Figure 7.

Calcium depletion promotes a more reducing ER. (A) Time-dependent changes in fluorescence lifetime (FLT) of the redox reporter ERroGFPiE or the ER calcium reporter D1ER cameleon before, during, and after exposure of AR42j cells to a pulse of cholecystokinin (CCK, 2 µM). Shown are temporally superimposed typical measurements reproduced three times in cells expressing either reporter. (B) Fluorescent emission intensity (grayscale) and lifetime (color-coded) images of ERroGFPiE or D1ER cameleon in AR42j cells before, at the peak of CCK action, and after washout of CCK (as in A). (C) Traces of time-dependent changes in cytosolic calcium concentration measured by Indo 1-AM emission ratios before and after exposure to thapsigargin (2 µM). Where indicated, the thapsigargin-induced cytosolic calcium spike was buffered by 50 µM BAPTA-AM. (D) Bar diagram showing mean ± SD (n > 5) fluorescence lifetime of the ER-localized calcium probe, D1ER cameleon, or the redox probe, ERroGFPiE, in cells exposed to thapsigargin, BAPTA-AM, or both (as in C; note that D1ER cameleon’s lifetime is inversely related to ER calcium). (E) A time series of fluorescence lifetime measurements of roGFPiE dithiol (SH), disulfide (S-S), or a mixture of the two that approximates the redox state of ERroGFPiE in untreated cells (S-S/SH in vivo like) exposed in vitro to a solution whose calcium concentration was increased at regular intervals by addition of calcium (gray trace). Note that calcium concentration does not affect fluorescence lifetime of roGFPiE in vitro. Shown are representative experiments reproduced twice.