Time-lapse Microscopy and Fluorescence Resonance Energy Transfer to Analyze the Dynamics and Interactions of Nucleolar Proteins in Living Cells.
Résumé
The dynamics of proteins play a key role in the organization and control of nuclear functions. Techniques were developed recently to observe the movement and interactions of proteins in living cells; time-lapse microscopy using fluorescent-tagged proteins gives access to observations of nuclear protein trafficking over time, and fluorescence resonance energy transfer (FRET) is used to investigate protein interactions in the time-lapse mode. In this chapter, we describe the application of these two approaches to follow the recruitment of nucleolar processing proteins at the time of nucleolar assembly. We question the role of prenucleolar bodies (PNB) during migration of the processing proteins from the chromosome periphery to sites of ribosomal genes (rDNA) transcription. The order of recruitment of different processing proteins into nucleoli is the consequence of differential sorting from the same PNBs. The dynamics of the interactions between processing proteins in PNBs suggest that PNBs are preassembly platforms for ribosomal RNA (rRNA) processing complexes.