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A role for nuclear lamins in nuclear envelope assembly.

Lopez-Soler RI, Moir RD, Spann TP, Stick R, Goldman RD - J. Cell Biol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA.

ABSTRACT
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.

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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.
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fig1: 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.

Mentions: The role of nuclear lamins in nuclear envelope assembly was investigated using LB3T, a lamin fragment consisting of the COOH-terminal nonhelical domain of Xenopus LB3. The effects of LB3T were assayed by adding demembranated sperm chromatin (1,000 sperm heads/μl) to Xenopus interphase extracts containing different concentrations of LB3T (see Materials and methods). In control reactions, equivalent amounts of wild-type LB3 or an equal volume of protein buffer (PB) (see Materials and methods) was added. The morphological features of the resulting nuclei were examined 2 h later. We observed a concentration-dependent effect of LB3T on nuclear size, and determined that the minimal effective concentration of LB3T was 10 μM (equivalent to approximately 10-fold molar concentration of the endogenous LB3). At this concentration, sperm chromatin remained small and highly condensed (Fig. 1, A and E) . Higher concentrations of LB3T had no additional effects on nuclear size; therefore, this concentration of LB3T was used throughout the study.


A role for nuclear lamins in nuclear envelope assembly.

Lopez-Soler RI, Moir RD, Spann TP, Stick R, Goldman RD - J. Cell Biol. (2001)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196852&req=5

fig1: 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.
Mentions: The role of nuclear lamins in nuclear envelope assembly was investigated using LB3T, a lamin fragment consisting of the COOH-terminal nonhelical domain of Xenopus LB3. The effects of LB3T were assayed by adding demembranated sperm chromatin (1,000 sperm heads/μl) to Xenopus interphase extracts containing different concentrations of LB3T (see Materials and methods). In control reactions, equivalent amounts of wild-type LB3 or an equal volume of protein buffer (PB) (see Materials and methods) was added. The morphological features of the resulting nuclei were examined 2 h later. We observed a concentration-dependent effect of LB3T on nuclear size, and determined that the minimal effective concentration of LB3T was 10 μM (equivalent to approximately 10-fold molar concentration of the endogenous LB3). At this concentration, sperm chromatin remained small and highly condensed (Fig. 1, A and E) . Higher concentrations of LB3T had no additional effects on nuclear size; therefore, this concentration of LB3T was used throughout the study.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus