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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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Analysis of mAb-π in living cells. HeLa  cells were injected with rhodamine-labeled mAb-π and analyzed at 30-min intervals. All cells in  this field were injected in the nucleus, except one  that was injected in the cytoplasm (A and B, arrowheads). The time between injection and the  first observation was ∼20 min. Note that shortly  after injection, the antibody injected in the cytoplasm (A and B, arrowheads) labels coiled bodies  (A–D, arrows). At 1.5 h after the first observation, one cell initially injected in the nucleus has  entered mitosis (arrowhead), and 3 h later coiled  bodies are labeled in the daughter cell nuclei (E,  arrows). At 6 h after the first observation, another cell is in mitosis and contains a labeled mitotic coiled body in the cytoplasm (F, arrowhead). Bar, 10 μm.
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Figure 7: Analysis of mAb-π in living cells. HeLa cells were injected with rhodamine-labeled mAb-π and analyzed at 30-min intervals. All cells in this field were injected in the nucleus, except one that was injected in the cytoplasm (A and B, arrowheads). The time between injection and the first observation was ∼20 min. Note that shortly after injection, the antibody injected in the cytoplasm (A and B, arrowheads) labels coiled bodies (A–D, arrows). At 1.5 h after the first observation, one cell initially injected in the nucleus has entered mitosis (arrowhead), and 3 h later coiled bodies are labeled in the daughter cell nuclei (E, arrows). At 6 h after the first observation, another cell is in mitosis and contains a labeled mitotic coiled body in the cytoplasm (F, arrowhead). Bar, 10 μm.

Mentions: Injection of rhodamine-labeled mAb-π into the nucleus reveals intensely labeled coiled bodies, which do not appear to change their relative positions during the time-lapse observations (Fig. 7, A–F). Injection of the antibody in the cytoplasm is rapidly followed by transport to the nucleus and incorporation into coiled bodies (Fig. 7, A–D, arrows). Importantly, microinjected cells undergo mitosis (Fig. 7, C and F, arrowheads) and labeled coiled bodies are seen in the daughter cells (Fig. 7 E, arrows). This demonstrates that, first, microinjection is not perturbing normal cell physiology and, second, binding of mAb-π to coilin is not preventing de novo assembly of 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)

Analysis of mAb-π in living cells. HeLa  cells were injected with rhodamine-labeled mAb-π and analyzed at 30-min intervals. All cells in  this field were injected in the nucleus, except one  that was injected in the cytoplasm (A and B, arrowheads). The time between injection and the  first observation was ∼20 min. Note that shortly  after injection, the antibody injected in the cytoplasm (A and B, arrowheads) labels coiled bodies  (A–D, arrows). At 1.5 h after the first observation, one cell initially injected in the nucleus has  entered mitosis (arrowhead), and 3 h later coiled  bodies are labeled in the daughter cell nuclei (E,  arrows). At 6 h after the first observation, another cell is in mitosis and contains a labeled mitotic coiled body in the cytoplasm (F, arrowhead). Bar, 10 μm.
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Related In: Results  -  Collection

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

Figure 7: Analysis of mAb-π in living cells. HeLa cells were injected with rhodamine-labeled mAb-π and analyzed at 30-min intervals. All cells in this field were injected in the nucleus, except one that was injected in the cytoplasm (A and B, arrowheads). The time between injection and the first observation was ∼20 min. Note that shortly after injection, the antibody injected in the cytoplasm (A and B, arrowheads) labels coiled bodies (A–D, arrows). At 1.5 h after the first observation, one cell initially injected in the nucleus has entered mitosis (arrowhead), and 3 h later coiled bodies are labeled in the daughter cell nuclei (E, arrows). At 6 h after the first observation, another cell is in mitosis and contains a labeled mitotic coiled body in the cytoplasm (F, arrowhead). Bar, 10 μm.
Mentions: Injection of rhodamine-labeled mAb-π into the nucleus reveals intensely labeled coiled bodies, which do not appear to change their relative positions during the time-lapse observations (Fig. 7, A–F). Injection of the antibody in the cytoplasm is rapidly followed by transport to the nucleus and incorporation into coiled bodies (Fig. 7, A–D, arrows). Importantly, microinjected cells undergo mitosis (Fig. 7, C and F, arrowheads) and labeled coiled bodies are seen in the daughter cells (Fig. 7 E, arrows). This demonstrates that, first, microinjection is not perturbing normal cell physiology and, second, binding of mAb-π to coilin is not preventing de novo assembly of 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