A pH-correctable, DNA-based fluorescent reporter for organellar calcium

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 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, it functions as a pHcorrectable, 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 C. elegans homolog of ATP13A2, was responsible for lysosomal Ca 2+ accumulation-the first example of a lysosome-specific Ca 2+ importer in animals. By enabling the facile quantification of compartmentalized Ca 2+ , CalipHluor can expand our understanding of subcellular Ca 2+ importers.

Dynamic DNA Structures

“…This switch was used as a fluorescent reporter to measure and map intracellular pH changes in living cells with spatial and temporal resolution. They later developed a series of DNA nanomachines that can sense and measure important physiological ions (e.g., Ca 2+ , Cl −1 ) in intracellular compartments . In addition to ions, biological molecules in living cells such as ATP may be probed as well.…”

A Molecular Hero Suit for In Vitro and In Vivo DNA Nanostructures

“…Though in some cases, applications are not necessarily dependent on the complexity of the DNA nanostructure. Rather, simple, reconfigurable DNA structures and devices are closer to real‐world applications, such as monitoring in vivo pH, and detecting chloride, calcium, or cellular microRNA . In the case of drug delivery, there are a variety of nanocages that can host different drug molecules.…”

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology

“…After an insightful introductiono nt he rational design of DNA nanodevices as fluorescent reportersf or quantitatively imaging analytes in specific organelles, Krishnan highlighted two of these nanodevicesd eveloped in her lab and their applications. First, CalipHluor was presented as ap H-correctable Ca 2 + reporter [28] to monitor changes in Ca 2 + concentrations during endosomal maturation, and to investigate the regulation of Ca 2 + in lysosomes. These studies have led to identifying catp-6, ah omolog of ATP13A2,a saregulator for lysosomal storage of Ca 2 + entry.S econd, Voltair is aD NA-based voltmeter that is used to evaluatet ransmembrane potential of organelles, [28] and to study the contribution of V-ATPases and the effect of cell starvation on membrane potentials of distinct organelles.…”

“…These studies have led to identifying catp‐6, a homolog of ATP13A2, as a regulator for lysosomal storage of Ca 2+ entry. Second, Voltair is a DNA‐based voltmeter that is used to evaluate transmembrane potential of organelles, and to study the contribution of V‐ATPases and the effect of cell starvation on membrane potentials of distinct organelles …”