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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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The association of NEP-B78 and LBRx  with reforming nuclear envelopes in XLK-2 cells.  XLK-2 cells were fixed and stained with either rabbit anti-LBR followed by FITC goat anti–rabbit Ig  (a–h) or mAb 3E9 followed by FITC-goat anti– mouse Ig (i–m). Fluorescence images of cells in  metaphase (a, e, i, and m), anaphase (b, f, j, and n),  telophase (c, g, k, and o) or early G1 (d, h, i, and p)  were collected with a 12 bit CCD camera attached  to a Zeiss Axioskop microscope. In each pair of micrographs, the distribution of DNA is revealed by  DAPI staining (a–d and i–l). Bars, 10 μm.
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Figure 4: The association of NEP-B78 and LBRx with reforming nuclear envelopes in XLK-2 cells. XLK-2 cells were fixed and stained with either rabbit anti-LBR followed by FITC goat anti–rabbit Ig (a–h) or mAb 3E9 followed by FITC-goat anti– mouse Ig (i–m). Fluorescence images of cells in metaphase (a, e, i, and m), anaphase (b, f, j, and n), telophase (c, g, k, and o) or early G1 (d, h, i, and p) were collected with a 12 bit CCD camera attached to a Zeiss Axioskop microscope. In each pair of micrographs, the distribution of DNA is revealed by DAPI staining (a–d and i–l). Bars, 10 μm.

Mentions: We extended our observations concerning the timing of    recruitment of NEP-B78- and LBRx-containing membranes to the Xenopus cultured cell line, XLK-2. Cells were fixed using 4% paraformaldehyde and 0.1% Triton X-100 and analyzed by indirect immunofluorescence microscopy using antibodies against each protein. We found that in metaphase cells, both LBR and NEP-B78 antibodies reacted with reticular structures in the cytoplasm in regions peripheral to condensed chromosomes, although NEP-B78 staining was very weak (Fig. 4, a, e, i, and m). NEP-B78 association with chromatin was first observed in anaphase (Fig. 4, j and n) at which stage LBRx-containing membranes appeared to remain in the cytoplasm (Fig. 4, b and f). Labeling of the chromatin periphery by LBR antibodies could be observed at telophase with a substantial amount of label still decorating the cytoplasm (Fig. 4, c and g). In contrast, NEP-B78 showed predominantly perichromatin staining at this time (Fig. 4, k and o). Both antibodies gave rise to nuclear staining in early G1 cells (Fig. 4, d, h, l, and p). These data support and extend the observation that the association of NEP-B78-containing membranes with decondensing chromatin is one of the earliest events of NE assembly and is temporally distinct from the association of LBRx-containing membranes.


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)

The association of NEP-B78 and LBRx  with reforming nuclear envelopes in XLK-2 cells.  XLK-2 cells were fixed and stained with either rabbit anti-LBR followed by FITC goat anti–rabbit Ig  (a–h) or mAb 3E9 followed by FITC-goat anti– mouse Ig (i–m). Fluorescence images of cells in  metaphase (a, e, i, and m), anaphase (b, f, j, and n),  telophase (c, g, k, and o) or early G1 (d, h, i, and p)  were collected with a 12 bit CCD camera attached  to a Zeiss Axioskop microscope. In each pair of micrographs, the distribution of DNA is revealed by  DAPI staining (a–d and i–l). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: The association of NEP-B78 and LBRx with reforming nuclear envelopes in XLK-2 cells. XLK-2 cells were fixed and stained with either rabbit anti-LBR followed by FITC goat anti–rabbit Ig (a–h) or mAb 3E9 followed by FITC-goat anti– mouse Ig (i–m). Fluorescence images of cells in metaphase (a, e, i, and m), anaphase (b, f, j, and n), telophase (c, g, k, and o) or early G1 (d, h, i, and p) were collected with a 12 bit CCD camera attached to a Zeiss Axioskop microscope. In each pair of micrographs, the distribution of DNA is revealed by DAPI staining (a–d and i–l). Bars, 10 μm.
Mentions: We extended our observations concerning the timing of    recruitment of NEP-B78- and LBRx-containing membranes to the Xenopus cultured cell line, XLK-2. Cells were fixed using 4% paraformaldehyde and 0.1% Triton X-100 and analyzed by indirect immunofluorescence microscopy using antibodies against each protein. We found that in metaphase cells, both LBR and NEP-B78 antibodies reacted with reticular structures in the cytoplasm in regions peripheral to condensed chromosomes, although NEP-B78 staining was very weak (Fig. 4, a, e, i, and m). NEP-B78 association with chromatin was first observed in anaphase (Fig. 4, j and n) at which stage LBRx-containing membranes appeared to remain in the cytoplasm (Fig. 4, b and f). Labeling of the chromatin periphery by LBR antibodies could be observed at telophase with a substantial amount of label still decorating the cytoplasm (Fig. 4, c and g). In contrast, NEP-B78 showed predominantly perichromatin staining at this time (Fig. 4, k and o). Both antibodies gave rise to nuclear staining in early G1 cells (Fig. 4, d, h, l, and p). These data support and extend the observation that the association of NEP-B78-containing membranes with decondensing chromatin is one of the earliest events of NE assembly and is temporally distinct from the association of LBRx-containing membranes.

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