Fig. 1: Fluorescence lifetime-based probing system for real-time monitoring of protein aggregation.
From: Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP
a Schematic representation of the HT-aggr probing system. b Normalised autocorrelation function (G(τ) − 1) from fluorescence correlation spectroscopy measurements of purified, native or heat-aggregated HT-aggr (K73T) probe or (c) HaloTag wild type (HT-WT), labelled with the solvatochromic fluorophore P114. Solid lines represent best fits of the autocorrelation function considering a single species for HT-aggr and HT-WT, and two species for heat-aggregated HT-WT, as shown by the hydrodynamic radius values in the inset. d, e Fluorescence lifetime histograms of samples in (b, c), respectively. f A time series of live-cell fluorescence lifetime micrographs (FLIM) of HT-aggr (K73T) transiently expressed in the ER or cytoplasm of COS7 cells, before and after exposure to a heat-shock treatment (43 °C), representative images from three independent experiments. g Fluorescence lifetime histograms from image-series shown in f, (green, yellow, orange and pink traces represent the time points before, 5-, 15- and 35-min heat shock, respectively). h fluorescence lifetime values of individual cells as in (f).
