Association of cytoplasmic free Ca2+ gradients with subcellular organelles.
Previous investigations have identified gradients of intracellular free (Ca2+)i (Ca2+i) in the cytoplasm of human fibroblasts. In this study we have compared the spatial distribution of these gradients with the subcellular distribution of cytoplasmic organelles. Using the Ca(2+)-sensitive dye fura-2 and organelle-specific fluorescent dyes, we have found that the highest Ca2+ concentrations are found in the perinuclear cytoplasm and that these regions co-localize with the Golgi apparatus. The area occupied by the endoplasmic reticulum, which includes the Golgi region plus an adjacent area, is also significantly elevated above the average cellular (Ca2+)i. Most mitochondria are located in regions different from those with the highest (Ca2+)i. A variety of phenomena which could have given rise to artifactual (Ca2+)i gradients have been ruled out, including compartmentalization of fura-2 in subcellular organelles, incomplete hydrolysis of fura-2AM esters, and the presence of pH gradients which might change the Ca2+ binding characteristics of fura-2. The existence of gradients in (Ca2+)i between ER and Golgi containing regions of the cytoplasm supports the hypothesis (Sambrook: Cell 61:197-199, 1990) that the traffic of membrane bound vesicles from ER to Golgi is directed by local variations in (Ca2+)i.
Wahl
M
M
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
Sleight
R G
RG
Gruenstein
E
E
D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D003593:Cytoplasm / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000648:ultrastructure; D016257:Fura-2; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure
D004072:Digitonin; D002118:Calcium; D016257:Fura-2