Lopez-Soler RI , Moir RD , Spann TP , Stick R , Goldman RD .

CA31760 NCI NIH HHS , F31 GM020083 NIGMS NIH HHS , R01 CA031760 NCI NIH HHS

	Figure 1. Sperm chromatin was incubated in extracts containing LB3T (A, B, and E–H) and as a control, purified LB3 (C, D, and I–L). These preparations were stained with the DNA dye TOTO (A, C, E, and I), the mAb 414 directed against nucleoporins (B and D; NPC), an mAb directed against LB3 (F and J), and the lipophillic dye, DiOC6 (G and K; MEM). Chromatin in extracts containing LB3T remained highly condensed (A and E), in some cases assuming an elongated appearance, and remained surrounded by patches of fluorescence for all three envelope markers (B, F, and G). In controls, the chromatin was decondensed (C and I) and surrounded by rims of nuclear pore complex, lamin, and membrane fluorescence (D and J–L). All images are from confocal sections taken through the midregions of nuclei. Immunoblot analyses of chromatin confirmed the fluorescence studies. As compared with the control samples, the addition of LB3T resulted in a significant reduction in the amount of p62, the major nucleoporin recognized by mAb 414 (compare lanes M and N). In a 10-fold longer exposure to this antibody, traces of p62 could be detected in the LB3T-treated preparations (unpublished data). In controls, other 414-reactive bands were detected following longer exposures, and were barely detectable in the presence of LB3T (unpublished data). In the presence of LB3T there was also a large reduction in the amount of LB3 (lane P) associated with chromatin, as compared with controls (lane O). Bars (A–L), 10 μm.
	Figure 2. Electron microscopy analysis of LB3T-treated and control nuclei. In the presence of LB3T, only membrane vesicles are found along the surface of condensed chromatin (A). In controls, decondensed chromatin masses surrounded by typical double membranes with nuclear pore complexes are seen (B, arrows). Bars, 2 μm.
	Figure 3. Sperm chromatin was incubated in normal interphase extracts (A and C) or in extracts containing LB3T (B and D). After incubation, the chromatin-associated proteins were separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting with the LBR antibody (58 kD; A and B), a fusogenic vesicle marker. LBR could not be detected in preparations containing LB3T (B). Samples were also blotted with an antibody directed against p78 (3E9), a marker for the nonfusogenic vesicles (C and D). This protein was present in both control and LB3T-treated chromatin samples.
	Figure 4. Chromatin was added to interphase extracts and samples were fixed 5 (A, E, and I), 10 (B, F, and J), 20 (C, G, and K), and 40 min (D, H, and L) after initiating assembly. Nuclei were then stained with either the LB3 mAb (A–D) or the membrane dye DiOC6 (E–H) and the 414 nucleoporin antibody (I–L). At 5 min, lamin appeared to coat the chromatin with a few foci of brighter fluorescence observed at the edges (A). At this same time point, patches of membrane fluorescence were associated with the surface of the chromatin (E), although very little nucleoporin staining was seen at this stage (I). After 10 min, patches of fluorescence for all three markers were detected around chromatin (B, F, and J). At this time, membrane and nuclear pore fluorescence were mainly coaligned (F and J). Normal rim staining patterns were observed at 40 min for each envelope marker (D, H, and L). Bars, 10 μm.
	Figure 6. Chromatin was incubated in HSS or HSS containing LB3T, washed, pelleted by centrifugation, and the associated proteins were separated by SDS-PAGE. Immunoblotting with the LB3 polyclonal antibody revealed both LB3T (22 kD) and LB3 (68 kD) in the presence of LB3T (B), whereas only LB3 bound to chromatin in the absence of LB3T (A). Chromatin pretreated in HSS with LB3T or HSS alone was added to interphase extracts containing no exogenous proteins. After 2 h, samples were fixed and stained with TOTO (C and E) and DiOC6 (D and F). The pretreated chromatin remained condensed (C) and contained only patches of membrane fluorescence (D). Chromatin incubated in HSS alone displayed normal decondensation and was surrounded by a rim of membrane fluorescence (E and F). Sperm chromatin preincubated in NWB containing LB3 and LB3T did not assemble nuclear envelopes when transferred to normal extracts (G and H; Materials and methods). In contrast, sperm chromatin pretreated with only LB3T assembled normally (I and J). Bar, 10 μm.
	Figure 5. Sperm chromatin incubated in extracts containing GST–LB3T (A and B) or GST as a control (C and D) was visualized with TOTO (A and C) and a monoclonal GST antibody (B and D). GST–LB3T was found in bright patches at the surface of chromatin, with less intense staining throughout the chromatin (B). GST alone had no apparent effect on chromatin decondensation (C), and was not detected in association with chromatin (D). λDNA was added to normal extracts (J–N) or LB3T-treated extracts (E–I). After 6 h, samples were fixed and stained with DiOC6, TOTO, and the LB3 and nucleoporin antibodies. In extracts containing LB3T, small patches of membrane (MEM) and LB3 fluorescence were seen at the edge of λDNA (E–H), but nucleoporin staining was difficult to detect (I). In controls, bright rims of fluorescence were observed for all three envelope markers (J–N). Bars, 10 μm.
	Figure 7. Isolated fusogenic and nonfusogenic membrane vesicles from Xenopus extracts were incubated in MWB containing LB3T. The pretreated membrane vesicles were washed, isolated, and combined with chromatin, HSS, and the complementary membrane vesicle. Samples were fixed and stained with DiOC6, TOTO, and the LB3 mAb. Pretreatment of the fusogenic membrane vesicles with LB3T yielded condensed chromatin structures (A) with patches of membrane (MEM) and LB3 fluorescence (B and C). In contrast, pretreatment of nonfusogenic membrane vesicles with LB3T resulted in normal nuclear membrane and lamin rim staining patterns surrounding decondensed chromatin (D–F). Bar, 10 μm.
	Figure 8. Bacterially expressed LB3 (A and B), LB3T (E and F), or a combination of LB3 and LB3T at a 1:3 molar ratio (C and D, respectively) were diluted into LAB, pelleted, and analyzed by SDS-PAGE. Under these conditions, ∼95% of the LB3 was detected in the pellet (A and B). However, when LB3 and LB3T were combined, most of the LB3 was detected in the supernatant (C and D). Under identical assembly conditions, most LB3T remained in the supernatant fraction (E and F). As a control, mixtures of LB3 and VIM-C in a 1:5 molar ratio, respectively, did not alter the assembly properties of LB3 (G–J). VIM-C runs to the bottom of the gel due to its low molecular weight (8.5 kD). Size markers are indicated on the left.

Aebi, The nuclear lamina is a meshwork of intermediate-type filaments. , Pubmed, Xenbase

Aebi, The nuclear lamina is a meshwork of intermediate-type filaments. , Pubmed , Xenbase
Boman, GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro. 1992, Pubmed , Xenbase
Burke, A cell free system to study reassembly of the nuclear envelope at the end of mitosis. 1986, Pubmed
Clements, Direct interaction between emerin and lamin A. 2000, Pubmed
Dabauvalle, Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin. 1991, Pubmed , Xenbase
Davis, Identification and characterization of a nuclear pore complex protein. 1986, Pubmed
Drummond, Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types. 1999, Pubmed , Xenbase
Ellis, GST-lamin fusion proteins act as dominant negative mutants in Xenopus egg extract and reveal the function of the lamina in DNA replication. 1997, Pubmed , Xenbase
Fawcett, On the occurrence of a fibrous lamina on the inner aspect of the nuclear envelope in certain cells of vertebrates. 1966, Pubmed
Furukawa, LAP2 binding protein 1 (L2BP1/BAF) is a candidate mediator of LAP2-chromatin interaction. 1999, Pubmed
Furukawa, Identification of the lamina-associated-polypeptide-2-binding domain of B-type lamin. 1998, Pubmed
Gajewski, Subcellular distribution of the Xenopus p58/lamin B receptor in oocytes and eggs. 1999, Pubmed , Xenbase
Gant, Nuclear assembly. 1997, Pubmed
Gant, Roles of LAP2 proteins in nuclear assembly and DNA replication: truncated LAP2beta proteins alter lamina assembly, envelope formation, nuclear size, and DNA replication efficiency in Xenopus laevis extracts. 1999, Pubmed , Xenbase
Gerace, Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution. 1978, Pubmed
Gerace, Organization and modulation of nuclear lamina structure. 1984, Pubmed
Gerace, The nuclear envelope lamina is reversibly depolymerized during mitosis. 1980, Pubmed
Glass, Lamins A and C bind and assemble at the surface of mitotic chromosomes. 1990, Pubmed
Goldberg, Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA. 1998, Pubmed
Goldberg, The tail domain of lamin Dm0 binds histones H2A and H2B. 1999, Pubmed
Heitlinger, Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions. 1991, Pubmed
Heitlinger, The role of the head and tail domain in lamin structure and assembly: analysis of bacterially expressed chicken lamin A and truncated B2 lamins. 1992, Pubmed
Hutchison, Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication. 1994, Pubmed
Jenkins, Nuclei that lack a lamina accumulate karyophilic proteins and assemble a nuclear matrix. 1993, Pubmed , Xenbase
Krohne, Localization of a nuclear envelope-associated protein by indirect immunofluorescence microscopy using antibodies against a major polypeptide from rat liver fractions enriched in nuclear envelope-associated material. 1978, Pubmed
Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. 1970, Pubmed
Lenz-Böhme, Insertional mutation of the Drosophila nuclear lamin Dm0 gene results in defective nuclear envelopes, clustering of nuclear pore complexes, and accumulation of annulate lamellae. 1997, Pubmed
Lohka, Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. 1984, Pubmed , Xenbase
Lourim, Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations. 1993, Pubmed , Xenbase
Lourim, Lamin-dependent nuclear envelope reassembly following mitosis: an argument. 1994, Pubmed
Macaulay, Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA. 1996, Pubmed , Xenbase
McKeon, Nuclear lamin proteins and the structure of the nuclear envelope: where is the function? 1987, Pubmed
Meier, The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs. 1991, Pubmed , Xenbase
Melchior, GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import. 1995, Pubmed
Moir, Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells. 2000, Pubmed
Moir, The dynamic properties and possible functions of nuclear lamins. 1995, Pubmed
Moir, Expression in Escherichia coli of human lamins A and C: influence of head and tail domains on assembly properties and paracrystal formation. 1991, Pubmed
Newmeyer, Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. 1988, Pubmed , Xenbase
Newport, A lamin-independent pathway for nuclear envelope assembly. 1990, Pubmed , Xenbase
Newport, Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. 1987, Pubmed , Xenbase
Newport, Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components. 1992, Pubmed , Xenbase
Paulin-Levasseur, The MAN antigens are non-lamin constituents of the nuclear lamina in vertebrate cells. 1996, Pubmed , Xenbase
Philpott, Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin. 1991, Pubmed , Xenbase
Schuler, Characterization of the human gene encoding LBR, an integral protein of the nuclear envelope inner membrane. 1994, Pubmed
Spann, Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis. 1997, Pubmed , Xenbase
Steen, Mistargeting of B-type lamins at the end of mitosis: implications on cell survival and regulation of lamins A/C expression. 2001, Pubmed
Stick, cDNA cloning of the developmentally regulated lamin LIII of Xenopus laevis. 1988, Pubmed , Xenbase
Stuurman, Intermediate filament protein polymerization: molecular analysis of Drosophila nuclear lamin head-to-tail binding. 1996, Pubmed
Sullivan, Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3 receptor function. 1993, Pubmed , Xenbase
Taniura, A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones. 1995, Pubmed
Ulitzur, Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract. 1992, Pubmed
Vigers, Regulation of nuclear envelope precursor functions during cell division. 1992, Pubmed , Xenbase
Vigers, A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs. 1991, Pubmed , Xenbase
Walter, Regulated chromosomal DNA replication in the absence of a nucleus. 1998, Pubmed , Xenbase
Wiese, Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event. 1997, Pubmed , Xenbase
Wilson, Lamins and disease: insights into nuclear infrastructure. 2001, Pubmed
Wilson, A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. 1988, Pubmed , Xenbase
Yang, A 300,000-mol-wt intermediate filament-associated protein in baby hamster kidney (BHK-21) cells. 1985, Pubmed
Yang, Integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis. 1997, Pubmed
Zhou, The complete sequence of the human intermediate filament chain keratin 10. Subdomainal divisions and model for folding of end domain sequences. 1988, Pubmed