Tracking protein oligomerization and aggregation in live cells by number and brightness

Abstract

Protein oligomerization is involved in a broad range of signal transduction pathways and regulatory cellular mechanisms, and therefore plays a role in several human diseases ranging from cancer to neurodegenerative diseases. It is a dynamic and often transient state change, showing spatial heterogeneity in a living cell. Despite the physiological importance of protein oligomerization and aggregation, its quantification in live cells with high spatial and temporal resolution remains a challenge. Number and Brightness (N&B) is a fluorescence fluctuation spectroscopy technique based on fast laser scanning microscopy and sensitive photon counting detection, that enables quantification of absolute protein counts and oligomeric state in each pixel of a light microscopy image. In this work, we implement a new N&B analysis tool in the VistaVision software and demonstrate its power in quantitatively measuring the protein oligomerization and aggregation states in live cells. We use this tool to demonstrate that the yeast G1/S transcription factor protein Mbp1 forms small oligomers in nitrogen-limited environments.