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 In a 10-fold longer exposure to this antibody, traces of p62 could be detected in the LB3T-treated preparations (unpublished data).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other 414-reactive bands were detected following longer exposures, and were barely detectable in the presence of LB3T (unpublished data).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.

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