Abstract
It is extremely challenging to quantitate lumenal Ca(2+) in acidic Ca(2+) stores of the cell because all Ca(2+) indicators are pH sensitive, and Ca(2+) transport is coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca(2+) simultaneously, CalipHluor functions as a pH-correctable Ca(2+) reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca(2+) changes during endosomal maturation and found a surge in lumenal Ca(2+) specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca(2+) accumulation-an example of a lysosome-specific Ca(2+) importer in animals. By enabling the facile quantification of compartmentalized Ca(2+), CalipHluor can expand the understanding of subcellular Ca(2+) importers.