Computer-based fluorescence quantification: a novel approach to study nucleolar biology

BACKGROUND: Nucleoli are composed of possibly several thousand different proteins and represent the most conspicuous compartments in the nucleus; they play a crucial role in the proper execution of many cellular processes. As such, nucleoli carry out ribosome biogenesis and sequester or associate with key molecules that regulate cell cycle progression, tumorigenesis, apoptosis and the stress response. Nucleoli are dynamic compartments that are characterized by a constant flux of macromolecules. Given the complex and dynamic composition of the nucleolar proteome, it is challenging to link modifications in nucleolar composition to downstream effects. RESULTS: In this contribution, we present quantitative immunofluorescence methods that rely on computer-based image analysis. We demonstrate the effectiveness of these techniques by monitoring the dynamic association of proteins and RNA with nucleoli under different physiological conditions. Thus, the protocols described by us were employed to study stress-dependent changes in the nucleolar concentration of endogenous and GFP-tagged proteins. Furthermore, our methods were applied to measure de novo RNA synthesis that is associated with nucleoli. We show that the techniques described here can be easily combined with automated high throughput screening (HTS) platforms, making it possible to obtain large data sets and analyze many of the biological processes that are located in nucleoli. CONCLUSIONS: Our protocols set the stage to analyze in a quantitative fashion the kinetics of shuttling nucleolar proteins, both at the single cell level as well as for a large number of cells. Moreover, the procedures described here are compatible with high throughput image acquisition and analysis using HTS automated platforms, thereby providing the basis to quantify nucleolar components and activities for numerous samples and experimental conditions. Together with the growing amount of information obtained for the nucleolar proteome, improvements in quantitative microscopy as they are described here can be expected to produce new insights into the complex biological functions that are orchestrated by the nucleolus.