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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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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.
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fig6: 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.

Mentions: Based on our results, it appeared that LB3T was preventing an early step in nuclear envelope assembly. However, previous results using Xenopus interphase extracts suggested that lamins become associated with chromatin only after nuclear envelope assembly is completed (Newport et al., 1990; Meier et al., 1991; Hutchison et al., 1994). In light of these conflicting results, we reexamined the earliest stages of normal envelope assembly using confocal microscopy. The assembly process was monitored by immunofluorescence at various times after adding chromatin to interphase extracts (see Materials and methods). Within 5 min, LB3 appeared to stain chromatin diffusely with a few brighter foci (Fig. 4 A). After 10–20 min, increasing amounts of lamin were observed at the periphery of the chromatin until an obvious rim of fluorescence formed at 40 min (Fig. 4, B–D). In a similar fashion, discontinuous patches of membrane fluorescence were detected with DiOC6 during the first 5 min, and by 40 min the decondensing chromatin was surrounded by a rim of membrane fluorescence (Fig. 4, E–H). In contrast, very little fluorescence was detected with the 414 nucleoporin antibody until 10 min after initiating nuclear envelope assembly, when a patchy fluorescent pattern coincident with membrane staining appeared (Fig. 4, compare J and F). After 40 min, the pore staining appeared as a rim along the surface of chromatin (Fig. 4, K and L, compare with Fig. 6, G and H) . These results indicate that both lamins and membranes associate with chromatin at very early time points in nuclear envelope assembly.


A role for nuclear lamins in nuclear envelope assembly.

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196852&req=5

fig6: 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.
Mentions: Based on our results, it appeared that LB3T was preventing an early step in nuclear envelope assembly. However, previous results using Xenopus interphase extracts suggested that lamins become associated with chromatin only after nuclear envelope assembly is completed (Newport et al., 1990; Meier et al., 1991; Hutchison et al., 1994). In light of these conflicting results, we reexamined the earliest stages of normal envelope assembly using confocal microscopy. The assembly process was monitored by immunofluorescence at various times after adding chromatin to interphase extracts (see Materials and methods). Within 5 min, LB3 appeared to stain chromatin diffusely with a few brighter foci (Fig. 4 A). After 10–20 min, increasing amounts of lamin were observed at the periphery of the chromatin until an obvious rim of fluorescence formed at 40 min (Fig. 4, B–D). In a similar fashion, discontinuous patches of membrane fluorescence were detected with DiOC6 during the first 5 min, and by 40 min the decondensing chromatin was surrounded by a rim of membrane fluorescence (Fig. 4, E–H). In contrast, very little fluorescence was detected with the 414 nucleoporin antibody until 10 min after initiating nuclear envelope assembly, when a patchy fluorescent pattern coincident with membrane staining appeared (Fig. 4, compare J and F). After 40 min, the pore staining appeared as a rim along the surface of chromatin (Fig. 4, K and L, compare with Fig. 6, G and H) . These results indicate that both lamins and membranes associate with chromatin at very early time points in nuclear envelope assembly.

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