Automated image analysis to quantify the subnuclear organization of transcriptional coregulatory protein complexes in living cell populations

Regulated gene transcription is dependent on the steady-state concentration of DNA-binding and coregulatory proteins assembled in distinct regions of the cell nucleus. For example, several different transcriptional coactivator proteins, such as the Glucocorticoid Receptor Interacting Protein (GRIP), localize to distinct spherical intranuclear bodies that vary from approximately 0.2-1 micron in diameter. We are using multi-spectral wide-field microscopy of cells expressing coregulatory proteins labeled with the fluorescent proteins (FP) to study the mechanisms that control the assembly and distribution of these structures in living cells. However, variability between cells in the population makes an unbiased and consistent approach to this image analysis absolutely critical. To address this challenge, we developed a protocol for rigorous quantification of subnuclear organization in cell populations. Cells transiently co-expressing a green FP (GFP)-GRIP and the monomeric red FP (mRFP) are selected for imaging based only on the signal in the red channel, eliminating bias due to knowledge of coregulator organization. The impartially selected images of the GFP-coregulatory protein are then analyzed using an automated algorithm to objectively identify and measure the intranuclear bodies. By integrating all these features, this combination of unbiased image acquisition and automated analysis facilitates the precise and consistent measurement of thousands of protein bodies from hundreds of individual living cells that represent the population.