Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development.

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.     

Remodeling the sperm nucleus into a male pronucleus at fertilization

After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.     

Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin.

Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin LIII did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.     

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

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.     

Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components

At the end of mitosis membrane vesicles are targeted to the surface of chromatin and fuse to form a continuous nuclear envelope. To investigate the molecular mechanisms underlying these steps in nuclear envelope assembly, we have developed a defined cell-free system in which the binding and fusion steps in nuclear envelope assembly can be examined separately. We have found that extensively boiled Xenopus egg extracts efficiently promote the decondensation of demembranated Xenopus sperm chromatin. When isolated membranes are added to this decondensed chromatin a specific subfraction of membrane vesicles (approximately 70 nM in diameter) bind to the chromatin, but these vesicles do not fuse to each other. Vesicle binding is independent of ATP and insensitive to N-ethylmalamide. Quantitative analysis of these sites by EM suggests that there is at least one vesicle binding site per 100 kb of chromosomal DNA. We show by tryptic digestion that vesicle-chromatin association requires proteins on both the vesicle and on the chromatin. In addition, we show that the vesicles bound under these conditions will fuse into an intact nuclear envelope when incubated with the soluble fraction of a Xenopus egg nuclear assembly extract. With respect to vesicle fusion, we have found that vesicles prebound to chromatin will fuse to each other when ATP and GTP are present in the boiled extract. These results indicate that nuclear envelope assembly is mediated by a subset of approximately 70-nM-diam vesicles which bind to chromatin sites spaced 100 kb apart and that fusion of these vesicles is regulated by membrane-associated GTP-binding proteins.     

Nuclear membrane vesicle targeting to chromatin in a Drosophila embryo cell-free system.

A Drosophila cell-free system was used to characterize proteins that are required for targeting vesicles to chromatin and for fusion of vesicles to form nuclear envelopes. Treatment of vesicles with 1 M NaCl abolished their ability to bind to chromatin. Binding of salt-treated vesicles to chromatin could be restored by adding the dialyzed salt extract. Lamin Dm is one of the peripheral proteins whose activity was required, since supplying interphase lamin isoforms Dm1, and Dm2 to the assembly extract restored binding. As opposed to the findings in Xenopus, okadaic acid had no effect on vesicle binding. Trypsin digestion of the salt-stripped vesicles eliminated their association with chromatin even in the presence of the dialyzed salt extract. One vesicles attached to chromatin surface, fusion events took place were found to be sensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). These chromatin-attached vesicles contained lamin Dm and otefin but not gp210. Thus, these results show that in Drosophila there are two populations of nuclear vesicles. The population that interacts first with chromatin contains lamin and otefin and requires both peripheral and integral membrane proteins, whereas fusion of vesicles requires GTPase activity.     

Ran及其结合与调控蛋白在核膜装配过程中的调控作用

核膜在细胞周期中呈现高度的动态性：在细胞分裂的前中期,核膜崩解并分散到细胞质中;在细胞分裂的后期,核膜开始在染色体的表面重新装配,最终形成完整的核膜结构。近期的研究发现,Ran GTP酶、物质转运蛋白importinβ、内层核膜蛋白LBR（lamin B receptor）以及核孔复合体蛋白nucleoporins在核膜重建的过程中起关键性调控作用,并受到细胞周期调控因子p34cdc2激酶的调节。LBR是一个八次跨膜的膜蛋白,主要定位于内层核膜。在细胞分裂的早期,随着核膜崩解,LBR与核膜崩解而生成的小膜泡一起分散到细胞质中;在细胞分裂的后期,通过LBR与importinβ相互结合,含有LBR的膜泡被importinβ携带至染色质的表面参与核膜重建。目前已知p34cdc2激酶对LBR与importinβ介导的核膜重建起重要调控作用。Nucleoporins是核孔复合体主要组分。随核膜崩解,核孔复合体解聚成nucleoporins,分散到细胞质中,或结合到其他亚细胞成分上。细胞分裂后期,核孔复合体伴随核膜装配而组装。     

Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery

Antibodies directed against nuclear envelope lamin proteins have been used in conjunction with three-dimensional light and electron microscope methodologies to determine the spatial organization of lamins in diploid interphase nuclei and to relate this organization to the positions of chromatin in the nuclear periphery. Using Drosophila early embryos, Drosophila Kc cells, and human HeLa cells, it is qualitatively and quantitatively observed that lamins are organized as a highly discontinuous, apparently fibrillar network that leaves large voids in the nuclear periphery containing little or no lamin. Using fluorescence microscopy to compare and quantitate the relationship between chromatin and the lamin network, it is found that although there is a strong tendency for the most peripheral chromatin to be positioned directly underneath a lamin fiber, only a small fraction of the chromatin in the nuclear periphery is sufficiently close to a lamin fiber to possibly be in direct contact.     

Nuclear reconstitution of plant (Orychophragmus violaceus) demembranated sperm in cell-free extracts from animal (Xenopus laevis) eggs

Cell-free extracts from animal Xenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. When incubated with Xenopus egg extracts, the demembranated sperm began to swell and then gradually decondensed. The assembly of the nuclear envelope in the reconstituted nuclei was visualized by means of electron microscopy and fluorescence microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nuclei, with a double membrane, was similar to that of nuclei after fertilization. The electron micrograph of the whole-mount prepared nuclear matrix--lamina showed the reconstituted nucleus to be filled with a dense network.     

Transmembrane proteins are not required for early stages of nuclear envelope assembly

All identified membrane fusion proteins are transmembrane proteins. In the present study, we explored the post-mitotic reassembly of the NE (nuclear envelope). The proteins that drive membrane rearrangements in NE assembly remain unknown. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on nuclear reconstitution in a cell-free system. Mixing of soluble interphase cytosolic extract and MV (membrane vesicles) from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced MV and cytosol with protein-free phosphatidylcholine LS (liposomes) that were pre-incubated with interphase cytosol. While later stages of NE assembly yielding functional nucleus did not proceed without integral proteins of MV, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus in contrast with post-fusion stages, fusion initiation in NE assembly, like membrane remodelling in budding and fission, does not require transmembrane proteins.     

Nuclear reconstitution in vitro: stages of assembly around protein-free DNA

We have developed a cell-free system derived from Xenopus eggs that reconstitutes nuclear structure around an added protein-free substrate (bacteriophage lambda DNA). Assembled nuclei are morphologically indistinguishable from normal eukaryotic nuclei: they are surrounded by a double membrane containing nuclear pores and are lined with a peripheral nuclear lamina. Nuclear assembly involves discrete intermediate steps, including nucleosome assembly, scaffold assembly, and nuclear membrane and lamina assembly, indicating that during reconstitution nuclear organization is assembled one level at a time. Topoisomerase II inhibitors block nuclear assembly. Lamin proteins and membrane vesicles bind to chromatin late in assembly, suggesting that these components do not interact with chromatin that is formed early in assembly. Reconstituted nuclei replicate their DNA; replication begins only after envelope formation has initiated, indicating that envelope attachment may be important for regulating replication.     

Stepwise reassembly of the nuclear envelope at the end of mitosis

The nuclear envelope consists of three distinct membrane domains: the outer membrane with the bound ribosomes, the inner membrane with the bound lamina, and the pore membrane with the bound pore complexes. Using biochemical and morphological methods, we observed that the nuclear membranes of HeLa cells undergoing mitosis are disassembled in a domain-specific manner, i.e., integral membrane proteins representing the inner nuclear membrane (the lamin B receptor) and the nuclear pore membrane (gp210) are segregated into different populations of mitotic vesicles. At the completion of mitosis, the inner nuclear membrane- derived vesicles associate with chromatin first, beginning in anaphase, whereas the pore membranes and the lamina assemble later, during telophase and cytokinesis. Our data suggest that the ordered reassembly of the nuclear envelope is triggered by the early attachment of inner nuclear membrane-derived vesicles to the chromatin.     

Detection of nuclear lamin B epitopes in oocyte nuclei from mice, sea urchins, and clams using a human autoimmune serum

Somatic nuclei typically contain two or three major proteins, the lamins A, B, and C or their antigenically related equivalents, interspersed between the chromatin and its attachment site, the inner nuclear membrane. The late oocyte nuclear envelopes of the previously investigated Xenopus and Spisula germinal vesicles, however, have no chromatin attached and only one lamin-like protein. Since mouse and sea urchin germinal vesicles have chromatin attached, we tested them for the possible presence of more than one lamin. In both species we found two different lamins incorporated in their nuclear envelope structure. One lamin is recognized by anti-lamin B and the other by anti-lamin AC antibodies. Spisula germinal vesicles were found to contain not only the nuclear envelope-bound lamin (clamin), but also a 65-kDa protein cross-reactive with anti-lamin B antibodies. This protein is present unattached to any structure and is apparently soluble. Our findings provide a possible explanation of the early presence of lamin B in pronuclei of mouse and sea urchin contrary to the late appearance of a lamin B equivalent in amphibian embryos. In Spisula, as in Xenopus, the presence of a lamin B equivalent could not be documented in the nuclear envelopes of early embryos, indicating that a separate lamin B equivalent is not essential for chromatin binding to the envelope in these species during early embryogenesis. The results also indicate that the nuclear complement of structural proteins might vary substantially in the same cell type of different species.     

利用非洲爪蟾精子染色质和卵提取物在体外重建细胞核

应用非洲爪蟾去膜精子染色质和卵提取物成功地进行了细胞核本外重建。当精子染色质加入卵提取物后,首先发生染色质去浓缩作用,染色质整体结构膨胀;膜泡在膨胀的染色质外周聚集并逐渐彼此融合成双层膜;核孔复合体以某种未知方式组装入双层膜而形成核膜结构,并逐渐完全覆盖膨大的染色质,最终形成典型的间期核结构。     

Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein

We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.     

GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.     

In vitro nuclear assembly with affinity-purified nuclear envelope precursor vesicle fractions, PV1 and PV2.

Nuclear envelope precursor vesicles were affinity purified from a Xenopus egg extract by a chromatin binding method. Vesicles bound to chromatin at 4 degrees C were dissociated with a high salt buffer and further fractionated into nuclear envelope precursor vesicle fractions 1 (PV1) and 2 (PV2) by differential centrifugation. PV1 contained larger vesicles. When chromatin was incubated in a Xenopus egg cytosol fraction supplemented with PV1, vesicles bound to chromatin, fused with each other, formed a bilayered nuclear envelope, and assembled into spherical small nuclei. However, the thus assembled nuclei did not grow to the normal size. Nuclear pore complexes were not found on the thus assembled nuclei. On the other hand, PV2 contained smaller vesicles. PV2 vesicles bound to chromatin, fused little with each other in the Xenopus egg cytosol fraction, and no nuclei were assembled. When PV1 supplemented with PV2 was used for the nuclear assembly reaction, the assembled nuclei grew to the normal size. Nuclear pore complexes existed in the thus assembled nuclear envelopes. These results suggested that 1) two vesicle populations, PV1 and PV2, are necessary for the assembly of normal sized nuclei, 2) PV1 contains a chromatin targeting molecule(s) and membrane fusion machinery, 3) PV2 contains a chromatin targeting molecule(s) and a molecule(s) necessary for nuclear pore complex assembly, and 4) PV1 has the ability to assemble a nuclear membrane, and PV2 is necessary for the assembly of nuclear pore complexes and for nuclei to grow to the normal size. An in vitro nuclear assembly system constituted with affinity-purified vesicle fractions, PV1 and PV2, was established.     

Nuclear envelope assembly around sperm chromatin in cell-free preparations from Drosophila embryos

Chicken sperm chromatin initiated an assembly of interphase-like nuclei in a cell-free cytoplasmic preparation from 1-6 h old Drosophila melanogaster embryos. The formation of these interphase-like nuclei from the condensed sperm chromatin happened in a series of distinct steps. Anti-Drosophila lamin monoclonal antibody stained the assembled nuclei in a pattern indistinguishable from normal Drosophila nuclei. This assembly process required an ATP regenerating system and could be blocked by the addition of novobiocin into the cell-free extract.     

A lamin-independent pathway for nuclear envelope assembly

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.