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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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Microinjection mAb-δ. (A) HeLa  cells were permeabilized with Triton  X-100, fixed with formaldehyde, and then  incubated with mAb-δ. (B and C) HeLa  cells were microinjected in the nucleus  with mAb-δ. The cells were then observed  immediately (B) or 24 h after injection (C).  (D–F) Purified mAb-δ was incubated for  1 h with 1:2 molar excess of a coilin peptide  containing the epitope for this antibody  (amino acids 363–481). The neutralized  mAb was then used for indirect immunofluorescence on fixed cells (D) or microinjected in the nucleus of living cells (E and  F). (E) Immediately after injection the  neutralized mAb is diffusely distributed  throughout the nucleoplasm with additional staining of a coiled body (arrow).  Note the bright pericellular staining which  is due to antibody–antigen complexes adsorbed to the cell membrane. (F) 24 h after  injection the staining is predominantly  concentrated in coiled bodies. (G and H)  HeLa cells were microinjected in the cytoplasm with mAb-δ and observed 1 h after  injection. The cells were either nontreated  (G) or treated with 10 μg/ml emetine for  2.5 h before injection (H). Bar, 10 μm.
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Figure 3: Microinjection mAb-δ. (A) HeLa cells were permeabilized with Triton X-100, fixed with formaldehyde, and then incubated with mAb-δ. (B and C) HeLa cells were microinjected in the nucleus with mAb-δ. The cells were then observed immediately (B) or 24 h after injection (C). (D–F) Purified mAb-δ was incubated for 1 h with 1:2 molar excess of a coilin peptide containing the epitope for this antibody (amino acids 363–481). The neutralized mAb was then used for indirect immunofluorescence on fixed cells (D) or microinjected in the nucleus of living cells (E and F). (E) Immediately after injection the neutralized mAb is diffusely distributed throughout the nucleoplasm with additional staining of a coiled body (arrow). Note the bright pericellular staining which is due to antibody–antigen complexes adsorbed to the cell membrane. (F) 24 h after injection the staining is predominantly concentrated in coiled bodies. (G and H) HeLa cells were microinjected in the cytoplasm with mAb-δ and observed 1 h after injection. The cells were either nontreated (G) or treated with 10 μg/ml emetine for 2.5 h before injection (H). Bar, 10 μm.

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)

Microinjection mAb-δ. (A) HeLa  cells were permeabilized with Triton  X-100, fixed with formaldehyde, and then  incubated with mAb-δ. (B and C) HeLa  cells were microinjected in the nucleus  with mAb-δ. The cells were then observed  immediately (B) or 24 h after injection (C).  (D–F) Purified mAb-δ was incubated for  1 h with 1:2 molar excess of a coilin peptide  containing the epitope for this antibody  (amino acids 363–481). The neutralized  mAb was then used for indirect immunofluorescence on fixed cells (D) or microinjected in the nucleus of living cells (E and  F). (E) Immediately after injection the  neutralized mAb is diffusely distributed  throughout the nucleoplasm with additional staining of a coiled body (arrow).  Note the bright pericellular staining which  is due to antibody–antigen complexes adsorbed to the cell membrane. (F) 24 h after  injection the staining is predominantly  concentrated in coiled bodies. (G and H)  HeLa cells were microinjected in the cytoplasm with mAb-δ and observed 1 h after  injection. The cells were either nontreated  (G) or treated with 10 μg/ml emetine for  2.5 h before injection (H). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Microinjection mAb-δ. (A) HeLa cells were permeabilized with Triton X-100, fixed with formaldehyde, and then incubated with mAb-δ. (B and C) HeLa cells were microinjected in the nucleus with mAb-δ. The cells were then observed immediately (B) or 24 h after injection (C). (D–F) Purified mAb-δ was incubated for 1 h with 1:2 molar excess of a coilin peptide containing the epitope for this antibody (amino acids 363–481). The neutralized mAb was then used for indirect immunofluorescence on fixed cells (D) or microinjected in the nucleus of living cells (E and F). (E) Immediately after injection the neutralized mAb is diffusely distributed throughout the nucleoplasm with additional staining of a coiled body (arrow). Note the bright pericellular staining which is due to antibody–antigen complexes adsorbed to the cell membrane. (F) 24 h after injection the staining is predominantly concentrated in coiled bodies. (G and H) HeLa cells were microinjected in the cytoplasm with mAb-δ and observed 1 h after injection. The cells were either nontreated (G) or treated with 10 μg/ml emetine for 2.5 h before injection (H). Bar, 10 μm.
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