Organization of the nucleolar RNP components in cultured rabbit fibroblasts following mild loosening of the nuclear content.

The organization of the ribonucleoprotein (RNP) components of the nucleolus was studied in ultrathin sections by a recent method of mild loosening of the nuclear content. This investigation was carried out in cultured rabbit fibroblasts in which the nucleoli contain clearly separated fibrillar and granular RNP components. In addition, the demarcation between these two particulate components was accentuated by infection with herpes simplex type 1 virus (HSV1), by recovery at 37 degrees C after a heat shock, and by actinomycin D treatment. Except in actinomycin D-treated cells, it was possible to identify two distinct components which are not visible in routinely prepared sections: (1) highly contrasted fibrillar clusters resulting from the loosening of the fibrillar component, (2) a granular network composed of nucleolar granules interconnected by a thin filament of about 5 nm in thickness. In actinomycin D-treated cells, only the granular zone was observed. High resolution autoradiography carried out both on standard fixed and on loosened cells following a 5 min labeling with tritiated uridine revealed that the fibrillar clusters correspond to sections of nucleolar transcription complexes. When the same labeling was followed by 3 h chase only the granular network was labeled indicating that it corresponds to the site of pre-ribosomal ribonucleic acid (pre-rRNA) maturation. Infection by HSV1 did not change this result. Preferential RNP staining and deoxyribonucleic acid (DNA) specific staining failed to reveal the thin filaments interconnecting the nucleolar granules. We suggest that the latter filaments serve as support for pre-rRNA processing and for storage of large pre-ribosomal subunits.