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Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells
R Benavente1, G Reimer, K M Rose
1Institute of Zoology I, University of Würzburg, Federal Republic of Germany.
Abstract:
After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtK2) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.
Insights
Antibodies targeting RNA polymerase I disrupt nucleolar structure, causing loss of the dense fibrillar component and formation of extranucleolar aggregates. This suggests rRNA gene transcription influences nucleolar organization.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The nucleolus is a dynamic nuclear substructure crucial for ribosome biogenesis and other cellular processes.
- Its structure comprises distinct components: fibrillar centers (FCs), dense fibrillar component (DFC), and granular component (GC).
- RNA polymerase I (Pol I) is the enzyme responsible for transcribing ribosomal RNA (rRNA) genes, a key function within the nucleolus.
Purpose of the Study:
- To investigate the effects of inhibiting RNA polymerase I activity on nucleolar structure and composition.
- To determine if modulating rRNA gene transcription impacts the organization of nucleolar components.
- To compare the observed nucleolar alterations with those induced by other cellular stressors.
Main Methods:
- Microinjection of antibodies against RNA polymerase I into cultured rat kangaroo (PtK2) and rat (RVF-SMC) cells.
- Analysis of nucleolar structure and composition using electron microscopy.
- Double-label immunofluorescence microscopy to detect specific nucleolar proteins, including fibrillarin.
Main Results:
- Microinjection of anti-RNA polymerase I antibodies led to progressive loss of the dense fibrillar component (DFC) from nucleoli.
- Within 4 hours, nucleoli transformed into structures with only granular component (GC) and attached fibrillar centers (FCs).
- Extranucleolar aggregates resembling DFC fragments, containing fibrillarin, appeared in the nucleoplasm.
Conclusions:
- Experimental modulation of RNA polymerase I activity can alter the topological distribution of nucleolar material, specifically the DFC.
- These changes are linked to the modulation of rRNA gene transcriptional activity.
- The observed structural alterations resemble early events of nucleolar disintegration during mitotic prophase, distinct from drug-induced nucleolar segregation.