Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.     

Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha- amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205- 217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.     

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.     

A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs

Extracts from Xenopus eggs capable of nuclear envelope assembly in vitro were fractionated by differential and density gradient centrifugation. Nuclear envelope assembly was found to require soluble components in the cytosol and two distinct particulate fractions, which we have called nuclear envelope precursor fractions A and B (NEP-A and NEP-B). Both NEP-A and NEP-B are sensitive to treatments with trypsin, sodium carbonate, and detergents, but can be distinguished from each other by their sensitivities to high salt and N-ethylmaleimide and by their levels of alpha-glucosidase activity. Vesicles in NEP-B bind to chromatin, whereas those in NEP-A do not. NEP-B may therefore be involved in the targeting of membranes to the surface of the chromatin, whereas NEP-A may provide a pool of vesicles that contributes many of the nuclear envelope membranes. NEP-B may also play a role in the assembly of nuclear pore complexes because the density of nuclear pores in the resulting envelope is dependent on the ratio of NEP-B to NEP-A in the reconstituted 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.     

Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor

We have investigated the mechanism which prevents reinitiation of DNA replication within a single cell cycle by exploiting the observation that intact G2 HeLa nuclei do not replicate in Xenopus egg extract, unless their nuclear membranes are first permeabilized (Leno et al., 1992). We have asked if nuclear membrane permeabilization allows escape of a negative inhibitor from the replicated nucleus or entry of a positive activator as proposed in the licensing factor hypothesis of Blow and Laskey (1988). We have distinguished these possibilities by repairing permeabilized nuclear membranes after allowing soluble factors to escape. Membrane repair of G2 nuclei reverses the effects of permeabilization arguing that escape of diffusible inhibitors is not sufficient to allow replication, but that entry of diffusible activators is required. Membrane repair has no significant effect on G1 nuclei. Pre-incubation of permeable G2 nuclei in the soluble fraction of egg extract before membrane repair allows semiconservative DNA replication of these nuclei when incubated in complete extract. Addition of the same fraction after membrane repair has no effect. Our results provide direct evidence for a positively acting "licensing" activity which is excluded form the interphase nucleus by the nuclear membrane. Nuclear membrane permeabilization and repair can be used as an assay for licensing activity which could lead to its purification and subsequent analysis of its action within the nucleus.     

Controlling pore assembly of staphylococcal gamma-haemolysin by low temperature and by disulphide bond formation in double-cysteine LukF mutants

Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double-cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C-T136C, Cap-Stem), or two beta-strands within the prestem domain (T117C-T136C, Stem-Stem) to control pore assembly of gamma-haemolysin at intermediate stages. The disulphide-trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap-Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem-Stem were stable in SDS and had ring-shaped structures similar to those of wild-type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2 degrees C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real-time transition of Stem-Stem from prepores to pores on ghost cells, visualized using a Ca2+-sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore-opening events. Taken together, these data indicate that the prepores are legitimate intermediates during gamma-haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for gamma-haemolysin pore assembly based on the demonstrated intermediates.     

Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs

A cell-free cytoplasmic preparation from activated Rana pipiens eggs could induce in demembranated Xenopus laevis sperm nuclei morphological changes similar to those seen during pronuclear formation in intact eggs. The condensed sperm chromatin underwent an initial rapid, but limited, dispersion. A nuclear envelope formed around the dispersed chromatin and the nuclei enlarged. The subcellular distribution of the components required for these changes was examined by separating the preparations into soluble (cytosol) and particulate fractions by centrifugation at 150,000 g for 2 h. Sperm chromatin was incubated with the cytosol or with the particulate material after it had been resuspended in either the cytosol, heat-treated (60 or 100 degrees C) cytosol or buffer. We found that the limited dispersion of chromatin occurred in each of these ooplasmic fractions, but not in the buffer alone. Nuclear envelope assembly required the presence of both untreated cytosol and particulate material. Ultrastructural examination of the sperm chromatin during incubation in the preparations showed that membrane vesicles of approximately 200 nm in diameter, found in the particulate fraction, flattened and fused together to contribute the membranous components of the nuclear envelope. The enlargement of the sperm nuclei occurred only after the nuclear envelope formed. The pronuclei formed in the cell-free preparations were able to incorporate [3H]dTTP into DNA. This incorporation was inhibited by aphidicolin, suggesting that the DNA synthesis by the pronuclei was dependent on DNA polymerase-alpha. When sperm chromatin was incubated greater than 3 h, the chromatin of the pronuclei often recondensed to form structures resembling mitotic chromosomes within the nuclear envelope. Therefore, it appeared that these ooplasmic preparations could induce, in vitro, nuclear changes resembling those seen during the first cell cycle in the zygote.     

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.     

Chromosome behaviour during meiotic prophase in the solanaceae

The molecular structure of chromatin during dogfish spermiogenesis was examined by electron microscopy after the dispersion of nuclei at low ionic strength. In early and late stages of differentiation (round and elongating spermatids), chromatin is globular, although basic nuclear proteins are different from those present in somatic nuclei. Three protein fractions are complexed with DNA in sperm nuclei. These fractions appear at the end of differentiation (elongated spermatids), subsequently undergoing a modification of their solubilization properties; only one protein fraction remains acid-soluble. Dispersed chromatin from sperm nuclei again shows a beads-on-a-string configuration both in the presence of the three specific sperm proteins and when the acid soluble fraction is extracted. Variations of the mean diameter of chromatin subunits during spermiogenesis appear rather limited compared to extensive modifications of chromatin superstructures.