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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 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.
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fig5: 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.

Mentions: To determine whether LB3T binds to chromatin, GST–LB3T was added to interphase extracts (see Materials and methods). The addition of GST-LB3T blocked chromatin decondensation and nuclear envelope assembly (Fig. 5 A). In addition, only weakly fluorescent patches of GST–LB3T were seen in the peripheral regions of chromatin (Fig. 5 B). Further microscopic analyses of these preparations showed that the distribution of lamins, membranes, and pores was abnormal and appeared to be identical to LB3T-treated chromatin (Fig. 1, A, B, and E–H). As a control, when equimolar amounts of purified GST were added to interphase extracts, there were no effects on nuclear envelope assembly, and no fluorescent staining of chromatin could be detected with the mAb against GST (Fig. 5, C and D). These results suggest that LB3T binds to chromatin in interphase extracts.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196852&req=5

fig5: 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.
Mentions: To determine whether LB3T binds to chromatin, GST–LB3T was added to interphase extracts (see Materials and methods). The addition of GST-LB3T blocked chromatin decondensation and nuclear envelope assembly (Fig. 5 A). In addition, only weakly fluorescent patches of GST–LB3T were seen in the peripheral regions of chromatin (Fig. 5 B). Further microscopic analyses of these preparations showed that the distribution of lamins, membranes, and pores was abnormal and appeared to be identical to LB3T-treated chromatin (Fig. 1, A, B, and E–H). As a control, when equimolar amounts of purified GST were added to interphase extracts, there were no effects on nuclear envelope assembly, and no fluorescent staining of chromatin could be detected with the mAb against GST (Fig. 5, C and D). These results suggest that LB3T binds to chromatin in interphase extracts.

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