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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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The effect of mAb injection on  coiled bodies. HeLa cells were microinjected in the nucleus with mAbs δ (A and  D), pδ (B and E), γ (C and F), π (G and J),  o (H and K), and φ (I and L). The cells  were observed either immediately after injection (A–C, G–I) or 24 h after injection  (D–F, J–L). For microscopical observation, the cells were permeabilized with Triton X-100, fixed, and incubated with a secondary antibody coupled to fluorescein.  Note that mAbs o and φ label both coiled  bodies (arrows) and nucleoli (arrowheads). Bar, 10 μm.
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Figure 4: The effect of mAb injection on coiled bodies. HeLa cells were microinjected in the nucleus with mAbs δ (A and D), pδ (B and E), γ (C and F), π (G and J), o (H and K), and φ (I and L). The cells were observed either immediately after injection (A–C, G–I) or 24 h after injection (D–F, J–L). For microscopical observation, the cells were permeabilized with Triton X-100, fixed, and incubated with a secondary antibody coupled to fluorescein. Note that mAbs o and φ label both coiled bodies (arrows) and nucleoli (arrowheads). Bar, 10 μm.

Mentions: Injection of mAbs pδ, γ, π, o, and φ into the nucleus of HeLa cells produce results similar to those observed with mAb-δ, i.e., rapid staining of the coiled body (Fig. 4, A–C and G–I). Intriguingly, mAbs o and φ stain nucleoli in addition to coiled bodies (Fig. 4, H and I, arrowheads). The intranucleolar staining produced by mAb-o is diffuse (Fig. 4 H, arrowheads), increases in intensity with time after microinjection (Fig. 4 K, arrowhead) and is never observed by indirect immunofluorescence, suggesting that it may reflect an unspecific accumulation of injected antibody in the nucleolus. In contrast, mAb-φ stains intranucleolar foci (Fig. 4 I, arrowheads) which are also detected by indirect immunofluorescence on fixed cells. Double-labeling experiments using antibodies directed to RNA polymerase I and UBF reveal that these intranucleolar foci correspond to sites of rRNA synthesis (data not shown; Jordan at al., 1996). Interestingly, the epitope recognized by mAb-φ is adjacent to a critical serine residue (serine 202) which when mutated to aspartate induces the formation of coiled body-like structures inside the nucleolus (Lyon et al., 1997; refer to Fig. 2 B). However, this type of intranucleolar labeling was never observed with anti-coilin polyclonal antibodies, making it difficult to conclude that it represents an additional localization of coilin within the nucleolus. Rather, it is more likely that mAb-φ recognizes an epitope common to coilin and another nuclear protein of ∼110 kD, as revealed by immunoblotting (refer to Fig. 1, A and B, lane 3).


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

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

The effect of mAb injection on  coiled bodies. HeLa cells were microinjected in the nucleus with mAbs δ (A and  D), pδ (B and E), γ (C and F), π (G and J),  o (H and K), and φ (I and L). The cells  were observed either immediately after injection (A–C, G–I) or 24 h after injection  (D–F, J–L). For microscopical observation, the cells were permeabilized with Triton X-100, fixed, and incubated with a secondary antibody coupled to fluorescein.  Note that mAbs o and φ label both coiled  bodies (arrows) and nucleoli (arrowheads). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: The effect of mAb injection on coiled bodies. HeLa cells were microinjected in the nucleus with mAbs δ (A and D), pδ (B and E), γ (C and F), π (G and J), o (H and K), and φ (I and L). The cells were observed either immediately after injection (A–C, G–I) or 24 h after injection (D–F, J–L). For microscopical observation, the cells were permeabilized with Triton X-100, fixed, and incubated with a secondary antibody coupled to fluorescein. Note that mAbs o and φ label both coiled bodies (arrows) and nucleoli (arrowheads). Bar, 10 μm.
Mentions: Injection of mAbs pδ, γ, π, o, and φ into the nucleus of HeLa cells produce results similar to those observed with mAb-δ, i.e., rapid staining of the coiled body (Fig. 4, A–C and G–I). Intriguingly, mAbs o and φ stain nucleoli in addition to coiled bodies (Fig. 4, H and I, arrowheads). The intranucleolar staining produced by mAb-o is diffuse (Fig. 4 H, arrowheads), increases in intensity with time after microinjection (Fig. 4 K, arrowhead) and is never observed by indirect immunofluorescence, suggesting that it may reflect an unspecific accumulation of injected antibody in the nucleolus. In contrast, mAb-φ stains intranucleolar foci (Fig. 4 I, arrowheads) which are also detected by indirect immunofluorescence on fixed cells. Double-labeling experiments using antibodies directed to RNA polymerase I and UBF reveal that these intranucleolar foci correspond to sites of rRNA synthesis (data not shown; Jordan at al., 1996). Interestingly, the epitope recognized by mAb-φ is adjacent to a critical serine residue (serine 202) which when mutated to aspartate induces the formation of coiled body-like structures inside the nucleolus (Lyon et al., 1997; refer to Fig. 2 B). However, this type of intranucleolar labeling was never observed with anti-coilin polyclonal antibodies, making it difficult to conclude that it represents an additional localization of coilin within the nucleolus. Rather, it is more likely that mAb-φ recognizes an epitope common to coilin and another nuclear protein of ∼110 kD, as revealed by immunoblotting (refer to Fig. 1, A and B, lane 3).

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