ER-Mitochondria Crosstalk during Cerebral Ischemia: Molecular Chaperones and ER-Mitochondrial Calcium Transfer
Figure 2
GRP78 retargets to mitochondria with glucose deprivation (GD). (a) Fluorescence photomicrographs were taken before and after 1 hr GD. Under normal conditions, the green fluorescence in eGFP-Grp78 transfected cells shows the normal perinuclear ER localization. After 1 hr GD this changes to a diffuse cytoplasmic pattern overlapping with the mitochondrial distribution visualized by partial overlap with Mito-tracker (red) fluorescence. Overlap is yellow. (b) After 3 hr GD, mitochondria were purified and analyzed by Western blotting using antibodies against GRP78, PDI (an ER-specific marker), or COXIV (a mitochondrial marker). The purified mitochondria do not show contamination with ER marker PDI but do have GRP78. Ctrl: control. Trans: transfected. (c) Submitochondrial localization of GRP78 in control and GD-stressed cells by immunoelectron microscopy. Arrows point to the localization of GRP78. The gold particles were associated exclusively with ER membrane in control cells (left panel). In contrast, immune-EM staining shows GRP78 within mitochondria from 3 hr GD-stressed cells, demonstrating significant mitochondrial labeling, with grains mainly decorating the inner mitochondrial membrane (right panel). M, mitochondrion. Scale bars, 100 nm.
