Diseases of the Nuclear Envelope

In the past decade, a wide range of fascinating monogenic diseases have been linked to mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins of the nuclear envelope. These diseases include dilated cardiomyopathy with variable muscular dystrophy, Dunnigan-type familial partial lipodystrophy, a Charcot-Marie-Tooth type 2 disease, mandibuloacral dysplasia, and Hutchinson-Gilford progeria syndrome. Several diseases are also caused by mutations in genes encoding B-type lamins and proteins that associate with the nuclear lamina. Studies of these so-called laminopathies or nuclear envelopathies, some of which phenocopy common human disorders, are providing clues about functions of the nuclear envelope and insights into disease pathogenesis and human aging.Mutations in LMNA encoding the A-type lamins cause a group of human disorders often collectively called laminopathies. The major A-type lamins, lamin A and lamin C, arise by alternative splicing of the LMNA pre-mRNA and are expressed in virtually all differentiated somatic cells. Although the A-type lamins are widely expressed, LMNA mutations are responsible for at least a dozen different clinically defined disorders with tissue-selective abnormalities. Mutations in genes encoding B-type lamins and lamin-associated proteins, most of which are similarly expressed in almost all somatic cells, also cause tissue-selective diseases.Research on the laminopathies has provided novel clues about nuclear envelope function. Recent studies have begun to shed light on how alterations in the nuclear envelope could explain disease pathogenesis. Along with basic research on nuclear structure, the nuclear lamins, and lamina-associated proteins, clinical research on the laminopathies will contribute to a complete understanding of the functions of the nuclear envelope in normal physiology and in human pathology.     

Regulatory Roles of the Nuclear Envelope

Roles of the nuclear envelope are considered in the regulation of nuclear protein import, ribonucleoprotein export, and coupling of DNA replication to the cell cycle. First, evidence is discussed that indicates that neutral and acidic amino acids can be important in nuclear localization signals as well as the widely acknowledged basic amino acids. Second, the recognition of nuclear localization signals by their receptor “importin” is discussed, focusing on the different roles of the two subunits of importin. Third, a role for the α subunit of importin in RNP export is considered together with the question of how the direction of traffic through nuclear pores is determined. The final part of this article considers evidence that the nuclear membrane prevents reinitiation of DNA replication in Xenopus eggs, by excluding a “licensing factor” that is essential for DNA replication. Replication licensing in Xenopus appears to involve several proteins including the MCM (minichromosome maintenance) complex and ORC, the origin recognition complex, which must bind before the MCM complex can bind to chromatin.     

Baculovirus Actin-Based Motility Drives Nuclear Envelope Disruption and Nuclear Egress

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Properties of Large-Conductance Cationic Channels in the Neuronal Nuclear Envelope

The genetic apparatus of the nucleus of eukaryotic cells is surrounded by the nuclear envelope containing ion channels with different biophysical properties. The channels responsible for calcium release from the nuclear envelope into the intranuclear space have attracted special attention. As we found, a great number of large-conductance ion channels selective with respect to monovalent cations and impermeable for bivalent cations are present in all membranes where inositol trisphosphate receptors are expressed. We showed that channels of this type are insensitive to blockers of potassium channels and, according to their properties, cannot be attributed to one well-known type of ion channels or another. In addition, since their blockers remain unknown, these channels at present cannot be isolated and cloned. Channels of this type deserve further investigation; we hypothesize that large-conductance cationic channels can be involved in the control of the duration of Ca²⁺ release from the calcium store.     

The Novel Nuclear Envelope Protein KAKU4 Modulates Nuclear Morphology in Arabidopsis

In animals, the nuclear lamina is a fibrillar meshwork on the inner surface of the nuclear envelope, composed of coiled-coil lamin proteins and lamin binding membrane proteins. Plants also have a meshwork on the inner surface of the nuclear envelope, but little is known about its composition other than the presence of members of the CROWDED NUCLEI (CRWN) protein family, possible plant lamin analogs. Here, we describe a candidate lamina component, based on two Arabidopsis thaliana mutants (kaku2 and kaku4) with aberrant nuclear morphology. The responsible gene in kaku2 encodes CRWN1, and the responsible gene in kaku4 encodes a plant-specific protein of unknown function (KAKU4) that physically interacts with CRWN1 and its homolog CRWN4. Immunogold labeling revealed that KAKU4 localizes at the inner nuclear membrane. KAKU4 deforms the nuclear envelope in a dose-dependent manner, in association with nuclear membrane invagination and stack formation. The KAKU4-dependent nuclear envelope deformation was enhanced by overaccumulation of CRWN1, although KAKU4 can deform the nuclear envelope even in the absence of CRWN1 and/or CRWN4. Together, these results suggest that plants have evolved a unique lamina-like structure to modulate nuclear shape and size.     

高等植物中LINC复合体的研究进展（英文）

在高等植物中发现了含SUN (Sad1/UNC84)结构域的蛋白质At SUN1和At SUN2后,人们发现了核骨架和细胞骨架复合物的连接体LINC (linker of nucleoskeleton and cytoskeleton)。虽然一些关键成分和相互作用还不清楚,但SUN结构域蛋白质在所有植物中高度保守。植物SUN结构域蛋白和新发现的KASH (Klarsicht/Anc/Syne-1)同源蛋白是构成植物LINC复合体的关键成分。WIPs (WPP域相互作用蛋白质)锚定在外核被膜(outer nuclear envelope,ONE)上。植物KASH蛋白的C末端锚定在植物核周质上。与opisthokonts中的PPPX相比,WIPs在C端具有高度保守的X-VPT序列。本文综述了近十年来连接植物内核膜和外核膜的相关植物核膜蛋白的研究进展。     

Genetic Analysis of Macromolecular Transport across the Nuclear Envelope

Numerous factors that promote movement of macromolecules in and out of the nucleus have now been identified. These include both soluble cytoplasmic and nucleoplasmic proteins and proteins of the nuclear pore complex (NPC). Genetic analyses of the nuclear transport process in the model organism, the budding yeastSaccharomyces cerevisiae,have revealed remarkable conservation of all of these factors. In addition, important clues as to how these factors promote the unique bidirectional movement across the NPC have emerged from studies of yeast. We summarize the characterization and genetic interactions of the soluble transport factors and present data to illustrate how genetic experiments can be used to further define the import and export pathways.     

Nuclear Envelope: Nanoarray Responsive to Aldosterone

Signalling between cytosol and nucleus is mediated by nuclear pores. These supramolecular complexes represent intelligent nanomachines regulated by a wide spectrum of factors. Among them, steroid hormones specifically interact with the pores and thus modify ion conductivity and macromolecule permeability of the nuclear envelope. In response to aldosterone the pores undergo dramatic changes in conformation, changes that depend on the nature of the transported cargo. Such changes can be imaged at the nanometer scale by using atomic force microscopy. Furthermore, steroid-induced macromolecule transport across the nuclear envelope causes osmotic water movements and nuclear swelling. Drugs that interact with intracellular steroid receptors (spironolactone) or with plasma membrane sodium channels (amiloride) inhibit swelling. Steroid hormone action is blocked when nuclear volume changes are prevented. This is shown in frog oocytes and human endothelial cells. In conclusion, nuclear pores serve as steroid-sensitive gates that determine nuclear activity.     

Further Observations on the Nuclear Envelope of the Animal Cell

The term pore complex is proposed for approximately cylindrical formations which are observed with the electron microscope to penetrate the nuclear envelope of cells. Cross-sections of the pore complex are somewhat annular in shape, but differ in appearance depending upon the level of the cross-section with respect to the nuclear surface. An explanation is offered for the apparent discrepancy between the width of pores in sections perpendicular to the nuclear envelope and the width of cross-sections of the pore complex in tangential sections. Channels associated with the pore complex extend deep into the nucleus. Although crescents and spirals of ribonucleoprotein particles were often seen in the immediate vicinity of the outer nuclear membrane, direct association with the pore complex was not observed. Many examples were found of pores that were not covered by a continuous membrane although the possibility of such a covering in some cases is not precluded.     

SEPT12/SPAG4/LAMINB1 Complexes Are Required for Maintaining the Integrity of the Nuclear Envelope in Postmeiotic Male Germ Cells

Male infertility affects approximately 50% of all infertile couples. The male-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile or immature sperm, and sperm with structural defects such as those caused by premature chromosomal condensation and DNA damage. Our previous studies based on a knockout mice model indicated that SEPT12 proteins are critical for the terminal morphological formation of sperm. SEPT12 mutations in men result in teratozospermia and oligozospermia. In addition, the spermatozoa exhibit morphological defects of the head and tail, premature chromosomal condensation, and nuclear damage. However, the molecular functions of SEPT12 during spermatogenesis remain unclear. To determine the molecular functions of SEPT12, we applied a yeast 2-hybrid system to identify SEPT12 interactors. Seven proteins that interact with SEPT12 were identified: SEPT family proteins (SEPT4 and SEPT6), nuclear or nuclear membrane proteins (protamine 2, sperm-associated antigen 4, and NDC1 transmembrane nucleoproine), and sperm-related structural proteins (pericentriolar material 1 and obscurin-like 1). Sperm-associated antigen 4 (SPAG4; also known as SUN4) belongs to the SUN family of proteins and acts as a linker protein between nucleoskeleton and cytoskeleton proteins and localizes in the nuclear membrane. We determined that SEPT12 interacts with SPAG4 in a male germ cell line through coimmunoprecipitation. During human spermiogenesis, SEPT12 is colocalized with SPAG4 near the nuclear periphery in round spermatids and in the centrosome region in elongating spermatids. Furthermore, we observed that SEPT12/SPAG4/LAMINB1 formed complexes and were coexpressed in the nuclear periphery of round spermatids. In addition, mutated SEPT12, which was screened from an infertile man, affected the integration of these nuclear envelope complexes through coimmunoprecipitation. This was the first study that suggested that SEPT proteins link to the SUN/LAMIN complexes during the formation of nuclear envelopes and are involved in the development of postmeiotic germ cells.     

Identification of Protein Transport Complexes in the Chloroplastic Envelope Membranes via Chemical Cross-Linking

Transport of cytoplasmically synthesized proteins into chloroplasts uses an import machinery present in the envelope membranes. To identify the components of this machinery and to begin to examine how these components interact during transport, chemical cross-linking was performed on intact chloroplasts containing precursor proteins trapped at a particular stage of transport by ATP limitation. Large crosslinked complexes were observed using three different reversible homobifunctional cross-linkers. Three outer envelope membrane proteins (OEP86, OEP75, and OEP34) and one inner envelope membrane protein (IEP110), previously reported to be involved in protein import, were identified as components of these complexes. In addition to these membrane proteins, a stromal member of the hsp100 family, ClpC, was also present in the complexes. We propose that ClpC functions as a molecular chaperone, cooperating with other components to accomplish the transport of precursor proteins into chloroplasts. We also propose that each envelope membrane contains distinct translocation complexes and that a portion of these interact to form contact sites even in the absence of precursor proteins.     

A Single Herpesvirus Protein Can Mediate Vesicle Formation in the Nuclear Envelope

Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission.     

Calcium Channels in the Nuclear Envelope of Pyramidal Neurons of the Rat Hippocampus

As is known, an increase in the concentration of Са²⁺ in the nuclei of nerve cells leads to activation of genes responsible for the formation of long-lasting postsynaptic changes; mechanisms of memory and learning are based on such changes. The pathways necessary for the entry of calcium into the nuclei of hippocampal pyramidal neurons remained unstudied. Using a patch-clamp technique, we studied what types of calcium channels exist in the membranes of isolated nuclei of pyramidal neurons of the hippocampal СА1 area. In the inner nuclear membrane of these cells, we, for the first time, found inositol trisphosphate receptors (IP₃Rs) activated by inositol trisphosphate applied in the concentration of ≥0.1 μM. The conductivity of single channels of such receptors was, on average, 366 pS; these channels were permeable for both monovalent and bivalent cations. Our data indicate that the nuclear envelope of pyramidal neurons of the hippocampal СА1 area can play the role of the calcium store from which Са²⁺ enter the cell nucleus directly. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 288–292, July–August, 2008.