高等动物细胞核膜和核纤层结构、功能及动态变化调控机制

细胞核是真核细胞中最大的细胞器．高等动物细胞核主要由双层核膜、核孔复合体、核纤层、染色质和核仁等组成．在细胞有丝分裂期,细胞核呈现去装配和再装配等动态变化．在细胞分裂间期,核膜、核孔复合体和核纤层构成细胞核的外周结构,为遗传物质在染色质和核仁中的代谢提供了一个相对稳定的环境,同时调控细胞核内外的物质转运,在细胞增殖、分化、个体发育和细胞衰老等许多方面发挥着重要作用．本文主要对高等动物细胞核膜和核纤层结构、功能及动态变化调控机制等方面的研究进展进行简要综述．     

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6–8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6–8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.     

Dynamic Assembly of Brambleberry Mediates Nuclear Envelope Fusion during Early Development

To accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, mitotic intermediates wherein individual chromatin masses are surrounded by nuclear envelope; the karyomeres then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion, resulting in formation of multiple micronuclei. As karyomeres form, Brambleberry protein localizes to the nuclear envelope, with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. brambleberry corresponds to an unannotated gene with similarity to Kar5p, a protein that participates in nuclear fusion in yeast. We also demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. Our studies provide insight into the machinery required for karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres.     

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.     

Redundancy of IL-1 Isoform Signaling and Its Implications for Arterial Remodeling

Aims

Mice deficient in IL-1 receptor 1 (hence unresponsive to both IL-1 isoforms α and β) have impaired expansive arterial remodeling due to diminished expression of matrix-degrading enzymes, especially MMP-3. Emergence of IL-1 as a target in cardiovascular disease prompted the investigation of the redundancy of IL-1α and IL-1β in the induction of MMP-3 and other matrix-remodeling enzymes in human cells.

Methods and Results

Human primary vascular smooth muscle cells (VSMCs) and carotid endarterectomy specimens were stimulated with equimolar concentrations of IL-1α or IL-1β and analyzed protease expression by immunoblot and ELISA. Either IL-1α or IL-1β increased the expression of pro-MMP-3 in VSMCs, facilitated VSMC migration through Matrigel, and induced MMP-3 production in specimens from atheromatous plaques. VSMCs also secreted MMP-1 and Cathepsin S (CatS) upon stimulation with IL-1α or IL-1β. IL-1 isoforms similarly increased MMP-1 and MMP-9 expression in carotid endarterectomy specimens. We examined the expression of MMP-3 and IL-1 isoforms by immunostaining of carotid atheromata, calculated the % positive areas, and tested associations by linear regression. MMP-3 colocalized with IL-1 isoforms in atheromata. MMP-3+ area in plaques positively associated with IL-1α+ (R2 = 0.61, P<0.001) and with IL-1β + areas (R2 = 0.68, P<0.001). MMP-3+ area within atheroma also associated with CD68+ area, but not with α-smooth muscle actin area.

Conclusions

Either IL-1α or IL-1β can induce the expression of enzymes implicated in remodeling of the arterial extracellular matrix, and facilitate human VSMC migration in vitro. Human atheromata contain both IL-1 isoforms in association with immunoreactive MMP-3. This redundancy of IL-1 isoforms suggests that selective blocking of one IL-1 isoform should not impair expansive arterial remodeling, a finding with important clinical implications for therapeutic targeting of IL-1 in atherosclerosis.     

Functional Dissection of YA, an Essential, Developmentally Regulated Nuclear Lamina Protein in Drosophila melanogaster

The Drosophila YA protein is a nuclear lamina component whose function is essential to initiate embryonic development. To identify regions of YA required for its action in its normal cellular context, we made targeted mutations in the YA protein and tested their consequences in flies and embryos in vivo. We found that critical amino acids are distributed along the length of the YA molecule, with functionally important regions including the N- and the C-terminal ends, the cysteine residues in YA’s two potential zinc fingers, a serine/threonine-rich region, and a potential maturation-promoting factor or mitogen-activated protein kinase phosphorylation target site, ITPIR. In addition, several Ya mutations showed intragenic complementation, with N-terminal mutations complementing C-terminal mutations, suggesting that YA proteins interact with one another. In support of this interaction, we demonstrated by immunoprecipitation that YA molecules are present in complexes with each other. Finally, we showed that the C-terminal 179 amino acids of YA are necessary to target, or retain, YA in the nuclear envelope.     

Structural and Functional Brain Remodeling during Pregnancy with Diffusion Tensor MRI and Resting-State Functional MRI

Although pregnancy-induced hormonal changes have been shown to alter the brain at the neuronal level, the exact effects of pregnancy on brain at the tissue level remain unclear. In this study, diffusion tensor imaging (DTI) and resting-state functional MRI (rsfMRI) were employed to investigate and document the effects of pregnancy on the structure and function of the brain tissues. Fifteen Sprague-Dawley female rats were longitudinally studied at three days before mating (baseline) and seventeen days after mating (G17). G17 is equivalent to the early stage of the third trimester in humans. Seven age-matched nulliparous female rats served as non-pregnant controls and were scanned at the same time-points. For DTI, diffusivity was found to generally increase in the whole brain during pregnancy, indicating structural changes at microscopic levels that facilitated water molecular movement. Regionally, mean diffusivity increased more pronouncedly in the dorsal hippocampus while fractional anisotropy in the dorsal dentate gyrus increased significantly during pregnancy. For rsfMRI, bilateral functional connectivity in the hippocampus increased significantly during pregnancy. Moreover, fractional anisotropy increase in the dentate gyrus appeared to correlate with the bilateral functional connectivity increase in the hippocampus. These findings revealed tissue structural modifications in the whole brain during pregnancy, and that the hippocampus was structurally and functionally remodeled in a more marked manner.     

Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser²²). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser²² is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.     

Integral Membrane Proteins of the Nuclear Envelope Are Dispersed throughout the Endoplasmic Reticulum during Mitosis

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC₆ and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.     

Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4–8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight into the cytogenetic mechanisms underlying their formation and the consequences for human fertility.     

Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.     

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.