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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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Immunoblotting analysis of NEP-B78, p65, and LBRx.  (A) Egg extracts (LSS) or isolated sperm pronuclei (NUC) were  resolved by 8% SDS-PAGE, transferred to nitrocellulose and  blotted with one of mAbs 3E9, 4G12, 16H12 or CEL5C. 3E9,  4G12 and 16H12 all detected a protein migrating with a Mr of 78 kD  (termed NEP-B78) in each fraction. CEL5C detected a single  band migrating at 65 kD (termed p65) in each fraction. The positions of prestained molecular mass markers are shown at the left-hand side of the panel. (B) Xenopus LSS was fractionated by  centrifugation at 200,000 g for 4 h to yield membrane-free supernatant (S200) and a nuclear membrane precursor fraction  (MEM) that is sufficient for complete NE assembly (Smythe and  Newport, 1991). Equivalent volumes of each fraction were resolved by SDS-PAGE and subjected to immunoblotting with the  indicated antibodies for NEP-B78, p65, or LBRx. (C) Oocyte  germinal vesicles were manually dissected from stage six oocytes  and incubated with egg lysis buffer (Buffer), 1 M KCl, 1 M Na2CO3  (1 M carbonate), 6 M Urea or 1% Triton X-100 (all in egg lysis  buffer). Soluble proteins (S) were separated from insoluble proteins (P) by centrifugation, resolved on 8% SDS-PAGE and blotted with either 4G12 or CEL5C. Only Triton X-100 was capable  of solubilizing NEP-B78 and p65.
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Figure 1: Immunoblotting analysis of NEP-B78, p65, and LBRx. (A) Egg extracts (LSS) or isolated sperm pronuclei (NUC) were resolved by 8% SDS-PAGE, transferred to nitrocellulose and blotted with one of mAbs 3E9, 4G12, 16H12 or CEL5C. 3E9, 4G12 and 16H12 all detected a protein migrating with a Mr of 78 kD (termed NEP-B78) in each fraction. CEL5C detected a single band migrating at 65 kD (termed p65) in each fraction. The positions of prestained molecular mass markers are shown at the left-hand side of the panel. (B) Xenopus LSS was fractionated by centrifugation at 200,000 g for 4 h to yield membrane-free supernatant (S200) and a nuclear membrane precursor fraction (MEM) that is sufficient for complete NE assembly (Smythe and Newport, 1991). Equivalent volumes of each fraction were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies for NEP-B78, p65, or LBRx. (C) Oocyte germinal vesicles were manually dissected from stage six oocytes and incubated with egg lysis buffer (Buffer), 1 M KCl, 1 M Na2CO3 (1 M carbonate), 6 M Urea or 1% Triton X-100 (all in egg lysis buffer). Soluble proteins (S) were separated from insoluble proteins (P) by centrifugation, resolved on 8% SDS-PAGE and blotted with either 4G12 or CEL5C. Only Triton X-100 was capable of solubilizing NEP-B78 and p65.

Mentions: The antigen recognized by four of the mAbs was characterized by immunoblotting. Egg extract (LSS) and whole, isolated, in vitro assembled nuclei were resolved on SDS-PAGE and blotted against one of mAbs 3E9, 4G12, 16H12, or CEL5C (Fig. 1 A). mAbs 3E9, 4G12 and 16H12 each detected a protein of Mr ∼78 kD in LSS and isolated nuclei, which in some gels could be resolved into a closely migrating doublet (e.g., Fig. 1 A, 16H12). In contrast, mAb CEL5C detected a single band in LSS and isolated nuclei, migrating with a Mr of 65 kD. LSS was then fractionated into a nuclear envelope precursor membrane fraction and a membrane-free supernatant (Smythe and Newport, 1991). Each fraction was resolved on SDS-PAGE and blotted with the mAbs as well as affinity-purified antibodies to LBR (Fig. 1 B). Both the 78- and the 65-kD protein were only detected in the membrane fraction and were absent from the membrane-free supernatant, indicating that both antigens were membrane proteins (Fig. 1 B). As expected, affinity-purified antibodies to LBR (Fig. 1 B) recognized a single, major ∼56-kD protein exclusively in the membrane fraction (Fig. 1 B) and we have designated this protein, LBRx, for LBR-cross reacting protein. Since mAbs 3E9, 16H12, and 4G12 were all raised against a nuclear envelope precursor vesicle fraction (termed NEP-B; Vigers and Lohka, 1991) that binds to chromatin in vitro, and each antibody detects the same 78-kD membrane protein, we adopted the term NEP-B78 to describe this protein. The 65-kD protein is present in two membrane fractions (see below) and is hereafter referred to as p65.


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)

Immunoblotting analysis of NEP-B78, p65, and LBRx.  (A) Egg extracts (LSS) or isolated sperm pronuclei (NUC) were  resolved by 8% SDS-PAGE, transferred to nitrocellulose and  blotted with one of mAbs 3E9, 4G12, 16H12 or CEL5C. 3E9,  4G12 and 16H12 all detected a protein migrating with a Mr of 78 kD  (termed NEP-B78) in each fraction. CEL5C detected a single  band migrating at 65 kD (termed p65) in each fraction. The positions of prestained molecular mass markers are shown at the left-hand side of the panel. (B) Xenopus LSS was fractionated by  centrifugation at 200,000 g for 4 h to yield membrane-free supernatant (S200) and a nuclear membrane precursor fraction  (MEM) that is sufficient for complete NE assembly (Smythe and  Newport, 1991). Equivalent volumes of each fraction were resolved by SDS-PAGE and subjected to immunoblotting with the  indicated antibodies for NEP-B78, p65, or LBRx. (C) Oocyte  germinal vesicles were manually dissected from stage six oocytes  and incubated with egg lysis buffer (Buffer), 1 M KCl, 1 M Na2CO3  (1 M carbonate), 6 M Urea or 1% Triton X-100 (all in egg lysis  buffer). Soluble proteins (S) were separated from insoluble proteins (P) by centrifugation, resolved on 8% SDS-PAGE and blotted with either 4G12 or CEL5C. Only Triton X-100 was capable  of solubilizing NEP-B78 and p65.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132889&req=5

Figure 1: Immunoblotting analysis of NEP-B78, p65, and LBRx. (A) Egg extracts (LSS) or isolated sperm pronuclei (NUC) were resolved by 8% SDS-PAGE, transferred to nitrocellulose and blotted with one of mAbs 3E9, 4G12, 16H12 or CEL5C. 3E9, 4G12 and 16H12 all detected a protein migrating with a Mr of 78 kD (termed NEP-B78) in each fraction. CEL5C detected a single band migrating at 65 kD (termed p65) in each fraction. The positions of prestained molecular mass markers are shown at the left-hand side of the panel. (B) Xenopus LSS was fractionated by centrifugation at 200,000 g for 4 h to yield membrane-free supernatant (S200) and a nuclear membrane precursor fraction (MEM) that is sufficient for complete NE assembly (Smythe and Newport, 1991). Equivalent volumes of each fraction were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies for NEP-B78, p65, or LBRx. (C) Oocyte germinal vesicles were manually dissected from stage six oocytes and incubated with egg lysis buffer (Buffer), 1 M KCl, 1 M Na2CO3 (1 M carbonate), 6 M Urea or 1% Triton X-100 (all in egg lysis buffer). Soluble proteins (S) were separated from insoluble proteins (P) by centrifugation, resolved on 8% SDS-PAGE and blotted with either 4G12 or CEL5C. Only Triton X-100 was capable of solubilizing NEP-B78 and p65.
Mentions: The antigen recognized by four of the mAbs was characterized by immunoblotting. Egg extract (LSS) and whole, isolated, in vitro assembled nuclei were resolved on SDS-PAGE and blotted against one of mAbs 3E9, 4G12, 16H12, or CEL5C (Fig. 1 A). mAbs 3E9, 4G12 and 16H12 each detected a protein of Mr ∼78 kD in LSS and isolated nuclei, which in some gels could be resolved into a closely migrating doublet (e.g., Fig. 1 A, 16H12). In contrast, mAb CEL5C detected a single band in LSS and isolated nuclei, migrating with a Mr of 65 kD. LSS was then fractionated into a nuclear envelope precursor membrane fraction and a membrane-free supernatant (Smythe and Newport, 1991). Each fraction was resolved on SDS-PAGE and blotted with the mAbs as well as affinity-purified antibodies to LBR (Fig. 1 B). Both the 78- and the 65-kD protein were only detected in the membrane fraction and were absent from the membrane-free supernatant, indicating that both antigens were membrane proteins (Fig. 1 B). As expected, affinity-purified antibodies to LBR (Fig. 1 B) recognized a single, major ∼56-kD protein exclusively in the membrane fraction (Fig. 1 B) and we have designated this protein, LBRx, for LBR-cross reacting protein. Since mAbs 3E9, 16H12, and 4G12 were all raised against a nuclear envelope precursor vesicle fraction (termed NEP-B; Vigers and Lohka, 1991) that binds to chromatin in vitro, and each antibody detects the same 78-kD membrane protein, we adopted the term NEP-B78 to describe this protein. The 65-kD protein is present in two membrane fractions (see below) and is hereafter referred to as p65.

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