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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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Electron microscope analysis of vesicle binding and fusion. MP2 was incubated with sperm chromatin  and cytosol alone (A–C) or  with sperm chromatin, cytosol, and MP1 (D–E) as described in Materials and  Methods. Incubations for the  samples displayed were for  2 h at room temperature after  which time the extracts were  fixed and sectioned for TEM.  A–C show MP2 vesicles  bound to the surface of partially decondensed sperm  chromatin. White arrows show  details of membrane morphology at sites of interaction with  chromatin (B). Black arrows  show typical membrane morphology at sites adjacent to  other vesicles (C). D–F show  nuclear envelope structures  typically observed in fully reconstituted extracts. Double  unit membranes formed a continuous boundary around decondensed chromatin (D) and  contained structures resembling nuclear pores (E) and  were studded with ribosome-like particles (F, arrowheads).  INM, inner nuclear membrane; ONM, outer nuclear  membrane. Bars: (A and D) 1  μm; (B and E) 100 nm; (C and  F) 200 nm.
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Figure 6: Electron microscope analysis of vesicle binding and fusion. MP2 was incubated with sperm chromatin and cytosol alone (A–C) or with sperm chromatin, cytosol, and MP1 (D–E) as described in Materials and Methods. Incubations for the samples displayed were for 2 h at room temperature after which time the extracts were fixed and sectioned for TEM. A–C show MP2 vesicles bound to the surface of partially decondensed sperm chromatin. White arrows show details of membrane morphology at sites of interaction with chromatin (B). Black arrows show typical membrane morphology at sites adjacent to other vesicles (C). D–F show nuclear envelope structures typically observed in fully reconstituted extracts. Double unit membranes formed a continuous boundary around decondensed chromatin (D) and contained structures resembling nuclear pores (E) and were studded with ribosome-like particles (F, arrowheads). INM, inner nuclear membrane; ONM, outer nuclear membrane. Bars: (A and D) 1 μm; (B and E) 100 nm; (C and F) 200 nm.

Mentions: To do this, samples from each of the assays described above were therefore fixed and sectioned for visualization by electron microscopy (Fig. 6) as described in Materials and Methods. Fig. 6 A shows typical structures resulting from incubations of sperm with MP2 and cytosol. The decondensed chromatin was extensively covered with a single layer of vesicles. At higher resolution, vesicles appeared distorted in two distinct ways. At points of contact with chromatin, vesicles appeared crenulated (white arrows, Fig. 6 B), while vesicles with adjacent surfaces appeared to adopt complementary shapes (black arrows, Fig. 6 C). However, even vesicles tightly juxtaposed did not fuse with one another (shown in detail in Fig. 6, B and C).


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)

Electron microscope analysis of vesicle binding and fusion. MP2 was incubated with sperm chromatin  and cytosol alone (A–C) or  with sperm chromatin, cytosol, and MP1 (D–E) as described in Materials and  Methods. Incubations for the  samples displayed were for  2 h at room temperature after  which time the extracts were  fixed and sectioned for TEM.  A–C show MP2 vesicles  bound to the surface of partially decondensed sperm  chromatin. White arrows show  details of membrane morphology at sites of interaction with  chromatin (B). Black arrows  show typical membrane morphology at sites adjacent to  other vesicles (C). D–F show  nuclear envelope structures  typically observed in fully reconstituted extracts. Double  unit membranes formed a continuous boundary around decondensed chromatin (D) and  contained structures resembling nuclear pores (E) and  were studded with ribosome-like particles (F, arrowheads).  INM, inner nuclear membrane; ONM, outer nuclear  membrane. Bars: (A and D) 1  μm; (B and E) 100 nm; (C and  F) 200 nm.
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Figure 6: Electron microscope analysis of vesicle binding and fusion. MP2 was incubated with sperm chromatin and cytosol alone (A–C) or with sperm chromatin, cytosol, and MP1 (D–E) as described in Materials and Methods. Incubations for the samples displayed were for 2 h at room temperature after which time the extracts were fixed and sectioned for TEM. A–C show MP2 vesicles bound to the surface of partially decondensed sperm chromatin. White arrows show details of membrane morphology at sites of interaction with chromatin (B). Black arrows show typical membrane morphology at sites adjacent to other vesicles (C). D–F show nuclear envelope structures typically observed in fully reconstituted extracts. Double unit membranes formed a continuous boundary around decondensed chromatin (D) and contained structures resembling nuclear pores (E) and were studded with ribosome-like particles (F, arrowheads). INM, inner nuclear membrane; ONM, outer nuclear membrane. Bars: (A and D) 1 μm; (B and E) 100 nm; (C and F) 200 nm.
Mentions: To do this, samples from each of the assays described above were therefore fixed and sectioned for visualization by electron microscopy (Fig. 6) as described in Materials and Methods. Fig. 6 A shows typical structures resulting from incubations of sperm with MP2 and cytosol. The decondensed chromatin was extensively covered with a single layer of vesicles. At higher resolution, vesicles appeared distorted in two distinct ways. At points of contact with chromatin, vesicles appeared crenulated (white arrows, Fig. 6 B), while vesicles with adjacent surfaces appeared to adopt complementary shapes (black arrows, Fig. 6 C). However, even vesicles tightly juxtaposed did not fuse with one another (shown in detail in Fig. 6, B and C).

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