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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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Effect of NEP-B78 and p65 mAbs on vesicle recruitment and nuclear assembly. Aliquots of MP2 membranes were  incubated with either anti-cytokeratin 18 mAb (CK-18) as control or anti-NEP-B78 mAB 3E9 or the anti-p65 mAb CEL5C and  reisolated as described in Materials and Methods. (A) Vesicle recruitment assays were performed using demembranated sperm  chromatin and cytosol supplemented with indicated antibody-  treated MP2 alone. (B and C) NE assembly assays were performed using sperm chromatin, cytosol plus MP1 and indicated  antibody-treated MP2. In A and B, DNA was stained with Hoechst  33258 (left-hand panels) and membranes are stained with DHCC  (right-hand panels). In (C) MP2 previously treated with control  CK-18 mAb or 3E9 was incubated with cytosol, MP1 and sperm  chromatin, fixed for immunofluorescence and probed for the  presence of LBRx using an FITC-labeled secondary antibody.  Left-hand panels show DNA stained with DAPI with corresponding fluorescein-labeled images to the right.
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Figure 10: Effect of NEP-B78 and p65 mAbs on vesicle recruitment and nuclear assembly. Aliquots of MP2 membranes were incubated with either anti-cytokeratin 18 mAb (CK-18) as control or anti-NEP-B78 mAB 3E9 or the anti-p65 mAb CEL5C and reisolated as described in Materials and Methods. (A) Vesicle recruitment assays were performed using demembranated sperm chromatin and cytosol supplemented with indicated antibody- treated MP2 alone. (B and C) NE assembly assays were performed using sperm chromatin, cytosol plus MP1 and indicated antibody-treated MP2. In A and B, DNA was stained with Hoechst 33258 (left-hand panels) and membranes are stained with DHCC (right-hand panels). In (C) MP2 previously treated with control CK-18 mAb or 3E9 was incubated with cytosol, MP1 and sperm chromatin, fixed for immunofluorescence and probed for the presence of LBRx using an FITC-labeled secondary antibody. Left-hand panels show DNA stained with DAPI with corresponding fluorescein-labeled images to the right.

Mentions: The data presented in Figs. 3, 7, and 9 indicated that both p65 and NEP-B78 are present in vesicles that are targeted to chromatin in the first step of NE reassembly. To determine whether either of these proteins played any role in the process of NE reassembly, we incubated aliquots of MP2 fraction either with mAb 3E9 or mAb CEL5C. After reisolation of membranes, vesicle recruitment was assayed by the addition of cytosol and chromatin (Fig. 10 A), while NE assembly was assayed by the readdition of cytosol, chromatin, and the essential MP1 fraction (Fig. 10 B). In all cases, membranes were visualized using the membrane dye, DHCC. Preincubation of MP2 membranes with mAb 3E9, but not CEL5C or an irrelevant control antibody (CK18), abolished recruitment of membranes present in the MP2 fraction to chromatin periphery. Similarly, treatment of MP2 membranes with the NEP-B78 specific mAb also blocked recruitment of MP1-derived membrane and nuclear envelope reassembly failed to occur (Fig. 10 B). Antibodies to p65 had no effect on NE reassembly, nor did Fab fragments derived from mAb 3E9 (not shown). These results were confirmed by indirect immunofluorescence assays. Nuclei assembled in the presence of MP1, MP2, and cytosol, with and without prior treatment with 3E9 were assayed for recruitment of LBRx-containing vesicles. 3E9 treatment of MP2 vesicles completely inhibited subsequent incorporation of LBRx into the NE in this reconstituted extract.


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)

Effect of NEP-B78 and p65 mAbs on vesicle recruitment and nuclear assembly. Aliquots of MP2 membranes were  incubated with either anti-cytokeratin 18 mAb (CK-18) as control or anti-NEP-B78 mAB 3E9 or the anti-p65 mAb CEL5C and  reisolated as described in Materials and Methods. (A) Vesicle recruitment assays were performed using demembranated sperm  chromatin and cytosol supplemented with indicated antibody-  treated MP2 alone. (B and C) NE assembly assays were performed using sperm chromatin, cytosol plus MP1 and indicated  antibody-treated MP2. In A and B, DNA was stained with Hoechst  33258 (left-hand panels) and membranes are stained with DHCC  (right-hand panels). In (C) MP2 previously treated with control  CK-18 mAb or 3E9 was incubated with cytosol, MP1 and sperm  chromatin, fixed for immunofluorescence and probed for the  presence of LBRx using an FITC-labeled secondary antibody.  Left-hand panels show DNA stained with DAPI with corresponding fluorescein-labeled images to the right.
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Related In: Results  -  Collection

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Figure 10: Effect of NEP-B78 and p65 mAbs on vesicle recruitment and nuclear assembly. Aliquots of MP2 membranes were incubated with either anti-cytokeratin 18 mAb (CK-18) as control or anti-NEP-B78 mAB 3E9 or the anti-p65 mAb CEL5C and reisolated as described in Materials and Methods. (A) Vesicle recruitment assays were performed using demembranated sperm chromatin and cytosol supplemented with indicated antibody- treated MP2 alone. (B and C) NE assembly assays were performed using sperm chromatin, cytosol plus MP1 and indicated antibody-treated MP2. In A and B, DNA was stained with Hoechst 33258 (left-hand panels) and membranes are stained with DHCC (right-hand panels). In (C) MP2 previously treated with control CK-18 mAb or 3E9 was incubated with cytosol, MP1 and sperm chromatin, fixed for immunofluorescence and probed for the presence of LBRx using an FITC-labeled secondary antibody. Left-hand panels show DNA stained with DAPI with corresponding fluorescein-labeled images to the right.
Mentions: The data presented in Figs. 3, 7, and 9 indicated that both p65 and NEP-B78 are present in vesicles that are targeted to chromatin in the first step of NE reassembly. To determine whether either of these proteins played any role in the process of NE reassembly, we incubated aliquots of MP2 fraction either with mAb 3E9 or mAb CEL5C. After reisolation of membranes, vesicle recruitment was assayed by the addition of cytosol and chromatin (Fig. 10 A), while NE assembly was assayed by the readdition of cytosol, chromatin, and the essential MP1 fraction (Fig. 10 B). In all cases, membranes were visualized using the membrane dye, DHCC. Preincubation of MP2 membranes with mAb 3E9, but not CEL5C or an irrelevant control antibody (CK18), abolished recruitment of membranes present in the MP2 fraction to chromatin periphery. Similarly, treatment of MP2 membranes with the NEP-B78 specific mAb also blocked recruitment of MP1-derived membrane and nuclear envelope reassembly failed to occur (Fig. 10 B). Antibodies to p65 had no effect on NE reassembly, nor did Fab fragments derived from mAb 3E9 (not shown). These results were confirmed by indirect immunofluorescence assays. Nuclei assembled in the presence of MP1, MP2, and cytosol, with and without prior treatment with 3E9 were assayed for recruitment of LBRx-containing vesicles. 3E9 treatment of MP2 vesicles completely inhibited subsequent incorporation of LBRx into the NE in this reconstituted extract.

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