Impaired PDI1A mobility in the calcium-depleted ER. (A) Fluorescent lifetime imaging of ER roGFPiE in untreated and thapsigargin-treated COS7 cells. Note the decline in mean lifetime, from 1.903 ns to 1.693 ns, reflecting a reductive shift in the probe. A histogram of the distribution of lifetimes in the cells is provided (right) noting the mean ± SD (vertical white lines) lifetime, on the background of the color code corresponding to the pseudocolored image. A vertical dashed line extending through both panels is provided for alignment of the two measurements. (B) Photomicrographs of immunostaining of endogenous BiP (an ER marker, green), PDI1A-mCherry fluorescence (red) and an overlay of the two (yellow) in a COS7 cell transfected with a plasmid encoding an ER-localized PDI1A-mCherry (Pearson’s coefficient of BiP/PDI1A-mCherry co-localization r = 0.89). The purple Hoechst stains the nucleus. (C) Trace of time-dependent changes in the fluorescence intensity of PDI1A-mCherry before and after photobleaching a small patch of transfected COS7 cell volume. The green trace is of an untreated cell and the orange of a cell exposed to thapsigargin 10 minutes before data acquisition. (D) Photomicrographs of the PDI1A-mCherry fluorescence in the time-dependent series quantified in ‘C’ above. (E) As in ‘C’, except that cells were transfected with an expression plasmid encoding ER-localized mCherry. (F) As in ‘D’ but from the time series of the ER mCherry-expressing cells. (G) As in ‘C’, but cells were treated with the glycosylation inhibitor tunicamycin to promote unfolded protein stress in the ER, without affecting ER Ca2+ levels. (H) Time series of the sample shown in (G). The mean ± SD of the half-time to recovery of the two samples is provided. The size bar is 20 μm. ER, endoplasmic reticulum; PDI1A, protein disulfide isomerase 1A; standard deviation.