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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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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.
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fig7: 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.

Mentions: The inhibition of nuclear envelope assembly by LB3T could also be mediated by factors associated with membrane vesicles. To test this possibility, either fusogenic or nonfusogenic vesicles were pretreated with LB3T in membrane wash buffer (MWB) for 20 min at 22°C, washed, and then transferred to HSS containing chromatin and the complementary vesicles required for nuclear envelope assembly (see Materials and methods). After 3 h, samples were stained with TOTO, DiOC6, and the mAb against LB3 (see Materials and methods). Pretreatment of the fusogenic vesicles with LB3T blocked chromatin decondensation and the formation of both the nuclear membrane and lamina (Fig. 7 , A–C). Nuclear pore assembly was also inhibited (unpublished data). In contrast, when nonfusogenic vesicles were pretreated with LB3T, normal membrane, lamin, and nuclear pore staining patterns were detected, indicating that normal assembly had taken place (Fig. 7, D–F, and unpublished data). Since LB3 is found only in the fusogenic vesicle fraction, these results suggest that LB3T may be interacting with LB3 to prevent vesicle binding to chromatin. Alternatively, LB3T may be interacting with LAPs such as LBR and LAP2β to block their association with chromatin and to prevent vesicle binding to chromatin. To test this possibility, membrane fractions were treated with detergent using the method of Steen et al. (2000) (see Materials and methods) in order to release LBR and LAP2β from the membranes. These detergent-treated preparations were incubated with sperm chromatin with or without LB3T. Immunoblotting analyses of these preparations showed that similar amounts of LBR and LAP2β bound to chromatin both in the presence and absence of LB3T (unpublished data). Therefore, LB3T does not appear to interact with LBR or LAP2β to block vesicle binding to chromatin.


A role for nuclear lamins in nuclear envelope assembly.

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

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196852&req=5

fig7: 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.
Mentions: The inhibition of nuclear envelope assembly by LB3T could also be mediated by factors associated with membrane vesicles. To test this possibility, either fusogenic or nonfusogenic vesicles were pretreated with LB3T in membrane wash buffer (MWB) for 20 min at 22°C, washed, and then transferred to HSS containing chromatin and the complementary vesicles required for nuclear envelope assembly (see Materials and methods). After 3 h, samples were stained with TOTO, DiOC6, and the mAb against LB3 (see Materials and methods). Pretreatment of the fusogenic vesicles with LB3T blocked chromatin decondensation and the formation of both the nuclear membrane and lamina (Fig. 7 , A–C). Nuclear pore assembly was also inhibited (unpublished data). In contrast, when nonfusogenic vesicles were pretreated with LB3T, normal membrane, lamin, and nuclear pore staining patterns were detected, indicating that normal assembly had taken place (Fig. 7, D–F, and unpublished data). Since LB3 is found only in the fusogenic vesicle fraction, these results suggest that LB3T may be interacting with LB3 to prevent vesicle binding to chromatin. Alternatively, LB3T may be interacting with LAPs such as LBR and LAP2β to block their association with chromatin and to prevent vesicle binding to chromatin. To test this possibility, membrane fractions were treated with detergent using the method of Steen et al. (2000) (see Materials and methods) in order to release LBR and LAP2β from the membranes. These detergent-treated preparations were incubated with sperm chromatin with or without LB3T. Immunoblotting analyses of these preparations showed that similar amounts of LBR and LAP2β bound to chromatin both in the presence and absence of LB3T (unpublished data). Therefore, LB3T does not appear to interact with LBR or LAP2β to block vesicle binding to chromatin.

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