An Imaging Platform for Evaluating Complex Formation in Real Time Applied to Translation Initiation in Living Cells

Elucidating the formation of molecular complexes in living cells is key for understanding cellular homeostasis, but has not been amenable to quantitative microscopy. Here we established a platform for real-time quantification of molecular interactions to monitor the formation of complexes that results in a differential diffusion from their components. We have applied this to one of the most dynamic processes: the regulation of mRNA translation. We observe that diffusion of initiation factors (IFs) changes markedly upon their association with mRNA. Quantifying their binding dynamics revealed the sequence of IFs assembly and disassembly in cell lines and the clustering of translation in neurons. This methodology revealed translation regulation at unprecedented spatial and temporal resolution and can be applied to the formation of any endogenous complex that results in a measurable shift in diffusion.