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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 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.
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fig3: 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.

Mentions: It has been shown that during the early stages of nuclear envelope assembly, nonfusogenic vesicles bind to the surface of chromatin. The nonfusogenic vesicles are unable to fuse to form the nuclear membrane without a second type of vesicle known as the fusogenic vesicle (Vigers and Lohka, 1991; Drummond et al., 1999). In the presence of LB3T, relatively few vesicles were seen associated with condensed chromatin (Fig. 2 A), as compared with the many vesicles seen during the early steps of normal nuclear membrane assembly (Macaulay and Forbes, 1996; Wiese et al., 1997). Based on these observations, we determined whether LB3T prevented either the fusogenic or nonfusogenic vesicles from binding to chromatin. Equivalent amounts of chromatin were added to control and LB3T-containing extracts. After 2 h the chromatin was pelleted, washed, and analyzed by immunoblotting with antibodies against the 58-kD lamin B receptor (LBR), a marker for fusogenic vesicles, and a 78-kD protein associated with nonfusogenic vesicles (Drummond et al., 1999; see Materials and methods). Under these conditions, LBR was not detected in the LB3T-treated preparations (Fig. 3 , compare A and B). In contrast, p78 was detected in both control and LB3T-treated preparations (Fig. 3, C and D). These results suggest that LB3T inhibits the binding of only fusogenic vesicles 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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: 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.
Mentions: It has been shown that during the early stages of nuclear envelope assembly, nonfusogenic vesicles bind to the surface of chromatin. The nonfusogenic vesicles are unable to fuse to form the nuclear membrane without a second type of vesicle known as the fusogenic vesicle (Vigers and Lohka, 1991; Drummond et al., 1999). In the presence of LB3T, relatively few vesicles were seen associated with condensed chromatin (Fig. 2 A), as compared with the many vesicles seen during the early steps of normal nuclear membrane assembly (Macaulay and Forbes, 1996; Wiese et al., 1997). Based on these observations, we determined whether LB3T prevented either the fusogenic or nonfusogenic vesicles from binding to chromatin. Equivalent amounts of chromatin were added to control and LB3T-containing extracts. After 2 h the chromatin was pelleted, washed, and analyzed by immunoblotting with antibodies against the 58-kD lamin B receptor (LBR), a marker for fusogenic vesicles, and a 78-kD protein associated with nonfusogenic vesicles (Drummond et al., 1999; see Materials and methods). Under these conditions, LBR was not detected in the LB3T-treated preparations (Fig. 3 , compare A and B). In contrast, p78 was detected in both control and LB3T-treated preparations (Fig. 3, C and D). These results suggest that LB3T inhibits the binding of only fusogenic vesicles 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