Revealing nucleolar architecture by low ionic strength treatment

Abstract

The internal nucleolar architecture of HeLa cells was revealed after a short hypotonic treatment. The response of nucleoli to a gradual reduction in the ionic strength of incubation buffer was assessed by immunofluorescence, confocal microscopy using human autoimmune sera monospecific for antigens present in different nucleolar components, and electron microscopy. The granular component dispersed first, followed by the dense fibrillar component, leaving distinct fibrillar centers remaining. This demonstrates differential sensitivity to low ionic strength treatment in the transcription and the maturation territories of the nucleoli. The changes described develop in only a few minutes and this approach can reveal momentary in situ intranucleolar arrangements. We suggest that the fibrillar centers provide a structural support for RNA-polymerase I complexes and are possibly also attached to a nuclear skeleton. The evidence presented implicates the fibrillar centers as the core elements of nucleoli and that functional nucleoli arise around them.