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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, p65, and LBRx to chromatin using isolated membrane fractions. Nuclear assembly assays were performed in which demembranated sperm was incubated in cytosol (S2004h) with MP1, MP2, MP1 + MP2 or with no additions for 90 min  and prepared for immunofluorescence microscopy (see Materials and Methods). (A) Samples were diluted in 0.33× extract buffer containing EGS, incubated at 37°C for 30 min and centrifuged onto coverslips before incubation with antibodies as described in Materials  and Methods. Assays were probed for the presence of NEP-B78 (left-hand micrographs) using mAb 4G12, p65 (center micrographs) or  LBRx (right-hand micrographs), and in each case the left-hand column shows DNA stained with DAPI and the right-hand column  shows the corresponding fluorescein-labeled images. (B) Samples were diluted in 0.33× extract buffer, and centrifuged onto coverslips  before incubation with EGS followed by antibodies for double immunofluorescence microscopy as described in Materials and Methods.  DNA was stained with DAPI (left-hand panels), NEP-B78 was detected using FITC-labeled secondary antibodies (middle panels) and  LBRx was detected using TRITC-labeled secondary antibodies (right-hand panels). Bars, 20 μm.
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Figure 9: Recruitment of NEP-B78, p65, and LBRx to chromatin using isolated membrane fractions. Nuclear assembly assays were performed in which demembranated sperm was incubated in cytosol (S2004h) with MP1, MP2, MP1 + MP2 or with no additions for 90 min and prepared for immunofluorescence microscopy (see Materials and Methods). (A) Samples were diluted in 0.33× extract buffer containing EGS, incubated at 37°C for 30 min and centrifuged onto coverslips before incubation with antibodies as described in Materials and Methods. Assays were probed for the presence of NEP-B78 (left-hand micrographs) using mAb 4G12, p65 (center micrographs) or LBRx (right-hand micrographs), and in each case the left-hand column shows DNA stained with DAPI and the right-hand column shows the corresponding fluorescein-labeled images. (B) Samples were diluted in 0.33× extract buffer, and centrifuged onto coverslips before incubation with EGS followed by antibodies for double immunofluorescence microscopy as described in Materials and Methods. DNA was stained with DAPI (left-hand panels), NEP-B78 was detected using FITC-labeled secondary antibodies (middle panels) and LBRx was detected using TRITC-labeled secondary antibodies (right-hand panels). Bars, 20 μm.

Mentions: We then used the isolated membrane fractions separately and in combination in nuclear assembly assays that we analyzed by indirect immunofluorescence microscopy using all three antibodies as probes. Fig. 9, a shows the results obtained when incubation mixtures containing cytosol plus MP1 alone, cytosol plus MP2 alone, cytosol plus MP1 and MP2 or cytosol alone were incubated in the presence of demembranated sperm chromatin, and an energy regeneration system. The results show that NEP-B78 was recruited to the surface of chromatin when MP2 vesicles alone were used (Fig. 9 A, MP2), but not when MP1 vesicles alone were present (Fig. 9 A, MP1), and consequently was found in the complete nuclear envelope that is formed when MP1 and MP2 were present together (Fig. 9 A, MP1 + MP2). Interestingly, recruitment of p65 was also only observed if MP2 vesicles were included in the assembly incubation. Importantly, recruitment of LBRx was not observed when incubations contained either MP2 alone or MP1 alone, but as expected, recruitment of LBRx was observed in the reconstituted mixture giving a morphologically normal nucleus. These results were obtained using a standard protocol for the detection by indirect immunofluorescence of nuclear antigens in the Xenopus cell-free system (Hutchison, 1994) and involves cross-linking with EGS before dilution and centrifugation of assembled nuclei onto coverslips. The absence of p65 and LBRx staining after incubation of chromatin and MP1 alone indicated that nonspecific cross-linking of membranes to chromatin with EGS was unlikely. However, we repeated this experiment with an altered protocol in which chromatin, after incubation with cytosol and the relevant membrane fraction, was diluted and pelleted onto coverslips before cross-linking with EGS to eliminate this possibility. In this experiment, NEP-B78 and LBRx were visualized after double immunofluorescence labeling with mAb 3E9 and anti-LBR antibodies. Colocalization between these antibodies was observed only in fully reconstituted nuclei (MP1 + MP2; Fig. 9 B). We have also performed colocalization between lamin B3 and LBRx again with the result that colocalization was only observed in fully reconstituted nuclei (data not shown). The results in Fig. 9 B, confirm that recruitment of LBRx from the MP1 fraction is only observed in incubations also containing MP2.


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, p65, and LBRx to chromatin using isolated membrane fractions. Nuclear assembly assays were performed in which demembranated sperm was incubated in cytosol (S2004h) with MP1, MP2, MP1 + MP2 or with no additions for 90 min  and prepared for immunofluorescence microscopy (see Materials and Methods). (A) Samples were diluted in 0.33× extract buffer containing EGS, incubated at 37°C for 30 min and centrifuged onto coverslips before incubation with antibodies as described in Materials  and Methods. Assays were probed for the presence of NEP-B78 (left-hand micrographs) using mAb 4G12, p65 (center micrographs) or  LBRx (right-hand micrographs), and in each case the left-hand column shows DNA stained with DAPI and the right-hand column  shows the corresponding fluorescein-labeled images. (B) Samples were diluted in 0.33× extract buffer, and centrifuged onto coverslips  before incubation with EGS followed by antibodies for double immunofluorescence microscopy as described in Materials and Methods.  DNA was stained with DAPI (left-hand panels), NEP-B78 was detected using FITC-labeled secondary antibodies (middle panels) and  LBRx was detected using TRITC-labeled secondary antibodies (right-hand panels). Bars, 20 μm.
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Related In: Results  -  Collection

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Figure 9: Recruitment of NEP-B78, p65, and LBRx to chromatin using isolated membrane fractions. Nuclear assembly assays were performed in which demembranated sperm was incubated in cytosol (S2004h) with MP1, MP2, MP1 + MP2 or with no additions for 90 min and prepared for immunofluorescence microscopy (see Materials and Methods). (A) Samples were diluted in 0.33× extract buffer containing EGS, incubated at 37°C for 30 min and centrifuged onto coverslips before incubation with antibodies as described in Materials and Methods. Assays were probed for the presence of NEP-B78 (left-hand micrographs) using mAb 4G12, p65 (center micrographs) or LBRx (right-hand micrographs), and in each case the left-hand column shows DNA stained with DAPI and the right-hand column shows the corresponding fluorescein-labeled images. (B) Samples were diluted in 0.33× extract buffer, and centrifuged onto coverslips before incubation with EGS followed by antibodies for double immunofluorescence microscopy as described in Materials and Methods. DNA was stained with DAPI (left-hand panels), NEP-B78 was detected using FITC-labeled secondary antibodies (middle panels) and LBRx was detected using TRITC-labeled secondary antibodies (right-hand panels). Bars, 20 μm.
Mentions: We then used the isolated membrane fractions separately and in combination in nuclear assembly assays that we analyzed by indirect immunofluorescence microscopy using all three antibodies as probes. Fig. 9, a shows the results obtained when incubation mixtures containing cytosol plus MP1 alone, cytosol plus MP2 alone, cytosol plus MP1 and MP2 or cytosol alone were incubated in the presence of demembranated sperm chromatin, and an energy regeneration system. The results show that NEP-B78 was recruited to the surface of chromatin when MP2 vesicles alone were used (Fig. 9 A, MP2), but not when MP1 vesicles alone were present (Fig. 9 A, MP1), and consequently was found in the complete nuclear envelope that is formed when MP1 and MP2 were present together (Fig. 9 A, MP1 + MP2). Interestingly, recruitment of p65 was also only observed if MP2 vesicles were included in the assembly incubation. Importantly, recruitment of LBRx was not observed when incubations contained either MP2 alone or MP1 alone, but as expected, recruitment of LBRx was observed in the reconstituted mixture giving a morphologically normal nucleus. These results were obtained using a standard protocol for the detection by indirect immunofluorescence of nuclear antigens in the Xenopus cell-free system (Hutchison, 1994) and involves cross-linking with EGS before dilution and centrifugation of assembled nuclei onto coverslips. The absence of p65 and LBRx staining after incubation of chromatin and MP1 alone indicated that nonspecific cross-linking of membranes to chromatin with EGS was unlikely. However, we repeated this experiment with an altered protocol in which chromatin, after incubation with cytosol and the relevant membrane fraction, was diluted and pelleted onto coverslips before cross-linking with EGS to eliminate this possibility. In this experiment, NEP-B78 and LBRx were visualized after double immunofluorescence labeling with mAb 3E9 and anti-LBR antibodies. Colocalization between these antibodies was observed only in fully reconstituted nuclei (MP1 + MP2; Fig. 9 B). We have also performed colocalization between lamin B3 and LBRx again with the result that colocalization was only observed in fully reconstituted nuclei (data not shown). The results in Fig. 9 B, confirm that recruitment of LBRx from the MP1 fraction is only observed in incubations also containing MP2.

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