proteins play a pivotal role in cellular differentiation and animal development. In this commentary, we discuss how nucleoskeleton-based regulatory mechanisms orchestrate nuclear dynamics.

Before their delivery to and degradation by the 26S proteasome, misfolded transmembrane proteins of the endoplasmic reticulum (ER) and inner-nuclear membrane (INM) must be extracted from lipid bilayers. This extraction process, known as retrotranslocation, requires both quality-control E3 ubiquitin ligases and dislocation factors that diminish the energetic cost of dislodging the transmembrane segments of a protein. Recently, we showed that retrotranslocation of all ER transmembrane proteins requires the Dfm1 rhomboid pseudoprotease. However, we did not investigate whether Dfm1 also mediated retrotranslocation of transmembrane substrates in the INM, which is contiguous with the ER but functionally separated from it by nucleoporins. Here, we show that canonical retrotranslocation occurs during INM-associated degradation (INMAD) but proceeds independently of Dfm1. Despite this independence, ER-associated degradation (ERAD)-M and INMAD cooperate to mitigate proteotoxicity. We show a novel misfolded-transmembrane-protein toxicity that elicits genetic suppression, demonstrating the cell's ability to tolerate a toxic burden of misfolded transmembrane proteins without functional INMAD or ERAD-M. This strikingly contrasted the suppression of the dfm1Δ null, which leads to the resumption of ERAD-M through HRD-complex remodeling. Thus, we conclude that INM retrotranslocation proceeds through a novel, private channel that can be studied by virtue of its role in alleviating membrane-associated proteotoxicity.

Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of glucose transporter 4 (GLUT4) from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling module is required for insulin-stimulated GLUT4 translocation to the PM, although where it intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to control GLUT4 mobilization from a trans-Golgi network (TGN) storage compartment, establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN from which newly synthesized lysosomal proteins are targeted to the late endosomes and the ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not mobilize ATP7A nor does copper mobilize GLUT4, and RAB10 is not required for copper-elicited ATP7A mobilization. Consequently, GLUT4 intracellular sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. Our results define the GLUT4-containing region of the TGN as a sorting and storage site from which different cargo are mobilized by distinct signals through unique molecular machinery.

The functional organization of the plant nuclear envelope is gaining increasing attention through new connections made between nuclear envelope-associated proteins and important plant biological processes. Animal nuclear envelope proteins play roles in nuclear morphology, nuclear anchoring and movement, chromatin tethering and mechanical signaling. However, how these roles translate to functionality in a broader biological context is often not well understood. A surprising number of plant nuclear envelope-associated proteins are plant-unique, suggesting that separate functionalities evolved after the split of Opisthokonta and Streptophyta. Significant progress has now been made in discovering broader biological roles of plant nuclear envelope proteins, increasing the number of known plant nuclear envelope proteins, and connecting known proteins to chromatin organization, gene expression, and the regulation of nuclear calcium. The interaction of viruses with the plant nuclear envelope is another emerging theme. Here, we survey the recent developments in this still relatively new, yet rapidly advancing field.

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 extra chromosomal 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.

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.

In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2–3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear lamina architecture and nuclear morphology and caused DNA damage and abnormal mitotic spindles, depending on the N2–3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development.

The major constituent of the nuclear envelope is the nuclear lamina. A fibrous meshwork of lamin filaments spanning underneath the nuclear membrane provides mechanical support to the nucleus. Lamins, type V intermediate filament proteins, are also involved in many other nuclear activities such as DNA repair and transcription. Recent structural studies provide new insight into how lamins assemble into ∼3.5nm thick filaments, which is in contrast with cytoplasmic intermediate filaments. The thinnest component of the cell cytoskeleton exhibits surprising mechanical properties. Here, we critically review and discuss structural and mechanical aspects of single lamin filaments.

Nuclear envelope proteins influence cell cytoarchitecure by poorly understood mechanisms. Here we show that small interfering RNA-mediated silencing of lamin A/C (LMNA) promotes contrasting stress fiber assembly and disassembly in individual cells and within cell populations. We show that LMNA-deficient cells have elevated myosin-II bipolar filament accumulations, irregular formation of actin comet tails and podosome-like adhesions, increased steady state nuclear localization of the mechanosensitive transcription factors MKL1 and YAP, and induced expression of some MKL1/serum response factor-regulated genes such as that encoding myosin-IIA (MYH9). Our studies utilizing live cell imaging and pharmacological inhibition of myosin-II support a mechanism of deregulated myosin-II self-organizing activity at the nexus of divergent actin cytoskeletal aberrations resulting from LMNA loss. In light of our results, we propose a model of how the nucleus, via linkage to the cytoplasmic actomyosin network, may act to control myosin-II contractile behavior through both mechanical and transcriptional feedback mechanisms.

Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302–332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function., Graphical Abstract, The heterozygous in-frame GAG deletion in the TOR1A gene leads to the movement disorder DYT1 dystonia. Cruz et al. established a CRISPR strategy to selectively disrupt the mutant allele, aiming to restore torsinA function. They used the variant Cas9-VRQR, which recognizes a PAM site created by the GAG deletion.

International audience; Untangling chromatin interactions The plant nucleus contains myriad subdomains that define and are defined by the composition of their chromatin. On a whole nucleus scale, this includes regions of active euchromatin and inactive heterochromatin, although the boundaries between these states are blurred. Different loci constantly enter and exit nuclear structures including the nucleolus and transient regulatory nuclear bodies. Chromatin is defined by its complex combinations of epigenetic modifications and variation in its constituent histone proteins. Here we preview articles that describe many of the mechanisms that regulate this dynamic chromatin.

Skeletal muscle weakness is an important determinant of age-related declines in independence and quality of life but its causes remain unclear. Accelerated ageing syndromes such as Hutchinson–Gilford Progerin Syndrome, caused by mutations in genes encoding nuclear envelope proteins, have been extensively studied to aid our understanding of the normal biological ageing process. Like several other pathologies associated with genetic defects to nuclear envelope proteins including Emery–Dreifuss muscular dystrophy, Limb–Girdle muscular dystrophy and congenital muscular dystrophy, these disorders can lead to severe muscle dysfunction. Here, we first describe the structure and function of nuclear envelope proteins, and then review the mechanisms by which mutations in genes encoding nuclear envelope proteins induce premature ageing diseases and muscle pathologies. In doing so, we highlight the potential importance of such genes in processes leading to skeletal muscle weakness in old age.

International audience; MBoC | ARTICLE Centrosome-nuclear axis repositioning drives the assembly of a bipolar spindle scaffold to ensure mitotic fidelity ABSTRACT During the initial stages of cell division, the cytoskeleton is extensively reorganized so that a bipolar mitotic spindle can be correctly assembled. This process occurs through the action of molecular motors, cytoskeletal networks, and the nucleus. How the combined activity of these different components is spatiotemporally regulated to ensure efficient spindle assembly remains unclear. To investigate how cell shape, cytoskeletal organization, and molecular motors cross-talk to regulate initial spindle assembly, we use a combination of micropatterning with high-resolution imaging and 3D cellular reconstruction. We show that during prophase, centrosomes and nucleus reorient so that centrosomes are positioned on the shortest nuclear axis at nuclear envelope (NE) breakdown. We also find that this orientation depends on a combination of centrosome movement controlled by Arp2/3-mediated regulation of microtubule dynamics and Dynein-generated forces on the NE that regulate nuclear reorientation. Finally, we observe this centrosome configuration favors the establishment of an initial bipolar spindle scaffold, facilitating chromosome capture and accurate segregation, without compromising division plane orientation.

Micronuclei are extra-nuclear bodies mainly derived from ana-telophase lagging chromosomes/chromatins (LCs) that are not incorporated into primary nuclei at mitotic exit. Unlike primary nuclei, most micronuclei are enclosed by nuclear envelope (NE) that is highly susceptible to spontaneous and irreparable rupture. Ruptured micronuclei act as triggers of chromothripsis-like chaotic chromosomal rearrangements and cGAS-mediated innate immunity and inflammation, raising the view that micronuclei play active roles in human aging and tumorigenesis. Thus, understanding the ways in which micronuclear envelope (mNE) goes awry acquires increased importance. Here, we review the data to present a general framework for this question. We firstly describe NE reassembly after mitosis and NE repair during interphase. Simultaneously, we briefly discuss how mNE is organized and how mNE rupture controls the fate of micronuclei and micronucleated cells. As a focus of this review, we highlight current knowledge about why mNE is rupture-prone and irreparable. For this, we survey observations from a series of elegant studies to provide a systematic overview. We conclude that the birth of rupture-prone and irreparable micronuclei may be the cumulative effects of their intracellular geographic origins, biophysical properties, and specific mNE features. We propose that DNA damage and immunogenicity in micronuclei increase stepwise from altered mNE components, mNE rupture, and refractory to repair. Throughout our discussion, we note interesting issues in mNE fragility that have yet to be resolved.

Nuclear movement and positioning play a role in developmental processes throughout life. Nuclear movement and positioning are mediated primarily by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes are comprised of the inner nuclear membrane SUN proteins and the outer nuclear membrane (ONM) KASH proteins. In Arabidopsis pollen tubes, the vegetative nucleus (VN) maintains a fixed distance from the pollen tube tip during growth, and the VN precedes the sperm cells (SCs). In pollen tubes of wit12 and wifi, mutants deficient in the ONM component of a plant LINC complex, the SCs precede the VN during pollen tube growth and the fixed VN distance from the tip is lost. Subsequently, pollen tubes frequently fail to burst upon reception. In this study, we sought to determine if the pollen tube reception defect observed in wit12 and wifi is due to decreased sensitivity to reactive oxygen species (ROS). Here, we show that wit12 and wifi are hyposensitive to exogenous H2O2, and that this hyposensitivity is correlated with decreased proximity of the VN to the pollen tube tip. Additionally, we report the first instance of nuclear Ca2+ peaks in growing pollen tubes, which are disrupted in the wit12 mutant. In the wit12 mutant, nuclear Ca2+ peaks are reduced in response to exogenous ROS, but these peaks are not correlated with pollen tube burst. This study finds that VN proximity to the pollen tube tip is required for both response to exogenous ROS, as well as internal nuclear Ca2+ fluctuations.

The nuclear envelope (NE) is continuous with the endoplasmic reticulum (ER), yet the NE carries out many functions distinct from those of bulk ER. This functional specialization depends on a unique protein composition that defines NE identity and must be both established and actively maintained. The NE undergoes extensive remodeling in interphase and mitosis, so mechanisms that seal NE holes and protect its unique composition are critical for maintaining its functions. New evidence shows that closure of NE holes relies on regulated de novo lipid synthesis, providing a link between lipid metabolism and generating and maintaining NE identity. Here, we review regulation of the lipid bilayers of the NE and suggest ways to generate lipid asymmetry across the NE despite its direct continuity with the ER. We also discuss the elusive mechanism of membrane fusion during nuclear pore complex (NPC) biogenesis. We propose a model in which NPC biogenesis is carefully controlled to ensure that a permeability barrier has been established before membrane fusion, thereby avoiding a major threat to compartmentalization.

The nucleus is one of the membrane-bound organelles that are a distinguishing feature between eukaryotes and prokaryotes. During meiosis, the nuclear envelope takes on functions beyond separating the nucleoplasm from the cytoplasm. These include associations with meiotic chromosomes to mediate pairing, being a sensor for recombination progression, and supportive of enormous nuclear growth during oocyte formation. In this review, we highlight recent results that have contributed to our understanding of meiotic nuclear envelope function and dynamics.

The nuclear envelope (NE) continues to the endoplasmic reticulum (ER). Proper partitioning of NE and ER is crucial for cellular activity, but the key factors maintaining the boundary between NE and ER remain to be elucidated. Here we show that the conserved membrane proteins Lem2 and Lnp1 cooperatively play a crucial role in maintaining the NE-ER membrane boundary in fission yeast Schizosaccharomyces pombe. Cells lacking both Lem2 and Lnp1 caused severe growth defects associated with aberrant expansion of the NE/ER membranes, abnormal leakage of nuclear proteins, and abnormal formation of vacuolar-like structures in the nucleus. Overexpression of the ER membrane protein Apq12 rescued the growth defect associated with membrane disorder caused by the loss of Lem2 and Lnp1. Genetic analysis showed that Apq12 had overlapping functions with Lnp1. We propose that a membrane protein network with Lem2 and Lnp1 acts as a critical factor to maintain the NE-ER boundary., Hirano et al find that deletion of Lem2 and Lnp1, localised to the nuclear envelope (NE) and the endoplasmic reticulum (ER) respectively, causes growth defects in S. pombe that are associated with aberrant expansion of NE/ER membranes and leakage of nuclear proteins. This study sheds light on the involvement of these membrane proteins in the maintenance of the nuclear membrane barrier.

CTP:phosphocholine cytidylyltransferase-alpha (CCTα) and CCTβ catalyze the rate-limiting step in phosphatidylcholine (PC) biosynthesis. CCTα is activated by association of its α-helical M-domain with nuclear membranes, which is negatively regulated by phosphorylation of the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we developed phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the P-domain, respectively. Oleate treatment of cultured cells triggered CCTα translocation to the nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Removal of oleate led to dissociation of CCTα from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCTα translocation to the NE and S319 dephosphorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addition and removal, and CCTα-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCTα-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCTα recruitment to nuclear membranes.

At the nuclear periphery, associations of chromatin with the nuclear lamina through lamina-associated domains (LADs) aid functional organization of the genome. We review the organization of LADs and provide evidence of LAD heterogeneity from cell ensemble and single-cell data. LADs are typically repressive environments in the genome; nonetheless, we discuss findings of lamin interactions with regulatory elements of active genes, and the role lamins may play in genome regulation. We address the relationship between LADs and other genome organizers, and the involvement of LADs in laminopathies. The current data lay the basis for future studies on the significance of lamin-chromatin interactions in health and disease.

During the nuclear export of nascent nucleocapsids of herpesviruses, the nucleocapsids bud through the inner nuclear membrane (INM) by acquiring the INM as a primary envelope (primary envelopment). We recently reported that herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), which consists of UL34 and UL31, interacts with an endosomal sorting complex required for transport III (ESCRT-III) adaptor ALIX and recruits ESCRT-III machinery to the INM for efficient primary envelopment. In this study, we identified a cluster of six arginine residues in the disordered domain of UL34 as a minimal region required for the interaction with ALIX, as well as the recruitment of ALIX and an ESCRT-III protein CHMP4B to the INM in HSV-1-infected cells. Mutations in the arginine cluster exhibited phenotypes similar to those with ESCRT-III inhibition reported previously, including the mislocalization of NEC, induction of membranous invagination structures containing enveloped virions, aberrant accumulation of enveloped virions in the invaginations and perinuclear space, and reduction of viral replication. We also showed that the effect of the arginine cluster in UL34 on HSV-1 replication was dependent primarily on ALIX. These results indicated that the arginine cluster in the disordered domain of UL34 was required for the interaction with ALIX and the recruitment of ESCRT-III machinery to the INM to promote primary envelopment. IMPORTANCE Herpesvirus UL34 homologs contain conserved amino-terminal domains that mediate vesicle formation through interactions with UL31 homologs during primary envelopment. UL34 homologs also comprise other domains adjacent to their membrane-anchoring regions, which differ in length, are variable in herpesviruses, and do not form distinguished secondary structures. However, the role of these disordered domains in infected cells remains to be elucidated. In this study, we present data suggesting that the arginine cluster in the disordered domain of HSV-1 UL34 mediates the interaction with ALIX, thereby leading to the recruitment of ESCRT-III machinery to the INM for efficient primary envelopment. This is the first study to report the role of the disordered domain of a UL34 homolog in herpesvirus infections.

Little is known about how the observed fat-specific pattern of 3D-spatial genome organisation is established. Here we report that adipocyte-specific knockout of the gene encoding nuclear envelope transmembrane protein Tmem120a disrupts fat genome organisation, thus causing a lipodystrophy syndrome. Tmem120a deficiency broadly suppresses lipid metabolism pathway gene expression and induces myogenic gene expression by repositioning genes, enhancers and miRNA-encoding loci between the nuclear periphery and interior. Tmem120a−/− mice, particularly females, exhibit a lipodystrophy syndrome similar to human familial partial lipodystrophy FPLD2, with profound insulin resistance and metabolic defects that manifest upon exposure to an obesogenic diet. Interestingly, similar genome organisation defects occurred in cells from FPLD2 patients that harbour nuclear envelope protein encoding LMNA mutations. Our data indicate TMEM120A genome organisation functions affect many adipose functions and its loss may yield adiposity spectrum disorders, including a miRNA-based mechanism that could explain muscle hypertrophy in human lipodystrophy., Little is known how spatial genome organization is directed in fat; however, key proadipogenic genes reposition from the nuclear envelope (NE) to the interior during adipogenesis. Here the authors show that deletion of NE protein Tmem120a in adipocytes disrupts fat genome organization, which results in suppression of lipid metabolism pathways and induces myogenic gene expression.

Endoplasmic reticulum (ER) stress occurs when the abundance of unfolded proteins in the ER exceeds the capacity of the folding machinery. Despite the expanding cadre of characterized cellular adaptations to ER stress, knowledge of the effects of ER stress on cellular physiology remains incomplete. We investigated the impact of ER stress on ER and inner nuclear membrane protein quality control mechanisms in Saccharomyces cerevisiae. We analyzed the turnover of substrates of four ubiquitin ligases (Doa10, Rkr1/Ltn1, Hrd1, and the Asi complex) and the metalloprotease Ste24 in induced models of ER stress. ER stress did not substantially impact Doa10 or Rkr1 substrates. However, Hrd1-mediated destruction of a protein that aberrantly engages the translocon (Deg1-Sec62) and substrates with luminal degradation signals was markedly impaired by ER stress; by contrast, Hrd1-dependent degradation of proteins with intramembrane degrons was largely unperturbed by ER stress. ER stress impaired the degradation of one of two Asi substrates analyzed and caused a translocon-clogging Ste24 substrate to accumulate in a form consistent with persistent translocon occupation. Degradation of Deg1-Sec62 in the absence of stress and stabilization during ER stress were independent of four ER stress–sensing pathways. Our results indicate ER stress differentially impacts degradation of protein quality control substrates, including those mediated by the same ubiquitin ligase. These observations suggest the existence of additional regulatory mechanisms dictating substrate selection during ER stress.

Nuclear pore complexes (NPCs) are huge protein assemblies within the nuclear envelope (NE) that serve as selective gates for macromolecular transport between nucleus and cytoplasm. When higher eukaryotic cells prepare for division, they rapidly disintegrate NPCs during NE breakdown such that nuclear and cytoplasmic components mix to enable the formation of a cytoplasmic mitotic spindle. At the end of mitosis, reassembly of NPCs is coordinated with the establishment of the NE around decondensing chromatin. We review recent progress on mitotic NPC disassembly and reassembly, focusing on vertebrate cells. We highlight novel mechanistic insights into how NPCs are rapidly disintegrated into conveniently reusable building blocks, and put divergent models of (post-)mitotic NPC assembly into a spatial and temporal context., Trends in Cell Biology, 31 (12), ISSN:0962-8924, ISSN:0968-0039, ISSN:1879-3088