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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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Immunogold electron microscopy reveals the distribution of NEP-B78, LBRx, and p65 between the inner and outer  nuclear envelope. Germinal vesicles (GVs) were isolated manually from stage VI oocytes and spread on silicon chips. Using this  approach, areas of inner and outer nuclear membrane are both  revealed and can be readily distinguished by reference to nuclear  pore morphology (Goldberg and Allen, 1992). Spread GVs were  labeled with mAb 3E9 (A), rabbit anti-LBR (B), or mAb CEL5C  (C) followed by 10-nm gold-conjugated secondary antibodies.  Using this approach the distribution of gold labeled antibodies  can be detected either in backscatter (revealing white spots corresponding to the position of 10-nm gold; right-hand panels in  A–C) or using the secondary electron detector (revealing antibody gold particles of ∼20-nm diam, arrows in left-hand panels in  A–C). Bars, 200 nm.
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Figure 2: Immunogold electron microscopy reveals the distribution of NEP-B78, LBRx, and p65 between the inner and outer nuclear envelope. Germinal vesicles (GVs) were isolated manually from stage VI oocytes and spread on silicon chips. Using this approach, areas of inner and outer nuclear membrane are both revealed and can be readily distinguished by reference to nuclear pore morphology (Goldberg and Allen, 1992). Spread GVs were labeled with mAb 3E9 (A), rabbit anti-LBR (B), or mAb CEL5C (C) followed by 10-nm gold-conjugated secondary antibodies. Using this approach the distribution of gold labeled antibodies can be detected either in backscatter (revealing white spots corresponding to the position of 10-nm gold; right-hand panels in A–C) or using the secondary electron detector (revealing antibody gold particles of ∼20-nm diam, arrows in left-hand panels in A–C). Bars, 200 nm.

Mentions: To further characterize these antibodies and their cognate antigens in the Xenopus system, we carried out immunogold labeling of isolated Xenopus oocyte germinal vesicles in conjunction with FEISEM. Germinal vesicles (GVs) were isolated manually from stage VI oocytes, spread on silicon chips and labeled with mAbs 3E9 or CEL5C, or rabbit anti-LBR followed by 10-nm gold–conjugated secondary antibodies (Fig. 2). Using this approach, the nucleoplasmic face of the inner membrane and cytoplasmic face of the outer nuclear membrane are both revealed (Goldberg and Allen, 1992). The distribution of gold-labeled antibodies can be detected either in backscatter, revealing white spots corresponding to the position of 10-nm gold or using the secondary electron detector to reveal antibody-gold particles directly on the membrane surface. Importantly, nuclear membrane topology can be readily distinguished by reference to nuclear pore morphology (Goldberg and Allen, 1992).Thus the area shown in Fig. 2, A and C (left-hand panels) can be distinguished as the cytoplasmic face of the outer nuclear membrane because all of the NPCs in the surrounding area display cytoplasmic ring structures while the area displayed in Fig. 2 B is distinguished as the nucleoplasmic face of inner nuclear membrane by the presence of nuclear pore baskets. In this experiment, NEP-B78 was observed in clusters between NPCs at the outer nuclear membrane (Fig. 2 A) but not at the inner nuclear membrane (data not shown). In contrast, specific LBRx labeling was observed in clusters between nuclear pores on the inner nuclear envelope (Fig. 2 B) but not at the outer nuclear membrane (data not shown). Labeling of the inner nuclear membrane with rabbit anti-LBR was prevented when incubations were performed in the presence of excess recombinant human LBR1-214 (data not shown). Finally, p65 was densely distributed between NPCs on the outer nuclear membrane (Fig. 2 C) but not the inner nuclear membrane (data not shown). The data suggest that NEP-B78 and p65 are both located on the outer nuclear membrane while LBRx is located on the inner nuclear membrane of oocyte germinal 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)

Immunogold electron microscopy reveals the distribution of NEP-B78, LBRx, and p65 between the inner and outer  nuclear envelope. Germinal vesicles (GVs) were isolated manually from stage VI oocytes and spread on silicon chips. Using this  approach, areas of inner and outer nuclear membrane are both  revealed and can be readily distinguished by reference to nuclear  pore morphology (Goldberg and Allen, 1992). Spread GVs were  labeled with mAb 3E9 (A), rabbit anti-LBR (B), or mAb CEL5C  (C) followed by 10-nm gold-conjugated secondary antibodies.  Using this approach the distribution of gold labeled antibodies  can be detected either in backscatter (revealing white spots corresponding to the position of 10-nm gold; right-hand panels in  A–C) or using the secondary electron detector (revealing antibody gold particles of ∼20-nm diam, arrows in left-hand panels in  A–C). Bars, 200 nm.
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Related In: Results  -  Collection

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Figure 2: Immunogold electron microscopy reveals the distribution of NEP-B78, LBRx, and p65 between the inner and outer nuclear envelope. Germinal vesicles (GVs) were isolated manually from stage VI oocytes and spread on silicon chips. Using this approach, areas of inner and outer nuclear membrane are both revealed and can be readily distinguished by reference to nuclear pore morphology (Goldberg and Allen, 1992). Spread GVs were labeled with mAb 3E9 (A), rabbit anti-LBR (B), or mAb CEL5C (C) followed by 10-nm gold-conjugated secondary antibodies. Using this approach the distribution of gold labeled antibodies can be detected either in backscatter (revealing white spots corresponding to the position of 10-nm gold; right-hand panels in A–C) or using the secondary electron detector (revealing antibody gold particles of ∼20-nm diam, arrows in left-hand panels in A–C). Bars, 200 nm.
Mentions: To further characterize these antibodies and their cognate antigens in the Xenopus system, we carried out immunogold labeling of isolated Xenopus oocyte germinal vesicles in conjunction with FEISEM. Germinal vesicles (GVs) were isolated manually from stage VI oocytes, spread on silicon chips and labeled with mAbs 3E9 or CEL5C, or rabbit anti-LBR followed by 10-nm gold–conjugated secondary antibodies (Fig. 2). Using this approach, the nucleoplasmic face of the inner membrane and cytoplasmic face of the outer nuclear membrane are both revealed (Goldberg and Allen, 1992). The distribution of gold-labeled antibodies can be detected either in backscatter, revealing white spots corresponding to the position of 10-nm gold or using the secondary electron detector to reveal antibody-gold particles directly on the membrane surface. Importantly, nuclear membrane topology can be readily distinguished by reference to nuclear pore morphology (Goldberg and Allen, 1992).Thus the area shown in Fig. 2, A and C (left-hand panels) can be distinguished as the cytoplasmic face of the outer nuclear membrane because all of the NPCs in the surrounding area display cytoplasmic ring structures while the area displayed in Fig. 2 B is distinguished as the nucleoplasmic face of inner nuclear membrane by the presence of nuclear pore baskets. In this experiment, NEP-B78 was observed in clusters between NPCs at the outer nuclear membrane (Fig. 2 A) but not at the inner nuclear membrane (data not shown). In contrast, specific LBRx labeling was observed in clusters between nuclear pores on the inner nuclear envelope (Fig. 2 B) but not at the outer nuclear membrane (data not shown). Labeling of the inner nuclear membrane with rabbit anti-LBR was prevented when incubations were performed in the presence of excess recombinant human LBR1-214 (data not shown). Finally, p65 was densely distributed between NPCs on the outer nuclear membrane (Fig. 2 C) but not the inner nuclear membrane (data not shown). The data suggest that NEP-B78 and p65 are both located on the outer nuclear membrane while LBRx is located on the inner nuclear membrane of oocyte germinal 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