Figure 4

Calcium depletion-dependent association of PDI1A and calreticulin in vivo . (A) Donor (cyan fluorescent protein) fluorescence (intensity) and donor lifetime images (FLT) of an ER calcium probe, D1ER Cameleon, in untreated and thapsigargin-treated COS7 cells. A histogram of the distribution of lifetimes in the cells is provided (right) noting the mean ± SD (vertical white lines) lifetime. Also shown is a pseudo-colored image of calcium concentration derived from Cameleon’s lifetime. Note the decline in mean [Ca²⁺]_ER from approximately 3 mM to 109 μM in the treated sample. (B) Donor (GFP) fluorescent lifetime of GFP transfected alongside mCherry, setting the lower limits of the FRET for this pair. The size bar is 20 μm. (C) Donor (GFP) fluorescent lifetime a fusion protein of GFP and mCherry, setting an upper limit of FRET for this pair. (D) Donor (GFP) fluorescent lifetime of CRT-GFP fusion protein transfected alongside an ER mCherry, as a FRET acceptor. Where indicated the cells were treated with thapsigargin (TG). (E) Donor (GFP) fluorescent lifetime of CRT-GFP fusion protein transfected alongside PDI1A-mCherry, as a FRET acceptor. Where indicated the cells were treated with thapsigargin (TG). (F) Bar diagram of fraction of GFP probe bound to mCherry probe derived from the FRET measurements in B-E (mean ± SEM, n >100, P <0.001). (G) Trace of time-dependent changes in the normalized intensity of CRT-GFP after photobleaching a small patch of transfected COS7 cell volume. The green trace is of untreated sample and the red of cells exposed to thapsigargin 10 minutes before data acquisition. The mean ± SD of the half-time to recovery of the two samples is provided. CRT, calreticulin; ER, endoplasmic reticulum; FRET, fluorescence resonance imaging measurement; SD, standard deviation; SEM, standard error of the mean.