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

Area of Science:

• Cellular Biology
• Biochemistry
• Molecular Biology

Background:

• Quantifying lumenal calcium (Ca²⁺) in acidic cellular organelles is challenging due to pH sensitivity of Ca²⁺ indicators and Ca²⁺ transport's coupling with pH.
• Existing methods struggle to accurately measure Ca²⁺ in acidic environments like the endolysosomal pathway.

Purpose of the Study:

• To develop a novel reporter for accurate measurement of lumenal Ca²⁺ in acidic organelles.
• To investigate Ca²⁺ dynamics within the endolysosomal pathway.
• To identify the molecular mechanisms underlying lysosomal Ca²⁺ accumulation.

Main Methods:

• Development of CalipHluor, a fluorescent DNA-based reporter for simultaneous ratiometric measurement of pH and Ca²⁺.
• Targeting CalipHluor to the endolysosomal pathway in Caenorhabditis elegans.
• Lysosomal proteomics and genetic analysis to identify Ca²⁺ transport proteins.

Main Results:

• CalipHluor successfully provided pH-correctable measurements of lumenal Ca²⁺.
• A significant surge in lumenal Ca²⁺ was observed specifically in lysosomes during endosomal maturation.
• The protein catp-6 (a homolog of ATP13A2) was identified as a key lysosomal Ca²⁺ importer in C. elegans.

Conclusions:

• CalipHluor enables facile quantification of compartmentalized Ca²⁺, overcoming pH-related limitations.
• The identification of catp-6 highlights a lysosome-specific Ca²⁺ importer in animals.
• This work expands the understanding of subcellular Ca²⁺ importers and lysosomal function.