REASSEMBLY OF FUNCTIONAL NUCLEOLI FOLLOWING IN-SITU UNRAVELING BY LOW-IONIC-STRENGTH TREATMENT OF CULTURED-MAMMALIAN-CELLS

In order to determine the most persistent components of the nucleolus that might serve as ''core'' nucleolar elements, we studied the reacti vity of nucleoli in living mammalian cells subjected to hypotonic buff er saline followed by the incubation of the cells in an isotonic mediu m. To document as precisely as possible the fine structural changes wh ich occurred, the cells were examined by video-enhanced optical micros copy, fluorescence confocal laser scanning microscopy, and electron mi croscopy combined with cytochemistry. Light microscopic autoradiograph y was used to demonstrate the transcriptional characteristics of the r eassembled nucleoli. It was shown that all the major compartments of t he intact nucleolus could be substantially affected by reduction of th e osmolarity of the environmental media. The dynamic events of the nuc leolar unraveling in low salt buffers occurred in the following order: dispersion of the nucleolar pars granulosa, disassociation of the fib rillar complexes into discrete fibrillar centers (FCs) and the dense f ibrillar component (DFC), and the almost complete unraveling of the DF C and FCs. At the terminal stages of nucleolar dispersion, the nuclear interior was mainly composed of a loose filamentous meshwork, and non e of the typically discerned nucleolar constituents was recognized. Ne vertheless, when hypotonically treated cells were returned to isotonic conditions, the nucleolar bodies rapidly began to reassemble. Within 1-2 h of cell incubation under isotonicity, the nucleoli not only beca me clearly visible, but also reconstituted to their initial size, shap e, and position within the nucleus. The ultrastructure and functional activity of the reassembled nucleoli were also found to be fully compa rable to those of the untreated controls. These data indicate that the architectural composition of the interphase nucleolus is strictly con trolled by the cell. As far as could be determined, none of the usual substructures of the intact nucleolus that could be substituted by com plete reassembly of the nucleolar bodies in normotonic conditions, inc luding FCs and the DFC, remained clearly preserved in the terminal sta ge of nucleolar unraveling. We concluded that the integrity of the nuc leolus was mainly preserved by the nuclear or nucleolar matrix system rather than by any other nucleolar structural domains. (C) 1997 Academ ic Press.