Fig. 2: Intra-ER accumulation of non-toxic aggregates of the metastable HT-aggr.
From: Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP
a Time series of FLIM micrographs of CHO-K1 cells post stable retroviral infection with ER-targeted HT-aggr probe (K73T), labelled with P1 fluorophore. ER-targeting of HT as in ref. 36. b Plot of mean fluorescence lifetime values of HT-aggrER/P1 for the time points in (a). c Plot of the percentage of cells with FLIM-detectable aggregates (lifetime > 5 ns, apparent as red puncta, the threshold set to visualise aggregates in nearly monochromatic) from image-series as in (a). d Frequency distribution of aggregates’ sizes extracted from FLIM time-series as in (a) using the Aggregates Detector, Icy algorithm. (shown in (b, c) are values of mean ± SEM, n = 7 independent fields of view with at least 20 cells each). e High-resolution fast 3D FLIM image projections of CHO-K1 cells with HT-aggrER/P1. f FLIM images of live CHO-K1 cells stably expressing HT-WTER or HT-aggrER variants with various degrees of metastability, as indicated by Gibbs free energy values of unfolding (ΔG0Unf), (h) inset. g Plot of relative cell area occupied by FLIM-detected aggregates (lifetime > 5 ns, red puncta) quantified for the HT-aggr variants from images as in (f). (mean ± SEM, n = 3 independent fields of view, 60, 42, 55 and 45 cells analysed for WT, K73T, L172Q and M21K F86L variants, respectively). h Plot of relative cell area with aggregates (a measure of aggregation from (g) for each variant) as a function of its metastability, represented by the equilibrium constant of unfolding, expressed as KUnf = exp(−ΔG0Unf/RT)21, where ΔG0Unf is the free energy of unfolding (inset). i Growth curves for CHO-K1 cells expressing the HT-aggrER variants (WT, cyan; K73T, yellow; L172Q, orange; M21KF86L, red) along with parental cells (magenta). Solid lines are the fits for the exponential growth according to the equation % conft = % conft0 2 (t/td), where td is the doubling time. Individual values and standard deviations of td calculated from triplicates are shown in the inset. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, unpaired T test (two-tailed).
