A biosensor based FLIM-FRET phasor approach to measure proteostasis capacity in cells

Moily NS, Hinde E and Hatters D

Department of Biochemistry and Molecular Biology, University of Melbourne, Australia.

The pool of quality control proteins that maintain protein-folding (proteostasis) is dynamic but can become quickly depleted in cellular stress and disease. The ability of these quality control chaperones to maintain the proteome in a folded state in health and response to stressors is not yet defined quantitatively. We have developed a family of barnase FRET-based biosensors with differing folding stabilities that engage primarily with HSP70 and HSP90 family proteins and modify its foldedness and aggregation[1]. Here we quantify the ability of these critical cellular chaperones to bind to the barnase biosensor using the phasor approach to FLIM analysis of FRET in a living cell. The phasor method is a fit free approach to fluorescence lifetime analysis that has the capacity to quantify the FRET efficiency of the barnase sensor in each pixel of a FLIM image and therefore spatially map protein foldedness in a living cell[2]. Using phasor FLIM-FRET analysis we are able to calculate the chaperone occupancy rates in folded and unfolded barnase biosensors fractions, thereby quantifying their holdase abilities in baseline conditions and when challenged through stress. By multiplexing this technology with image correlation spectroscopy, we also hope to understand the stickiness of unfolded proteins in cells by measuring barnase diffusion rates and predict how cells react to excess unfolded protein load and aggregate formation. References: 1. Wood, R.J., et al., A biosensor-based framework to measure latent proteostasis capacity. Nature Communications, 2018. 9(1): p. 287. 2. Hinde, E., et al., Biosensor FRET detection by the phasor approach to fluorescence lifetime imaging microscopy (FLIM). Microsc Res Tech, 2012. 75(3): p. 271-81.