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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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Reconstitution of NE assembly with purified particulate fractions. Demembranated sperm was incubated in cytosol  with the addition of MP1 (A), MP2 (B), MP1+ MP2 l (C), or no  additions (D) for 90 min at room temperature. Samples were processed for fluorescence microscopy (see Materials and Methods).  In each case the left-hand panel shows DNA visualized with  Hoechst 33258 and the right-hand panel shows the same sperm  stained with 3,3′-dihexyloxocarbocyanine (DHCC) to reveal  membranes. Bar, 10 μm.
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Figure 5: Reconstitution of NE assembly with purified particulate fractions. Demembranated sperm was incubated in cytosol with the addition of MP1 (A), MP2 (B), MP1+ MP2 l (C), or no additions (D) for 90 min at room temperature. Samples were processed for fluorescence microscopy (see Materials and Methods). In each case the left-hand panel shows DNA visualized with Hoechst 33258 and the right-hand panel shows the same sperm stained with 3,3′-dihexyloxocarbocyanine (DHCC) to reveal membranes. Bar, 10 μm.

Mentions: To investigate the possibility that two distinct vesicle populations mediate nuclear membrane assembly and that one population binds to chromatin before the other, we fractionated cell-free extracts of Xenopus eggs. Vigers and Lohka (1991) described the separation of two particulate fractions from LSS, which were required to reconstitute NE assembly, by differential and density dependent centrifugation. However, in the absence of biochemical markers, they were unable to unequivocally identify the nature of the components in each fraction that were necessary for NE reassembly. To test the hypothesis that NEP-B78 and LBRx defined the activities of two distinct NE-precursor vesicles, we repeated the fractionation procedure developed by Vigers and Lohka (1991). Two membrane containing fractions (termed MP1 and MP2) were separated and assayed individually or in combination for their ability to form a structurally and functionally competent NE when incubated with membrane-free cytosol (here termed S2004h) and demembranated sperm chromatin. These assays were analyzed by fluorescence microscopy using Hoechst 33258 to stain chromatin and DHCC for total membranes (Fig. 5, left- and right-hand columns, respectively). This revealed that MP1 did not contain membranes with the ability to bind to chromatin in the absence of MP2 (Fig. 5 A). In contrast, membranes did bind to chromatin upon incubation with MP2 alone (Fig. 5 B). The incubation of MP2 with cytosol and chromatin did not undergo any further morphological changes normally associated with complete NE reassembly (Wiese et al., 1997) even after prolonged incubation, and failed to initiate DNA replication (data not shown). When both vesicle-containing fractions were introduced into the assay system, full nuclear envelope assembly was supported as judged by continuous rim staining with DHCC and morphology characteristic of fully formed nuclei (Fig. 5 C) that underwent DNA replication (data not shown). No vesicles were observed upon incubation of demembranated sperm in S2004h (Fig. 5 D). These results indicate that the MP2 fraction was enriched in a vesicle type capable of binding to chromatin although, even after prolonged incubation, nuclear envelope formation did not occur and DNA replication was not supported. At the level of resolution of the light microscope, it was not possible to determine at which point NE assembly was arrested.


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)

Reconstitution of NE assembly with purified particulate fractions. Demembranated sperm was incubated in cytosol  with the addition of MP1 (A), MP2 (B), MP1+ MP2 l (C), or no  additions (D) for 90 min at room temperature. Samples were processed for fluorescence microscopy (see Materials and Methods).  In each case the left-hand panel shows DNA visualized with  Hoechst 33258 and the right-hand panel shows the same sperm  stained with 3,3′-dihexyloxocarbocyanine (DHCC) to reveal  membranes. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Reconstitution of NE assembly with purified particulate fractions. Demembranated sperm was incubated in cytosol with the addition of MP1 (A), MP2 (B), MP1+ MP2 l (C), or no additions (D) for 90 min at room temperature. Samples were processed for fluorescence microscopy (see Materials and Methods). In each case the left-hand panel shows DNA visualized with Hoechst 33258 and the right-hand panel shows the same sperm stained with 3,3′-dihexyloxocarbocyanine (DHCC) to reveal membranes. Bar, 10 μm.
Mentions: To investigate the possibility that two distinct vesicle populations mediate nuclear membrane assembly and that one population binds to chromatin before the other, we fractionated cell-free extracts of Xenopus eggs. Vigers and Lohka (1991) described the separation of two particulate fractions from LSS, which were required to reconstitute NE assembly, by differential and density dependent centrifugation. However, in the absence of biochemical markers, they were unable to unequivocally identify the nature of the components in each fraction that were necessary for NE reassembly. To test the hypothesis that NEP-B78 and LBRx defined the activities of two distinct NE-precursor vesicles, we repeated the fractionation procedure developed by Vigers and Lohka (1991). Two membrane containing fractions (termed MP1 and MP2) were separated and assayed individually or in combination for their ability to form a structurally and functionally competent NE when incubated with membrane-free cytosol (here termed S2004h) and demembranated sperm chromatin. These assays were analyzed by fluorescence microscopy using Hoechst 33258 to stain chromatin and DHCC for total membranes (Fig. 5, left- and right-hand columns, respectively). This revealed that MP1 did not contain membranes with the ability to bind to chromatin in the absence of MP2 (Fig. 5 A). In contrast, membranes did bind to chromatin upon incubation with MP2 alone (Fig. 5 B). The incubation of MP2 with cytosol and chromatin did not undergo any further morphological changes normally associated with complete NE reassembly (Wiese et al., 1997) even after prolonged incubation, and failed to initiate DNA replication (data not shown). When both vesicle-containing fractions were introduced into the assay system, full nuclear envelope assembly was supported as judged by continuous rim staining with DHCC and morphology characteristic of fully formed nuclei (Fig. 5 C) that underwent DNA replication (data not shown). No vesicles were observed upon incubation of demembranated sperm in S2004h (Fig. 5 D). These results indicate that the MP2 fraction was enriched in a vesicle type capable of binding to chromatin although, even after prolonged incubation, nuclear envelope formation did not occur and DNA replication was not supported. At the level of resolution of the light microscope, it was not possible to determine at which point NE assembly was arrested.

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