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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-δ in living cells. HeLa  cells were injected with rhodamine-labeled mAb-δ and analyzed at 30-min intervals. All cells in  this field were injected in the nucleus. The time  between injection and the first observation was  ∼20 min. Within 6 h after the first observation,  the coiled bodies progressively disappear (A–G).  At later time points coiled bodies are never detected in these cells (H–L). Note that one of the  cells has divided by 39 h (L, arrows). J and L depict phase-contrast images corresponding to I  and K, respectively. Bar, 10 μm.
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Figure 8: Analysis of mAb-δ in living cells. HeLa cells were injected with rhodamine-labeled mAb-δ and analyzed at 30-min intervals. All cells in this field were injected in the nucleus. The time between injection and the first observation was ∼20 min. Within 6 h after the first observation, the coiled bodies progressively disappear (A–G). At later time points coiled bodies are never detected in these cells (H–L). Note that one of the cells has divided by 39 h (L, arrows). J and L depict phase-contrast images corresponding to I and K, respectively. Bar, 10 μm.

Mentions: In contrast with the above observations, injection of rhodamine-labeled mAb-δ into the nucleus shows a progressive disappearance of coiled bodies over a period of ∼6 h (Fig. 8, A–G). However, coiled bodies are readily visible immediately after injection into the nucleus (Fig. 8 A) or shortly after injection into the cytoplasm (refer to Fig. 3 G). Thus, binding of mAb-δ is not preventing the assembly of coilin–antibody complexes into coiled bodies. Possibly, when complexes of mAb-δ with coilin are incorporated into a coiled body they block the subsequent assembly of new protein, leading to a progressive disappearance of the structure due to its normal turnover rate. Alternatively, the presence of mAb-δ/coilin complexes in a coiled body may somehow trigger its disassembly. After their disappearance, coiled bodies are no longer visible in cells injected with mAb-δ, indicating that the antibody continues to exert its effect for a period of at least 39 h (Fig. 8, H–L). During this period, the injected cells remain viable, as shown by their ability to divide (Fig. 8, K and L, arrows).


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-δ in living cells. HeLa  cells were injected with rhodamine-labeled mAb-δ and analyzed at 30-min intervals. All cells in  this field were injected in the nucleus. The time  between injection and the first observation was  ∼20 min. Within 6 h after the first observation,  the coiled bodies progressively disappear (A–G).  At later time points coiled bodies are never detected in these cells (H–L). Note that one of the  cells has divided by 39 h (L, arrows). J and L depict phase-contrast images corresponding to I  and K, respectively. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Analysis of mAb-δ in living cells. HeLa cells were injected with rhodamine-labeled mAb-δ and analyzed at 30-min intervals. All cells in this field were injected in the nucleus. The time between injection and the first observation was ∼20 min. Within 6 h after the first observation, the coiled bodies progressively disappear (A–G). At later time points coiled bodies are never detected in these cells (H–L). Note that one of the cells has divided by 39 h (L, arrows). J and L depict phase-contrast images corresponding to I and K, respectively. Bar, 10 μm.
Mentions: In contrast with the above observations, injection of rhodamine-labeled mAb-δ into the nucleus shows a progressive disappearance of coiled bodies over a period of ∼6 h (Fig. 8, A–G). However, coiled bodies are readily visible immediately after injection into the nucleus (Fig. 8 A) or shortly after injection into the cytoplasm (refer to Fig. 3 G). Thus, binding of mAb-δ is not preventing the assembly of coilin–antibody complexes into coiled bodies. Possibly, when complexes of mAb-δ with coilin are incorporated into a coiled body they block the subsequent assembly of new protein, leading to a progressive disappearance of the structure due to its normal turnover rate. Alternatively, the presence of mAb-δ/coilin complexes in a coiled body may somehow trigger its disassembly. After their disappearance, coiled bodies are no longer visible in cells injected with mAb-δ, indicating that the antibody continues to exert its effect for a period of at least 39 h (Fig. 8, H–L). During this period, the injected cells remain viable, as shown by their ability to divide (Fig. 8, K and L, arrows).

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