Wilhelm bernhard workshop on the cell nucleus

FUNCTIONAL ORGANIZATION OF THE POLYTENE CELL NUCLEOLUS

Rodríguez-Vilariño, V; Díez, J.L.; Medina, F.J.

Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
In polytene cells, DNA is amplified forming the polytene or giant chromosomes, as a result of lateral amplification of the homologous chromatids that remain associated throughout successive rounds of replication. Ribosomal genes result, therefore, amplified, and their activity gives rise to the formation of a giant nucleolus, which may be called “Polytene Cell Nucleolus” (PCN). We have studied some basic aspects of the organization of the PCN using as a model the polytene cells of salivary glands from Chironomus larvae, characterized by a high level of polyteny, and a nucleolus measuring as much as 10-15 µm in diameter. The organization of nucleolar subcomponents in these cells has been defined at the ultrastructural level, being composed of the dense fibrillar component (DFC) and the granular component (GC); the fact that fibrillar centers (FC) have never been identified in these nucleoli, either under physiological conditions or after the use of inhibitors, re-opens the earlier debate, probably closed with insufficient evidence, on the functional significance of these structures in the nucleolar activity. In general, modifications and rearrangements of nucleolar subcomponents have been studied in relation to the nucleolar activity. By means of immunocytochemistry and confocal microscopy we have studied the organization of DNA within the PCN. The results showed that this organization was highly dynamic, as indicated by drastic changes in the spatial distribution, associated to physiological changes. The use of FISH has made possible the definition of the location of rDNA in the context of total nucleolar DNA and its structural changes associated to the activity of the nucleolus. Under physiological conditions, no rDNA has been located in condensed chromosomal bands; on the contrary, different levels of decondensation have been appreciated, until the total extension of the innermost transcribing (or ready for transcription) segments. On the other hand, the immunodetection of fibrillarin and nucleolin, two nucleolar proteins involved in pre-rRNA processing, has been carried out, allowing us to differentiate specific subnucleolar domains. In general, the organization of the PCN suggests that it functions as a single entity, and not as the juxtaposition of the activity of multiple NORs. Finally, we have explored the possibilities of the nucleolus for the study of the location of proteins not related with ribosome biogenesis, giving account of the so-called “plurifunctional nucleolus”.