Mitochondrial sequestration of BCECF after ester loading in the giant alga Chara australis

Ratiometric fluorescent dyes are often used to monitor free ion concentrations in vivo, especially in cells that are recalcitrant to transformation with genetically encoded fluorescent markers. Although intracellular dye distributions are often found to be cytosolic, dye localisation has often not been examined in detail. We began exploring the use of BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to monitor pH in the giant alga Chara australis and discovered that younger leaf cells could be loaded using the acetoxymethyl ester of BCECF. However, we were puzzled to find in microphotometric measurements that the fluorescence ratio appeared insensitive to manipulations affecting cytosolic pH. Confocal imaging of C. australis cells loaded with BCECF showed an accumulation of the dye in two locations: (1) on the outside of the chloroplasts in irregularly shaped stationary bodies; (2) within 1-1.5 mu m structures that moved rapidly with the pericellular cytoplasmic streaming. Together with the streaming cytoplasm, these organelles were rendered stationary with 50 mu M cytochalasin D. Rhodamine 123, a mitochondrionspecific dye, highlighted organelles outside of the chloroplasts, similar to those shown by BCECF in location 1. We conclude that in the cytoplasmic compartment, BCECF was sequestered within cytoplasmic mitochondria in immature and fast-growing cells and within the cortical mitochondrial system in older and slowly growing cells. Thus, BCECF-AM is unsuitable for reporting changes in cytosolic pH in C. australis but might be employed in future to study pH changes in the mitochondria.