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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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Quantitative analysis of the effects of mAb injection on  coiled bodies. Cells were microinjected in the nucleus with mAbs  δ, pδ, γ, π, o, and φ. The cells were either immediately permeabilized and fixed (t0), or further incubated for 6 or 24 h. The injected mAb was detected using fluorescein-conjugated secondary  antibodies. Cells were double-labeled with a rabbit anti-coilin antiserum (204.3) detected using Texas red fluorescence. For each  mAb, the percentage of injected cells with coiled bodies was estimated at each time point (CB[tn]), and the values at time zero  (CB[t0]) were taken as reference. For each mAb, at least three independent microinjection experiments were performed. The total number of cells counted per mAb at each time point ranged  between 100 and 600.
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Figure 6: Quantitative analysis of the effects of mAb injection on coiled bodies. Cells were microinjected in the nucleus with mAbs δ, pδ, γ, π, o, and φ. The cells were either immediately permeabilized and fixed (t0), or further incubated for 6 or 24 h. The injected mAb was detected using fluorescein-conjugated secondary antibodies. Cells were double-labeled with a rabbit anti-coilin antiserum (204.3) detected using Texas red fluorescence. For each mAb, the percentage of injected cells with coiled bodies was estimated at each time point (CB[tn]), and the values at time zero (CB[t0]) were taken as reference. For each mAb, at least three independent microinjection experiments were performed. The total number of cells counted per mAb at each time point ranged between 100 and 600.

Mentions: To further characterize the effect of microinjected antibodies on the coiled body, a quantitative analysis was performed. Each mAb was injected into the nuclei of interphase HeLa cells and at 0, 6, and 24 h after injection the cells were fixed, incubated with fluorescein-conjugated secondary antibodies, and then double-labeled with a rabbit anti-coilin antiserum (204.3) detected using Texas red fluorescence. For each time point, the proportion of injected cells with coiled bodies was estimated and the values at time zero were taken as reference (Fig. 6). The results show that immediately after injection, the proportion of injected cells containing coiled bodies identified by antiserum 204.3 was similar to that of noninjected cells. At 6 and 24 h after injection, the proportion of noninjected cells containing coiled bodies remained unaltered, whereas injected cells showed significant changes. The mAbs δ, o, and φ induce a drastic disappearance of coiled bodies. The mAb pδ also reduces the proportion of cells with coiled bodies, although less efficiently than the previous ones. The mAb-γ induces a slight decrease in the proportion of injected cells containing coiled bodies at 6 h after injection, but this value remains roughly unchanged by 24 h after injection. In striking contrast with these results, mAb-π causes an increase in the relative numbers of injected cells containing coiled bodies.


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

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

Quantitative analysis of the effects of mAb injection on  coiled bodies. Cells were microinjected in the nucleus with mAbs  δ, pδ, γ, π, o, and φ. The cells were either immediately permeabilized and fixed (t0), or further incubated for 6 or 24 h. The injected mAb was detected using fluorescein-conjugated secondary  antibodies. Cells were double-labeled with a rabbit anti-coilin antiserum (204.3) detected using Texas red fluorescence. For each  mAb, the percentage of injected cells with coiled bodies was estimated at each time point (CB[tn]), and the values at time zero  (CB[t0]) were taken as reference. For each mAb, at least three independent microinjection experiments were performed. The total number of cells counted per mAb at each time point ranged  between 100 and 600.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132868&req=5

Figure 6: Quantitative analysis of the effects of mAb injection on coiled bodies. Cells were microinjected in the nucleus with mAbs δ, pδ, γ, π, o, and φ. The cells were either immediately permeabilized and fixed (t0), or further incubated for 6 or 24 h. The injected mAb was detected using fluorescein-conjugated secondary antibodies. Cells were double-labeled with a rabbit anti-coilin antiserum (204.3) detected using Texas red fluorescence. For each mAb, the percentage of injected cells with coiled bodies was estimated at each time point (CB[tn]), and the values at time zero (CB[t0]) were taken as reference. For each mAb, at least three independent microinjection experiments were performed. The total number of cells counted per mAb at each time point ranged between 100 and 600.
Mentions: To further characterize the effect of microinjected antibodies on the coiled body, a quantitative analysis was performed. Each mAb was injected into the nuclei of interphase HeLa cells and at 0, 6, and 24 h after injection the cells were fixed, incubated with fluorescein-conjugated secondary antibodies, and then double-labeled with a rabbit anti-coilin antiserum (204.3) detected using Texas red fluorescence. For each time point, the proportion of injected cells with coiled bodies was estimated and the values at time zero were taken as reference (Fig. 6). The results show that immediately after injection, the proportion of injected cells containing coiled bodies identified by antiserum 204.3 was similar to that of noninjected cells. At 6 and 24 h after injection, the proportion of noninjected cells containing coiled bodies remained unaltered, whereas injected cells showed significant changes. The mAbs δ, o, and φ induce a drastic disappearance of coiled bodies. The mAb pδ also reduces the proportion of cells with coiled bodies, although less efficiently than the previous ones. The mAb-γ induces a slight decrease in the proportion of injected cells containing coiled bodies at 6 h after injection, but this value remains roughly unchanged by 24 h after injection. In striking contrast with these results, mAb-π causes an increase in the relative numbers of injected cells containing coiled bodies.

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