Four mAbs are shown to induce a progressive disappearance of coiled bodies within 6 h after microinjection into the nucleus of HeLa cells. least 3 d. Epitope mapping reveals that this 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 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human -globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance. Keywords: coiled body, p80Ccoilin, splicing, spliceosomal snRNPs, nucleolus he intranuclear structure that is now known as the coiled body was first described in 1903 by the neurocytologist Ramon-y-Cajal. Cajal observed that neurons stained with silver contained spherical structures NNC 55-0396 0.5 m in diameter which were often associated with nucleoli, and called them nucleolar accessory bodies. Later, electron microscopists have rediscovered the accessory body of Cajal and introduced the name coiled body because when this structure is viewed with the electron microscope it resembles a tangle of coiled threads (Hardin et al., 1969; Monneron and Bernhard, 1969; Hervs et al., 1980). The next major advance in the study of coiled bodies came with the discovery of patient autoimmune sera that selectively stain these structures and react with a protein of 80 kD termed p80Ccoilin (Raska et al., 1990, 1991; Andrade et al., 1991). Anti-coilin antibodies proved to be a very convenient marker for identifying coiled bodies, and data from numerous laboratories indicate that similar or equivalent structures are present in nuclei from plants (Moreno Diaz de la Espina et al., 1980; Beven et al., 1995), flies (Yannoni and White, 1997), frogs (Gall et al., 1995; Roth, 1995), birds (Ochs et al., 1995), NNC 55-0396 and mammals (for review see Bohmann et al., 1995protein SPH-1 (Tuma et al., 1993). This protein is highly homologous to coilin at both its amino and carboxy termini, but shows much less homology in the internal domain (see Carmo-Fonseca et al., 1994). In the nucleus of amphibian oocytes SPH-1 is localized in spheres that are Rabbit Polyclonal to Cytochrome P450 2B6 thought to be equivalent to coiled bodies (for NNC 55-0396 review see Roth, 1995; Gall et al., 1995). The coilin sequence includes two motifs at amino acid positions 107C112 and 181C198 that closely match the consensus sequence of the simple and bipartite nuclear localization sequence (NLS)1, respectively (Bohmann et al., 1995and purified using an Ni-NTA agarose affinity column (Quiagen, Hilden, Germany), as previously described (Bohmann et al., 1995and purified as HisCtag fusion proteins by Ni-NTA affinity chromatography. Cell Culture HeLa cells were grown as monolayers in minimum essential medium (MEM) with Earle’s Salts supplemented with 2 mM l-glutamine, 1% MEM nonessential amino acids, 50 IU/ml penicillin and 50 mg/ml streptomycin, and 10% fetal calf serum (Int., Buckinghamshire, England, UK). Immunofluorescence For indirect immunofluorescence cells were grown on 10 10-mm glass coverslips. The cells were washed twice in PBS, fixed with 3.7% formaldehyde (freshly prepared from paraformaldehyde) in PBS for 10 min at room temperature, and subsequently permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature. Alternatively, cells were first permeabilized with 0.5% Triton X-100 in CSK buffer (100 mM NaCl, 300 mM sucrose, 10 mM Pipes, 3 mM MgCl2, 1 mM EGTA, pH 6.8; Fey et al., 1986) containing 0.1 mM PMSF for 1 min on ice, and subsequently fixed with 3.7% formaldehyde in CSK buffer, for 10 min at room temperature. NNC 55-0396 After fixation and permeabilization the cells were rinsed in PBS containing 0.05% Tween-20 (PBS-Tw), incubated for 30 min with primary antibodies diluted in PBS, washed in PBS-Tw, and then incubated for 30 min with the appropriate secondary antibodies conjugated to fluorescein (FITC), NNC 55-0396 Texas red, or indodicarbocyanine (Cy5) (Jackson ImmunoResearch Laboratories, West Grove, PA). Finally, the coverslips were mounted in VectaShield (Vector Laboratories, Peterborough, UK) and.
