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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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Coilin does not colocalize with transcription sites. (A)  HeLa cells were microinjected with Br-UTP in the cytoplasm,  fixed, and then immunolabeled with mAb-π. The sites of incorporated Br-uridine are stained green and the sites containing coilin are stained red. Note that coilin is present both in coiled  bodies (arrows) and in numerous nucleoplasmic microfoci (arrowheads). (B) HeLa cells were injected in the nucleus with  mAb-δ. 24 h later the cells were reinjected in the cytoplasm with  Br-UTP. Note that the coilin microfoci (stained red) do not colocalize with transcription sites (stained green). Bar, 10 μm.
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Figure 5: Coilin does not colocalize with transcription sites. (A) HeLa cells were microinjected with Br-UTP in the cytoplasm, fixed, and then immunolabeled with mAb-π. The sites of incorporated Br-uridine are stained green and the sites containing coilin are stained red. Note that coilin is present both in coiled bodies (arrows) and in numerous nucleoplasmic microfoci (arrowheads). (B) HeLa cells were injected in the nucleus with mAb-δ. 24 h later the cells were reinjected in the cytoplasm with Br-UTP. Note that the coilin microfoci (stained red) do not colocalize with transcription sites (stained green). Bar, 10 μm.

Mentions: Although coilin microfoci become more prominent after the disappearance of coiled bodies, similar structures are present in nontreated cells and we have previously demonstrated that at the electron microscopy level they correspond to microspherules of ∼0.05 μm in diameter (Rebelo et al., 1996). Furthermore, we show here that neither coiled bodies nor coilin microfoci colocalize with sites of incorporation of brominated UTP, indicating that they do not correspond to transcription sites (Fig. 5, A and B).


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

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

Coilin does not colocalize with transcription sites. (A)  HeLa cells were microinjected with Br-UTP in the cytoplasm,  fixed, and then immunolabeled with mAb-π. The sites of incorporated Br-uridine are stained green and the sites containing coilin are stained red. Note that coilin is present both in coiled  bodies (arrows) and in numerous nucleoplasmic microfoci (arrowheads). (B) HeLa cells were injected in the nucleus with  mAb-δ. 24 h later the cells were reinjected in the cytoplasm with  Br-UTP. Note that the coilin microfoci (stained red) do not colocalize with transcription sites (stained green). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132868&req=5

Figure 5: Coilin does not colocalize with transcription sites. (A) HeLa cells were microinjected with Br-UTP in the cytoplasm, fixed, and then immunolabeled with mAb-π. The sites of incorporated Br-uridine are stained green and the sites containing coilin are stained red. Note that coilin is present both in coiled bodies (arrows) and in numerous nucleoplasmic microfoci (arrowheads). (B) HeLa cells were injected in the nucleus with mAb-δ. 24 h later the cells were reinjected in the cytoplasm with Br-UTP. Note that the coilin microfoci (stained red) do not colocalize with transcription sites (stained green). Bar, 10 μm.
Mentions: Although coilin microfoci become more prominent after the disappearance of coiled bodies, similar structures are present in nontreated cells and we have previously demonstrated that at the electron microscopy level they correspond to microspherules of ∼0.05 μm in diameter (Rebelo et al., 1996). Furthermore, we show here that neither coiled bodies nor coilin microfoci colocalize with sites of incorporation of brominated UTP, indicating that they do not correspond to transcription sites (Fig. 5, A and B).

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