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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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Biochemical characterization of MP1 and MP2 fractions; localization of NEP-B78, p65, LBRx, and B-type lamins in  egg extract fractions. (A) Total egg extract was subjected to fractionation by centrifugation as described (Smythe and Newport,  1991) to produce a total NE precursor membrane fraction (tot)  or as described in (Vigers and Lohka, 1991) to yield MP1 and  MP2 fractions. Equal protein loadings of each fraction were subjected to SDS-PAGE and stained with Coomassie blue. (B) Total  egg extract (LSS) was subjected to fractionation by centrifugation as described (Vigers and Lohka, 1991) to yield MP1 and  MP2 fractions and a membrane-free cytosol (S2004h). Volumes  of each fraction, proportional to that found in a single egg, were  resolved on an 8% polyacrylamide gel and subjected to immunoblotting for NEP-B78 (top) using mAb 4G12, p65 (middle), or  LBRx (bottom). In each case, lane 1 contains low speed supernatant (LSS) and lanes 2–4 contain MP1, MP2 and cytosol, respectively. (C) MP1 and MP2 fractions were resolved on a 10% gel  and subjected to immunoblotting for B-type lamins using the  mAb L6 8A7.
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Figure 7: Biochemical characterization of MP1 and MP2 fractions; localization of NEP-B78, p65, LBRx, and B-type lamins in egg extract fractions. (A) Total egg extract was subjected to fractionation by centrifugation as described (Smythe and Newport, 1991) to produce a total NE precursor membrane fraction (tot) or as described in (Vigers and Lohka, 1991) to yield MP1 and MP2 fractions. Equal protein loadings of each fraction were subjected to SDS-PAGE and stained with Coomassie blue. (B) Total egg extract (LSS) was subjected to fractionation by centrifugation as described (Vigers and Lohka, 1991) to yield MP1 and MP2 fractions and a membrane-free cytosol (S2004h). Volumes of each fraction, proportional to that found in a single egg, were resolved on an 8% polyacrylamide gel and subjected to immunoblotting for NEP-B78 (top) using mAb 4G12, p65 (middle), or LBRx (bottom). In each case, lane 1 contains low speed supernatant (LSS) and lanes 2–4 contain MP1, MP2 and cytosol, respectively. (C) MP1 and MP2 fractions were resolved on a 10% gel and subjected to immunoblotting for B-type lamins using the mAb L6 8A7.

Mentions: To determine whether we could detect any biochemical differences between membrane fractions required for NE assembly, all fractions obtained by differential centrifugation were subjected to SDS-PAGE and either stained with Coomassie blue for protein or immunoblotted with antibodies to NEP-B78, LBR, p65, and B-type lamins. Coomassie blue staining of total and fractionated membranes indicated that MP1 and MP2 comprised different but partially overlapping sets of proteins (Fig. 7 A). Fractionation of a defined volume of eggs yields differing volumes of the three fractions of interest. Therefore to investigate the distribution of specific proteins between these fractions, volumes of each fraction, proportional to their ratios in a single egg, were examined (Fig. 7, B and C). Western blotting analysis (Fig. 7 B) revealed that antibodies to NEP-B78, LBR, and p65 detected their cognate proteins in an unfractionated extract (lane 1), and as expected for integral membrane proteins, were undetectable in the cytosolic fraction obtained by prolonged high speed centrifugation (lane 4). We found that p65 (Fig. 7 B, middle panels), was distributed approximately equally between the MP1 and MP2 fractions. Importantly, NEP-B78 was found exclusively in the MP2 fraction (Fig. 7 B, upper panels), while LBRx was observed only in the MP1 fraction (Fig. 7 B, lower panels). Isolated MP1 and MP2 fractions were also blotted for the presence of B-type lamins using the mAb L6 8A7 that detects lamins B1, B2, and B3. In Xenopus egg extracts, the major lamin isoform present is B3, which is largely soluble and present in the cytosolic fraction (Jenkins et al., 1993b; Meier et al., 1991). However, both lamin B3 and low levels of lamin B2 have been detected in isolated Xenopus membrane fractions (Lourim and Krohne, 1993). Using mAb L6 8A7 to detect both B3 and B2, we found that a band of the expected mobility for lamin B3 was indeed present in MP1 membranes but not in MP2. In addition, we observed an additional band with reduced electrophoretic mobility in both MP1 and MP2 (Fig. 7 C)   presumably corresponding to lamin B2 (Lourim and Krohne, 1993). The results indicate that the fractionation procedure gives rise to membrane preparations that are biochemically distinct.


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)

Biochemical characterization of MP1 and MP2 fractions; localization of NEP-B78, p65, LBRx, and B-type lamins in  egg extract fractions. (A) Total egg extract was subjected to fractionation by centrifugation as described (Smythe and Newport,  1991) to produce a total NE precursor membrane fraction (tot)  or as described in (Vigers and Lohka, 1991) to yield MP1 and  MP2 fractions. Equal protein loadings of each fraction were subjected to SDS-PAGE and stained with Coomassie blue. (B) Total  egg extract (LSS) was subjected to fractionation by centrifugation as described (Vigers and Lohka, 1991) to yield MP1 and  MP2 fractions and a membrane-free cytosol (S2004h). Volumes  of each fraction, proportional to that found in a single egg, were  resolved on an 8% polyacrylamide gel and subjected to immunoblotting for NEP-B78 (top) using mAb 4G12, p65 (middle), or  LBRx (bottom). In each case, lane 1 contains low speed supernatant (LSS) and lanes 2–4 contain MP1, MP2 and cytosol, respectively. (C) MP1 and MP2 fractions were resolved on a 10% gel  and subjected to immunoblotting for B-type lamins using the  mAb L6 8A7.
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Figure 7: Biochemical characterization of MP1 and MP2 fractions; localization of NEP-B78, p65, LBRx, and B-type lamins in egg extract fractions. (A) Total egg extract was subjected to fractionation by centrifugation as described (Smythe and Newport, 1991) to produce a total NE precursor membrane fraction (tot) or as described in (Vigers and Lohka, 1991) to yield MP1 and MP2 fractions. Equal protein loadings of each fraction were subjected to SDS-PAGE and stained with Coomassie blue. (B) Total egg extract (LSS) was subjected to fractionation by centrifugation as described (Vigers and Lohka, 1991) to yield MP1 and MP2 fractions and a membrane-free cytosol (S2004h). Volumes of each fraction, proportional to that found in a single egg, were resolved on an 8% polyacrylamide gel and subjected to immunoblotting for NEP-B78 (top) using mAb 4G12, p65 (middle), or LBRx (bottom). In each case, lane 1 contains low speed supernatant (LSS) and lanes 2–4 contain MP1, MP2 and cytosol, respectively. (C) MP1 and MP2 fractions were resolved on a 10% gel and subjected to immunoblotting for B-type lamins using the mAb L6 8A7.
Mentions: To determine whether we could detect any biochemical differences between membrane fractions required for NE assembly, all fractions obtained by differential centrifugation were subjected to SDS-PAGE and either stained with Coomassie blue for protein or immunoblotted with antibodies to NEP-B78, LBR, p65, and B-type lamins. Coomassie blue staining of total and fractionated membranes indicated that MP1 and MP2 comprised different but partially overlapping sets of proteins (Fig. 7 A). Fractionation of a defined volume of eggs yields differing volumes of the three fractions of interest. Therefore to investigate the distribution of specific proteins between these fractions, volumes of each fraction, proportional to their ratios in a single egg, were examined (Fig. 7, B and C). Western blotting analysis (Fig. 7 B) revealed that antibodies to NEP-B78, LBR, and p65 detected their cognate proteins in an unfractionated extract (lane 1), and as expected for integral membrane proteins, were undetectable in the cytosolic fraction obtained by prolonged high speed centrifugation (lane 4). We found that p65 (Fig. 7 B, middle panels), was distributed approximately equally between the MP1 and MP2 fractions. Importantly, NEP-B78 was found exclusively in the MP2 fraction (Fig. 7 B, upper panels), while LBRx was observed only in the MP1 fraction (Fig. 7 B, lower panels). Isolated MP1 and MP2 fractions were also blotted for the presence of B-type lamins using the mAb L6 8A7 that detects lamins B1, B2, and B3. In Xenopus egg extracts, the major lamin isoform present is B3, which is largely soluble and present in the cytosolic fraction (Jenkins et al., 1993b; Meier et al., 1991). However, both lamin B3 and low levels of lamin B2 have been detected in isolated Xenopus membrane fractions (Lourim and Krohne, 1993). Using mAb L6 8A7 to detect both B3 and B2, we found that a band of the expected mobility for lamin B3 was indeed present in MP1 membranes but not in MP2. In addition, we observed an additional band with reduced electrophoretic mobility in both MP1 and MP2 (Fig. 7 C)   presumably corresponding to lamin B2 (Lourim and Krohne, 1993). The results indicate that the fractionation procedure gives rise to membrane preparations that are biochemically distinct.

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