Nuclear acceptor sites for androgen-receptor complexes in seminal-vesicle epithelium.

An assay method in vitro was developed and applied to quantify acceptor binding of steroid-receptor complexes in nuclei from isolated epithelium of guinea-pig seminal vesicle. Steroid-receptor complex prepared from 1-day-castrated animals was incubated with purified nuclei from 1-28 day-castrated animals in a medium containing 0.15 M-KCl. Free and bound steroid-receptor complexes were measured and the data were submitted to Scatchard analysis. With nuclei from 1-day-castrated animals the Kd for binding of cytosolic [3H]dihydrotestosterone-receptor complexes was found to be 0.83 X 10(-10) M and the capacity for binding was 0.35 pmol/mg of nuclear DNA. Scatchard analysis consistently disclosed only a single line of constant slope and gave the same kinetic constants for nuclei obtained from animals castrated up to 28 days before assay. Administration of 2 mg of dihydrotestosterone, 3 alpha-androstanediol or androsterone or 100 microgram of oestradiol-17 beta 1 h before killing of the 1-day-castrated animals that provided the nuclei resulted in a significant decrease in nuclear acceptor binding of the steroid-receptor complex compared with untreated animals. Thus our assay method disclosed nuclear acceptor sites that may be involved in responses to androgens (and oestrogens) in vivo. We conclude that there is a class of nuclear accept or sites of high affinity and limited capacity that may be occupied by steroid-receptor complexes in vivo.     

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 dynamics.

The nuclear envelope (NE) provides a semi permeable barrier between the nucleus and cytoplasm and plays a central role in the regulation of macromolecular trafficking between these two compartments. In addition to this transport function, the NE is a key determinant of interphase nuclear architecture. Defects in NE proteins such as A-type lamins and the inner nuclear membrane protein, emerin, result in several human diseases that include cardiac and skeletal myopathies as well as lipodystrophy. Certain disease-linked A-type lamin defects cause profound changes in nuclear organization such as loss of peripheral heterochromatin and redistribution of other nuclear envelope components. While clearly essential in maintenance of nuclear integrity, the NE is a highly dynamic organelle. In interphase it is constantly remodeled to accommodate nuclear growth. During mitosis it must be completely dispersed so that the condensed chromosomes may gain access to the mitotic spindle. Upon completion of mitosis, dispersed NE components are reutilized in the assembly of nuclei within each daughter cell. These complex NE rearrangements are under precise temporal and spatial control and involve interactions with microtubules, chromatin, and a variety of cell-cycle regulatory molecules.     

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 breakdown proceeds by microtubule-induced tearing of the lamina.

The mechanism of nuclear envelope breakdown (NEBD) was investigated in live cells. Early spindle microtubules caused folds and invaginations in the NE up to one hour prior to NEBD, creating mechanical tension in the nuclear lamina. The first gap in the NE appeared before lamin B depolymerization, at the site of maximal tension, by a tearing mechanism. Gap formation relaxed this tension and dramatically accelerated the rate of chromosome condensation. The hole produced in the NE then rapidly expanded over the nuclear surface. NE fragments remaining on chromosomes were removed toward the centrosomes in a microtubule-dependent manner, suggesting a mechanism mediated by a minus-end-directed motor.     

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 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.     

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 influences on cell-cycle progression

The nuclear envelope is a complex double membrane system that serves as a dynamic interface between the nuclear and cytoplasmic compartments. Among its many roles is to provide an anchor for gene regulatory proteins on its nucleoplasmic surface and for the cytoskeleton on its cytoplasmic surface. Both sets of anchors are proteins called NETs (nuclear envelope transmembrane proteins), embedded respectively in the inner or outer nuclear membranes. Several lines of evidence indicate that the nuclear envelope contributes to cell-cycle regulation. These contributions come from both inner and outer nuclear membrane NETs and appear to operate through several distinct mechanisms ranging from sequestration of gene-regulatory proteins to activating kinase cascades.