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Microinjection of anti-coilin antibodies affects the structure of coiled bodies.

Almeida F, Saffrich R, Ansorge W, Carmo-Fonseca M - J. Cell Biol. (1998)

Bottom Line: After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d.Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence.Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts.

View Article: PubMed Central - PubMed

Affiliation: Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal.

ABSTRACT
The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.

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Immunoblot analysis of anti-coilin mAbs. An 8% acrylamide gel was loaded with either total protein extract from HeLa  cells (2 × 105 cells/well) (A) or a protein extract from isolated  HeLa cell nuclei (106 cell equivalents/well) (B). Immunoblot  analysis was performed using the following antibodies: rabbit antiserum 204.3 (diluted 1:20,000) (lane 1); mAb-o (25 μg/ml purified IgG) (lane 2); mAb-φ (10 μg/ml purified IgG) (lane 3);  mAb-γ (25 μg/ml purified IgG) (lane 4); mAb-π (5 μg/ml purified IgG) (lane 5); mAb-δ (0.08 μg/ml purified IgG) (lane 6);  mAb-pδ (15 μg/ml purified IgG) (lane 7); mouse antiserum 25.3  (diluted 1:5,000) (lane 8); and nonspecific polyclonal mouse IgG  (5 μg/ml purified IgG) (lane 9). Due to significant differences in  the binding affinity of each mAb, different antibody concentrations were used in order to produce a signal of similar intensity.  Antiserum 25.3 derives from the mouse used to produce hybridomas φ, γ, π, δ, and pδ. Nonspecific mouse IgG was purchased  from Sigma Chemical Co. Molecular mass markers (kD) are indicated on the left.
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Figure 1: Immunoblot analysis of anti-coilin mAbs. An 8% acrylamide gel was loaded with either total protein extract from HeLa cells (2 × 105 cells/well) (A) or a protein extract from isolated HeLa cell nuclei (106 cell equivalents/well) (B). Immunoblot analysis was performed using the following antibodies: rabbit antiserum 204.3 (diluted 1:20,000) (lane 1); mAb-o (25 μg/ml purified IgG) (lane 2); mAb-φ (10 μg/ml purified IgG) (lane 3); mAb-γ (25 μg/ml purified IgG) (lane 4); mAb-π (5 μg/ml purified IgG) (lane 5); mAb-δ (0.08 μg/ml purified IgG) (lane 6); mAb-pδ (15 μg/ml purified IgG) (lane 7); mouse antiserum 25.3 (diluted 1:5,000) (lane 8); and nonspecific polyclonal mouse IgG (5 μg/ml purified IgG) (lane 9). Due to significant differences in the binding affinity of each mAb, different antibody concentrations were used in order to produce a signal of similar intensity. Antiserum 25.3 derives from the mouse used to produce hybridomas φ, γ, π, δ, and pδ. Nonspecific mouse IgG was purchased from Sigma Chemical Co. Molecular mass markers (kD) are indicated on the left.

Mentions: To obtain monoclonal antibodies, hybridomas were derived by fusion of the mouse myeloma cell line Ag8.653 with spleen cells from Balb/c mice immunized with recombinant human coilin. Six clones (designated δ, pδ, γ, π, o, and φ) were isolated. The antibodies secreted by clones δ, pδ, o, and φ are of the IgG1 class, whereas mAb-γ is IgG2b and mAb-π is IgG2a (Table I). Immunoblot analysis reveals that all mAbs recognize a band of ∼80 kD in HeLa protein extracts (Fig. 1, A and B). Clones π and δ react specifically with p80–coilin, whereas the other clones cross-react with additional peptides. In addition to coilin, the mAbs o and γ recognize a major cytoplasmic protein band of ∼140 kD, mAb-φ strongly reacts with a nuclear protein of ∼110 kD, and mAb-pδ reveals a minor nuclear protein band of ∼115 kD. To map the epitopes recognized by each mAb, a series of His–coilin deletion mutants was generated and probed by immunoblot analysis (Fig. 2). From these data we conclude that the different clones react with epitopes distributed along the entire protein sequence. By immunofluorescence all mAbs labeled coiled bodies (Fig. 3 A and data not shown), as previously reported for clone δ (Rebelo et al., 1996).


Microinjection of anti-coilin antibodies affects the structure of coiled bodies.

Almeida F, Saffrich R, Ansorge W, Carmo-Fonseca M - J. Cell Biol. (1998)

Immunoblot analysis of anti-coilin mAbs. An 8% acrylamide gel was loaded with either total protein extract from HeLa  cells (2 × 105 cells/well) (A) or a protein extract from isolated  HeLa cell nuclei (106 cell equivalents/well) (B). Immunoblot  analysis was performed using the following antibodies: rabbit antiserum 204.3 (diluted 1:20,000) (lane 1); mAb-o (25 μg/ml purified IgG) (lane 2); mAb-φ (10 μg/ml purified IgG) (lane 3);  mAb-γ (25 μg/ml purified IgG) (lane 4); mAb-π (5 μg/ml purified IgG) (lane 5); mAb-δ (0.08 μg/ml purified IgG) (lane 6);  mAb-pδ (15 μg/ml purified IgG) (lane 7); mouse antiserum 25.3  (diluted 1:5,000) (lane 8); and nonspecific polyclonal mouse IgG  (5 μg/ml purified IgG) (lane 9). Due to significant differences in  the binding affinity of each mAb, different antibody concentrations were used in order to produce a signal of similar intensity.  Antiserum 25.3 derives from the mouse used to produce hybridomas φ, γ, π, δ, and pδ. Nonspecific mouse IgG was purchased  from Sigma Chemical Co. Molecular mass markers (kD) are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132868&req=5

Figure 1: Immunoblot analysis of anti-coilin mAbs. An 8% acrylamide gel was loaded with either total protein extract from HeLa cells (2 × 105 cells/well) (A) or a protein extract from isolated HeLa cell nuclei (106 cell equivalents/well) (B). Immunoblot analysis was performed using the following antibodies: rabbit antiserum 204.3 (diluted 1:20,000) (lane 1); mAb-o (25 μg/ml purified IgG) (lane 2); mAb-φ (10 μg/ml purified IgG) (lane 3); mAb-γ (25 μg/ml purified IgG) (lane 4); mAb-π (5 μg/ml purified IgG) (lane 5); mAb-δ (0.08 μg/ml purified IgG) (lane 6); mAb-pδ (15 μg/ml purified IgG) (lane 7); mouse antiserum 25.3 (diluted 1:5,000) (lane 8); and nonspecific polyclonal mouse IgG (5 μg/ml purified IgG) (lane 9). Due to significant differences in the binding affinity of each mAb, different antibody concentrations were used in order to produce a signal of similar intensity. Antiserum 25.3 derives from the mouse used to produce hybridomas φ, γ, π, δ, and pδ. Nonspecific mouse IgG was purchased from Sigma Chemical Co. Molecular mass markers (kD) are indicated on the left.
Mentions: To obtain monoclonal antibodies, hybridomas were derived by fusion of the mouse myeloma cell line Ag8.653 with spleen cells from Balb/c mice immunized with recombinant human coilin. Six clones (designated δ, pδ, γ, π, o, and φ) were isolated. The antibodies secreted by clones δ, pδ, o, and φ are of the IgG1 class, whereas mAb-γ is IgG2b and mAb-π is IgG2a (Table I). Immunoblot analysis reveals that all mAbs recognize a band of ∼80 kD in HeLa protein extracts (Fig. 1, A and B). Clones π and δ react specifically with p80–coilin, whereas the other clones cross-react with additional peptides. In addition to coilin, the mAbs o and γ recognize a major cytoplasmic protein band of ∼140 kD, mAb-φ strongly reacts with a nuclear protein of ∼110 kD, and mAb-pδ reveals a minor nuclear protein band of ∼115 kD. To map the epitopes recognized by each mAb, a series of His–coilin deletion mutants was generated and probed by immunoblot analysis (Fig. 2). From these data we conclude that the different clones react with epitopes distributed along the entire protein sequence. By immunofluorescence all mAbs labeled coiled bodies (Fig. 3 A and data not shown), as previously reported for clone δ (Rebelo et al., 1996).

Bottom Line: After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d.Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence.Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts.

View Article: PubMed Central - PubMed

Affiliation: Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal.

ABSTRACT
The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.

Show MeSH
Related in: MedlinePlus