0000000000481767
AUTHOR
Roger Y. Tsien
Organelle pH studies using targeted avidin and fluorescein–biotin
Abstract Background: Mammalian organelles of the secretory pathway are of differing pH. The pH values form a decreasing gradient: the endoplasmic reticulum (ER) is nearly neutral, the Golgi is mildly acidic and the secretory granules are more acidic still (∼pH 5). The mechanisms that regulate pH in these organelles are still unknown. Results: Using a novel method, we tested whether differences in H + ‘leak' and/or counterion conductances contributed to the pH difference between two secretory pathway organelles. A pH-sensitive, membrane-permeable fluorescein–biotin was targeted to endoplasmic-reticulum- and Golgi-localized avidin-chimera proteins in HeLa cells. In live, intact cells, ER pH (…
Cytochrome c is released in a single step during apoptosis
Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt…