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Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types.

Drummond S, Ferrigno P, Lyon C, Murphy J, Goldberg M, Allen T, Smythe C, Hutchison CJ - J. Cell Biol. (1999)

Bottom Line: In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system.Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells.The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation Unit, University of Dundee, Dundee DD1 4HN, Scotland, United Kingdom.

ABSTRACT
In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

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Recruitment of  NEP-B78, LBRx, and p65 to  chromatin periphery during  NE reassembly in vitro. Demembranated sperm chromatin was incubated in Xenopus  LSS and samples were taken  at the indicated time points  and processed for immunofluorescence (see Materials and  Methods). In each case DNA  was stained with DAPI and  proteins detected using fluorescein-labeled secondary  antibody. NEP-B78 (left-hand micrographs), LBRx  (center micrographs), or p65  (right-hand micrographs). Bar,  20 μm.
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Figure 3: Recruitment of NEP-B78, LBRx, and p65 to chromatin periphery during NE reassembly in vitro. Demembranated sperm chromatin was incubated in Xenopus LSS and samples were taken at the indicated time points and processed for immunofluorescence (see Materials and Methods). In each case DNA was stained with DAPI and proteins detected using fluorescein-labeled secondary antibody. NEP-B78 (left-hand micrographs), LBRx (center micrographs), or p65 (right-hand micrographs). Bar, 20 μm.

Mentions: In cell-free extracts derived from Xenopus eggs, replication competent nuclei are assembled over a 60-min period (Hutchison et al., 1987). Assembly involves the cytosol- dependent decondensation of chromatin followed by membrane recruitment to chromatin surfaces. Subsequent membrane fusion and surface area expansion is accompanied by the appearance of nuclear pores and the lamina, before the onset of DNA replication. Typically in these extracts, membrane recruitment to decondensing chromatin can be detected after 10–15 min. To compare the timing of recruitment of NEP-B78, p65, and LBRx, nuclear reassembly assays in unfractionated extract were analyzed by indirect immunofluorescence microscopy using antibodies against each protein (Fig. 3). Nuclei displaying rim staining with mAbs 4G12 (detecting NEP-B78; Fig. 3, left-hand panels) and CEL5C (detecting p65; Fig. 3, right-hand panels) were readily observed 15 min after the addition of sperm chromatin to Xenopus egg extracts. As the NEs expanded over the following 45-min period, the intensity of staining with both 4G12 and CEL5C increased. In contrast, LBRx was only detected in the NE of assembling nuclei some 30 min after the addition of demembranated sperm chromatin to LSS (Fig. 3, middle panels). These data suggest that the association of NEP-B78- and p65-containing vesicles with decondensing chromatin is one of the earliest events of NE assembly and is distinct from the association of LBRx-containing vesicles.


Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types.

Drummond S, Ferrigno P, Lyon C, Murphy J, Goldberg M, Allen T, Smythe C, Hutchison CJ - J. Cell Biol. (1999)

Recruitment of  NEP-B78, LBRx, and p65 to  chromatin periphery during  NE reassembly in vitro. Demembranated sperm chromatin was incubated in Xenopus  LSS and samples were taken  at the indicated time points  and processed for immunofluorescence (see Materials and  Methods). In each case DNA  was stained with DAPI and  proteins detected using fluorescein-labeled secondary  antibody. NEP-B78 (left-hand micrographs), LBRx  (center micrographs), or p65  (right-hand micrographs). Bar,  20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132889&req=5

Figure 3: Recruitment of NEP-B78, LBRx, and p65 to chromatin periphery during NE reassembly in vitro. Demembranated sperm chromatin was incubated in Xenopus LSS and samples were taken at the indicated time points and processed for immunofluorescence (see Materials and Methods). In each case DNA was stained with DAPI and proteins detected using fluorescein-labeled secondary antibody. NEP-B78 (left-hand micrographs), LBRx (center micrographs), or p65 (right-hand micrographs). Bar, 20 μm.
Mentions: In cell-free extracts derived from Xenopus eggs, replication competent nuclei are assembled over a 60-min period (Hutchison et al., 1987). Assembly involves the cytosol- dependent decondensation of chromatin followed by membrane recruitment to chromatin surfaces. Subsequent membrane fusion and surface area expansion is accompanied by the appearance of nuclear pores and the lamina, before the onset of DNA replication. Typically in these extracts, membrane recruitment to decondensing chromatin can be detected after 10–15 min. To compare the timing of recruitment of NEP-B78, p65, and LBRx, nuclear reassembly assays in unfractionated extract were analyzed by indirect immunofluorescence microscopy using antibodies against each protein (Fig. 3). Nuclei displaying rim staining with mAbs 4G12 (detecting NEP-B78; Fig. 3, left-hand panels) and CEL5C (detecting p65; Fig. 3, right-hand panels) were readily observed 15 min after the addition of sperm chromatin to Xenopus egg extracts. As the NEs expanded over the following 45-min period, the intensity of staining with both 4G12 and CEL5C increased. In contrast, LBRx was only detected in the NE of assembling nuclei some 30 min after the addition of demembranated sperm chromatin to LSS (Fig. 3, middle panels). These data suggest that the association of NEP-B78- and p65-containing vesicles with decondensing chromatin is one of the earliest events of NE assembly and is distinct from the association of LBRx-containing vesicles.

Bottom Line: In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system.Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells.The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation Unit, University of Dundee, Dundee DD1 4HN, Scotland, United Kingdom.

ABSTRACT
In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

Show MeSH
Related in: MedlinePlus