Automated Detection and Quantification of Granular Cell Compartments

Abstract

Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effective adaptation to physiological changes. Thus, in response to stress and disease, cells initiate protective mechanisms to restore homeostasis. Among these mechanisms are the arrest of translation and remodeling of ribonucleoprotein complexes into granular compartments in the cytoplasm, known as stress granules (SGs). To date, the analysis of SGs has relied on the manual demarcation and measurement of the compartment, making quantitative studies time-consuming, while preventing the efficient use of high-throughput technology. We developed the first fully automated, computer-based procedures that measure the association of fluorescent molecules with granular compartments. Our methods quantify automatically multiple granule parameters and generate data at the level of single cells or individual SGs. These techniques detect simultaneously in an automated fashion proteins and RNAs located in SGs. The effectiveness of our protocols is demonstrated by studies that reveal several of the unique biological and structural characteristics of SGs. In particular, we show that the type of stress determines granule size and composition, as illustrated by the concentration of poly(A)-RNA and a specific SG marker protein. Furthermore, we took advantage of the computer-based and automated methods to design assays suitable for high-throughput screening.