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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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Characterization of NEP-B78- and LBRx-containing  vesicles in MP2 and MP1 fractions by (A) immunogold labeling  and FEISEM and (B) sucrose density gradient centrifugation.  (A) MP1 (top panels) or MP2 vesicles (bottom panels) were isolated on silicon chips, fixed with 4% paraformaldehyde and 0.1%  glutaraldehyde and labeled with either mAb 3E9 followed by  10-nm gold–conjugated sheep anti–mouse Ig (left-hand panels)  or rabbit anti–LBR followed by 10-nm gold–conjugated sheep  anti–rabbit Ig (right-hand panels). Samples were imaged on a Topcon DS130F scanning electron microscope using both secondary  electron and backscatter detectors. The micrographs displayed in  A show backscatter images superimposed over secondary images. Before superimposition, the backscatter images were chromatically inverted so that 10-nm gold particles appear as black  dots. (B) Aliquots of MP1 and MP2 were overlaid with a sucrose  density gradient and subjected to centrifugation as described in  Materials and Methods. 14 fractions were retrieved, from the top  of the centrifuge tube (lane 1) to the bottom (lane 14), and analyzed by SDS-PAGE and Western blotting for the presence of  NEP-B78 (top) using mAb 3E9 or LBR (bottom). Bars, 200 nm.
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Figure 8: Characterization of NEP-B78- and LBRx-containing vesicles in MP2 and MP1 fractions by (A) immunogold labeling and FEISEM and (B) sucrose density gradient centrifugation. (A) MP1 (top panels) or MP2 vesicles (bottom panels) were isolated on silicon chips, fixed with 4% paraformaldehyde and 0.1% glutaraldehyde and labeled with either mAb 3E9 followed by 10-nm gold–conjugated sheep anti–mouse Ig (left-hand panels) or rabbit anti–LBR followed by 10-nm gold–conjugated sheep anti–rabbit Ig (right-hand panels). Samples were imaged on a Topcon DS130F scanning electron microscope using both secondary electron and backscatter detectors. The micrographs displayed in A show backscatter images superimposed over secondary images. Before superimposition, the backscatter images were chromatically inverted so that 10-nm gold particles appear as black dots. (B) Aliquots of MP1 and MP2 were overlaid with a sucrose density gradient and subjected to centrifugation as described in Materials and Methods. 14 fractions were retrieved, from the top of the centrifuge tube (lane 1) to the bottom (lane 14), and analyzed by SDS-PAGE and Western blotting for the presence of NEP-B78 (top) using mAb 3E9 or LBR (bottom). Bars, 200 nm.

Mentions: To further characterize the discrete vesicles involved in NE assembly in the cell-free system, we performed a morphological analysis of NEP-B78- and LBRx-containing vesicles by immunogold labeling of membranes in MP1 and MP2 fractions in conjunction with FEISEM. Membranes were treated with either mAb 3E9 (to detect NEP-B78) or anti-LBR antibodies, followed by gold-tagged secondary antibodies, and examined using FEISEM (Fig. 8 A). We detected LBRx in larger ∼600-nm-diam “rough” vesicles that were enriched in the MP1 fraction (Fig. 8 A, upper right-hand panel). Importantly, no gold particles were observed in MP1 treated with mAb 3E9 (Fig. 8 A, upper left-hand panel). Conversely, we found NEP-B78 in “smooth” vesicles and tubular structures with variable diameter, which were enriched in MP2. No labeling of MP2 vesicles was observed with anti-LBR antibodies (Fig. 8 A, lower panels).


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)

Characterization of NEP-B78- and LBRx-containing  vesicles in MP2 and MP1 fractions by (A) immunogold labeling  and FEISEM and (B) sucrose density gradient centrifugation.  (A) MP1 (top panels) or MP2 vesicles (bottom panels) were isolated on silicon chips, fixed with 4% paraformaldehyde and 0.1%  glutaraldehyde and labeled with either mAb 3E9 followed by  10-nm gold–conjugated sheep anti–mouse Ig (left-hand panels)  or rabbit anti–LBR followed by 10-nm gold–conjugated sheep  anti–rabbit Ig (right-hand panels). Samples were imaged on a Topcon DS130F scanning electron microscope using both secondary  electron and backscatter detectors. The micrographs displayed in  A show backscatter images superimposed over secondary images. Before superimposition, the backscatter images were chromatically inverted so that 10-nm gold particles appear as black  dots. (B) Aliquots of MP1 and MP2 were overlaid with a sucrose  density gradient and subjected to centrifugation as described in  Materials and Methods. 14 fractions were retrieved, from the top  of the centrifuge tube (lane 1) to the bottom (lane 14), and analyzed by SDS-PAGE and Western blotting for the presence of  NEP-B78 (top) using mAb 3E9 or LBR (bottom). Bars, 200 nm.
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Figure 8: Characterization of NEP-B78- and LBRx-containing vesicles in MP2 and MP1 fractions by (A) immunogold labeling and FEISEM and (B) sucrose density gradient centrifugation. (A) MP1 (top panels) or MP2 vesicles (bottom panels) were isolated on silicon chips, fixed with 4% paraformaldehyde and 0.1% glutaraldehyde and labeled with either mAb 3E9 followed by 10-nm gold–conjugated sheep anti–mouse Ig (left-hand panels) or rabbit anti–LBR followed by 10-nm gold–conjugated sheep anti–rabbit Ig (right-hand panels). Samples were imaged on a Topcon DS130F scanning electron microscope using both secondary electron and backscatter detectors. The micrographs displayed in A show backscatter images superimposed over secondary images. Before superimposition, the backscatter images were chromatically inverted so that 10-nm gold particles appear as black dots. (B) Aliquots of MP1 and MP2 were overlaid with a sucrose density gradient and subjected to centrifugation as described in Materials and Methods. 14 fractions were retrieved, from the top of the centrifuge tube (lane 1) to the bottom (lane 14), and analyzed by SDS-PAGE and Western blotting for the presence of NEP-B78 (top) using mAb 3E9 or LBR (bottom). Bars, 200 nm.
Mentions: To further characterize the discrete vesicles involved in NE assembly in the cell-free system, we performed a morphological analysis of NEP-B78- and LBRx-containing vesicles by immunogold labeling of membranes in MP1 and MP2 fractions in conjunction with FEISEM. Membranes were treated with either mAb 3E9 (to detect NEP-B78) or anti-LBR antibodies, followed by gold-tagged secondary antibodies, and examined using FEISEM (Fig. 8 A). We detected LBRx in larger ∼600-nm-diam “rough” vesicles that were enriched in the MP1 fraction (Fig. 8 A, upper right-hand panel). Importantly, no gold particles were observed in MP1 treated with mAb 3E9 (Fig. 8 A, upper left-hand panel). Conversely, we found NEP-B78 in “smooth” vesicles and tubular structures with variable diameter, which were enriched in MP2. No labeling of MP2 vesicles was observed with anti-LBR antibodies (Fig. 8 A, lower panels).

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