Nuclear envelope dynamics during male pronuclear development

Upon fertilization, the sperm nucleus undergoes reactivation. The poreless sperm nuclear envelope is replaced by a functional male pronuclear envelope and the highly compact male chromatin decondenses. Here some recent evidence is examined: that disassembly of the sperm lamina is required for chromatin decondensation, that remnant portions of the sperm nuclear envelope target the binding of egg membrane vesicles that form the male pronuclear envelope, that functional male pronuclear envelopes containing lamin B receptor assemble prior to lamin import and lamina formation, and that lamina assembly drives male pronuclear swelling. Several unresolved issues are discussed.     

Nuclear envelope structure.

The past 18 months have seen significant advances in our knowledge of the constituents of the nuclear envelope, their interactions during interphase and the mechanisms involved in their mitotic dynamics. Although most of the new data are in general agreement with, and contribute detail to, our traditional image of the nuclear envelope, a few observations appear to mark the beginning of new and important directions in research.     

Nuclear envelope inclusions demonstrated by freeze-fracture.

Inclusions in the perinuclear space of the nuclear envelope of human diploid (MRC-5) fibroblasts, limpet (Patella vulgata) haemocytes, and yeast (Saccharomyces cerevisiae) as observed with the freeze-fracture technique are described. The significance of these inclusions is discussed and it is tentatively concluded that they represent vesicles engaged in transporting macromolecules between nucleus and cytoplasm. Although the inclusions were infrequently observed, their demonstration in mammalian, invertebrate and lower eukaryotic cell types raises the possibility that this form of nucleocytoplasmic exchange may potentially be adopted under appropriate circumstances by the eukaryotic cell in general.     

Nuclear envelope of the seminal-vesicle epithelium.

The nuclear envelope of seminal-vesicle epithelium was isolated by a procedure involving enzymic digestion with deoxyribonuclease I, sonication in the presence of 0.34 M-sodium citrate, and centrifugation through sucrose density gradients. The mass of envelope DNA was only 0.8% of that of envelope protein, and by transmission electron microscopy the envelope was 98-99% pure. We showed that the envelope possess a protein kinase activity which is uninfluenced by cyclic nucleotides. Both lysine-rich histone and dephosphophosvitin as substrates gave a greater specific activity than did envelope protein itself. Optimum requirements with respect to Na+, Mg2+, pH and ATP were established for each substrate, and the influence of other factors on enzyme activity was investigated. Data, obtained mainly with the use of lysine-rich histone, are presented which indicate that nuclear envelope from intact and 96 h-castrated guinea pigs may have equal protein kinase activities and, in separate experiments, equal phosphoprotein phosphatase activities. Clarification of these initial observations must await identification of the natural substrates or the envelope's phosphorylation-dephosphorylation reactions.     

Nuclear envelope dynamics during plant cell division suggest common mechanisms between kingdoms

Behaviour of the NE (nuclear envelope) during open mitosis has been explored extensively in metazoans, but lack of native markers has limited similar investigations in plants. In the present study, carried out using living synchronized tobacco BY-2 suspension cultures, the non-functional NE marker LBR (lamin B receptor)-GFP (green fluorescent protein) and two native, functional NE proteins, AtSUN1 [Arapidopsis thaliana SUN (Sad1/UNC84) 1] and AtSUN2, we provide evidence that the ER (endoplasmic reticulum)-retention theory for NE membranes is applicable in plants. We also observe two apparently unique plant features: location of the NE-membrane components in close proximity to chromatin throughout division, and spatially distinct reformation of the NE commencing at the chromatin surface facing the spindle poles and concluding at the surface facing the cell plate. Mobility of the proteins was investigated in the interphase NE, during NE breakdown and reformation, in the spindle membranes and the cell plate. A role for AtSUN2 in nuclear envelope breakdown is suggested.     

Nuclear envelope dynamics during mammalian spermatogenesis: new insights on male fertility

The production of highly specialized spermatozoa from undifferentiated spermatogonia is a strictly organized and programmed process requiring extensive restructuring of the entire cell. One of the most remarkable cellular transformations accompanying the various phases of spermatogenesis is the profound remodelling of the nuclear architecture, in which the nuclear envelope (NE) seems to be crucially involved. In recent years, several proteins from the distinct layers forming the NE (i.e. the inner and outer nuclear membranes as well as the nuclear lamina) have been associated with meiosis and/or spermiogenesis in different mammalian species. Among these are A‐ and B‐type lamins, Dpy‐19‐like protein 2 (DPY19L2), lamin B receptor (LBR), lamina‐associated polypeptide 1 (LAP1), LAP2/emerin/MAN1 (LEM) domain‐containing proteins, spermatogenesis‐associated 46 (SPATA46) and diverse elements of the linker of nucleoskeleton and cytoskeleton (LINC) complex, namely Sad‐1/UNC‐84 homology (SUN) and Klarsicht/ANC‐1/Syne‐1 homology (KASH) domain‐containing proteins. Herein, we summarize the current state of the art on the cellular and subcellular distribution of NE proteins expressed during mammalian spermatogenesis, and discuss the latest research developments regarding their testis‐specific functions. This review provides a comprehensive and innovative overview of the NE network as a regulatory platform and as an essential determinant of efficient meiotic chromosome recombination as well as spermiogenesis‐associated nuclear remodelling and differentiation in mammalian male germline cells. Thus, this review provides important novel insights on the biological relevance of NE proteins for male fertility.     

Nuclear envelope dynamics in oocytes: from germinal vesicle breakdown to mitosis

We have recently gained new insight into the mechanisms involved in nuclear envelope breakdown, the irreversible step that commits a cell to the M phase. Results from mammalian cell and starfish oocyte studies suggest that mechanical forces of the cytoskeleton, as well as biochemical disassembly of nuclear envelope protein complexes, play important roles in this process.     

Nuclear envelope assembly after mitosis

In higher eukaryotes, the entire nucleus disassembles during prometaphase of the cell cycle and later reassembles around daughter chromosomes. Remarkably, the complex events that occur to create a functional nucleus in vivo can be duplicated in vitro by using cell-free extracts. Current experiments are aimed at understanding the molecular mechanisms of assembly and disassembly of the nuclear pore complexes and nuclear membranes, and the functional roles of four identified inner membrane proteins, two of which bind to both chromatin and the nuclear lamina.     

Nuclear envelope remodelling during mitosis

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.     

Nuclear envelope: nuclear pore complexity

A new study shows that the filamentous fungus, Aspergillus nidulans, which has a closed mitosis, does not maintain a continuous permeability barrier during mitosis. This work challenges current views of the differences between closed and open mitosis and has implications for understanding mitotic specific changes in the nuclear pore complex and Ran GTPase system in lower eukaryotes.     

Nuclear envelope lipids request border surveillance

In this issue, Thaller et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202004222) explore how the ESCRT protein Chm7 is recruited to sites of defective nuclear pore assembly. They show that a lipid, phosphatidic acid, is enriched at pathological nuclear envelope herniations, where it promotes Chm7 recruitment for membrane surveillance and repair.

The membranes of the nucleus form a protective boundary around the DNA, while nuclear pore complexes (NPCs) embedded in these membranes act as control gates, deciding what can pass (1). Maintaining the integrity of this boundary, called the nuclear envelope (NE), is essential for cell survival. Complications can arise if the NE or its pores become disrupted. Cells employ a sophisticated surveillance system that can rapidly recognize and fix any damage inflicted on the NE. How this damage is located and what activates its repair is still poorly understood.The integrity of the NE is compromised in a variety of conditions, including neurodegenerative diseases like amyotrophic lateral sclerosis and frontotemporal degeneration. An age-related decline in NE/NPC function has been observed (2). Moreover, a key feature of early onset dystonia, a disease that causes muscle spasms, is NE herniations that originate from NPC-like structures. Herniations are frequently observed in yeast cells with defects in NPC biogenesis. It is therefore thought that herniations result from defective NPC assembly.Embedding new NPCs into the NE is not trivial. Interphase NPC assembly likely occurs through an inside-out evagination of the inner nuclear membrane (INM) followed by a membrane fusion with the outer nuclear membrane (3). This process creates holes in the NE and poses a threat to NE integrity if not properly executed. Protection comes from an ESCRT-dependent surveillance system that is recruited by NE disruption or defective NPC assembly (4). In yeast, two key players are the ESCRT (endosomal sorting complexes required for transport) protein Chm7 and the LEM (LAP2-emerin-MAN1) domain protein Heh1. Heh1 and Chm7 are normally segregated to opposite sides of the NE. However, if the NE is damaged, Heh1 and Chm7 come in contact (5). Heh1 activates Chm7, which can then repair the damage to the NE by closing and sealing any gaps. Active Chm7/Heh1 may form a polymer similar to that of the human CHMP7–LEM2 complex (6). Several domains of Chm7 (the orthologue of mammalian CHMP7) contribute to its cellular localization and activity when NE surveillance is triggered. In this issue, Thaller et al. (7) shed light on the determinants controlling the timely recruitment of Chm7 to NE defect sites.The researchers characterized a conserved hydrophobic region of Chm7, which is predicted to form an amphipathic helix. Amphipathic helices are found in numerous proteins and are defined by the separation of hydrophobic and polar residues between the two faces of the helix. This separation enables these helices to bind at apolar/polar interfaces such as the lipid surfaces of cell organelles. Depending on the nature and distribution of the hydrophobic and polar residues as well as the length of the helix, amphipathic helices can be tuned into versatile molecular tools and for example, deform lipid bilayers, recognize specific lipids or sense membrane curvature.Chm7 is normally located in the cytosol but can be forced into the nucleus and into interaction with Heh1 by inhibiting its nuclear export. Interestingly, Thaller et al. observed that mutating the hydrophobic face of the amphipathic helix inhibited Chm7 recruitment to the INM, where Heh1 is located, suggesting the existence of a previously unknown membrane-binding activity in Chm7. The authors employed in vitro assays using liposomes to elucidate what attracts Chm7 to membranes. Chm7 showed enhanced liposome binding when the concentration of phosphatidic acid (PA) was increased, suggesting that Chm7 binds directly to PA-rich lipid bilayers. Chm7 preferred liposomes with a small diameter and, hence, high curvature over larger liposomes with an essentially flat surface. Given that Chm7 bound to PA-rich membranes in vitro, the authors then analyzed how altered cellular PA levels affect Chm7 distribution. Interestingly, elevated PA levels led to a redistribution of Chm7 from the cytoplasm to the membranes of the NE and the endoplasmic reticulum, which required Chm7’s amphipathic helix. Since increased PA levels can disrupt NE integrity, this raised the interesting possibility that Chm7 directly senses an instability in nuclear membranes via PA.A key question that follows is whether PA indeed accumulates at sites of Chm7 activity. To probe INM PA levels, Thaller et al. took advantage of an INM-specific PA biosensor (8). This sensor comprises an amphipathic helix that specifically binds to the phosphate moiety of PA by a three-finger grip of basic residues. The PA sensor was nucleoplasmic under normal growth conditions, indicating low PA levels at the INM. In contrast, the PA sensor relocalized to distinct INM foci when a constitutively active variant of Chm7 was expressed and colocalized with Chm7 at these foci. Thus, this hyperactive variant of Chm7 appears to affect INM PA levels, either by locally altering PA metabolism or through direct recruitment of PA, suggesting some positive feedback in PA-mediated Chm7 recruitment to membranes.Wild-type Chm7 is known to accumulate at the NE when de novo NPC assembly is perturbed. A hallmark of several NPC assembly mutants is the occurrence of NE herniations. These aberrant structures likely arise as a consequence of impaired NE remodeling. Notably, the PA sensor accumulated in distinct foci along the nuclear periphery in a nuclear pore mutant that exhibits such herniations. Thaller et al. found that Chm7 was dispensable for this PA sensor accumulation. This suggested that a local increase in PA concentration likely precedes Chm7 recruitment under conditions of NPC misassembly. Finally, through a series of elegant correlative light and electron microscopy experiments, the authors offered compelling ultrastructural evidence that the NPC misassembly-associated NE herniations can indeed recruit the PA sensor, indicative of high local PA concentrations at these sites (7).Thaller et al. propose a model in which a specific lipid, PA, can request NE surveillance by Chm7. PA accumulates at NE herniations, which are indicative of NE damage, and recruits Chm7 via its PA-sensing amphipathic helix. Chm7 then binds to Heh1, which reinforces the membrane recruitment and activates Chm7.This study is conceptually important and offers a lot of food for thought. First, because it adds a missing link—a lipid—to the complex hierarchy of signals that lead to NE surveillance and repair. Second, because it raises the question of which specific lipids surround NPCs in health and disease and how these lipids become locally enriched. And more generally, because it gives fresh insight into the poorly understood connection between lipid metabolism and the functional architecture of the nucleus (8, 9). Notably, Opi1, from which the PA sensor is derived, not only senses PA but also senses the lipid-packing density of a membrane, which is related to its lipid saturation state (10). Hence, the SOS call from PA that Thaller et al. have now detected may just be the tip of the iceberg, with other lipid surveillance codes remaining to be discovered.     

Nuclear envelope disease and chromatin organization

The fifth U.K. meeting on nuclear envelope disease and chromatin brought together international experts from across the field of nuclear envelope biology to discuss the advancements in a class of tissue-specific degenerative diseases called the laminopathies. Clinically, these range from relatively mild fat-wasting disorders to the severe premature aging condition known as Hutchinson-Gilford progeria syndrome. Since the first association of the nuclear envelope with human inherited disease in 1994, there has been an exponential increase in an unexpected variety of functions associated with nuclear envelope proteins, ranging from mechanical support and nucleocytoskeletal connections to regulation of chromatin organization and gene expression. This Biochemical Society Focused Meeting reinforced the functional complexity of nuclear-associated diseases, revealed new avenues to be investigated and highlighted the signalling pathways suitable as therapeutic targets.     

Nuclear envelope defects in muscular dystrophy

Muscular dystrophies are a heterogeneous group of disorders linked to defects in 20-30 different genes. Mutations in the genes encoding a pair of nuclear envelope proteins, emerin and lamin A/C, have been shown to cause the X-linked and autosomal forms respectively of Emery-Dreifuss muscular dystrophy. A third form of muscular dystrophy, limb girdle muscular dystrophy 1b, has also been linked to mutations in the lamin A/C gene. Given that these two genes are ubiquitously expressed, a major goal is to determine how they can be associated with tissue specific diseases. Recent results suggest that lamin A/C and emerin contribute to the maintenance of nuclear envelope structure and at the same time may modulate the expression patterns of certain mechanosensitive and stress induced genes. Both emerin and lamin A/C may play an important role in the response of cells to mechanical stress and in this way may help to maintain muscle cell integrity.