Generation and characterization of a lysosomally targeted, genetically encoded Ca2+-sensor

Distinct spatiotemporal Ca2 + signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2 + signals can be driven by release of Ca2 + from intracellular organelles that sequester Ca2 + such as the ER (endoplasmic reticulum) or through the opening of Ca2 +-permeable channels in the plasma membrane and influx of extracellular Ca2 +. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2 + to levels comparable with those found within the ER lumen. These organelles harbour ligand-gated Ca2 +-release channels and evidence indicates that they can operate as Ca2 +-signalling platforms. Lysosomes sequester Ca2 + to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide ‘trigger ’ release events that can subsequently elicit more extensive Ca2 + signals from stores including the ER. In order to investigate lysosomal-specific Ca2 + signalling a simple method for measuring lysosomal Ca2 + release is essential. In the present study we describe the generation and characterization of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2 + release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2 + handling on cellular physiology. Key words: cameleon, calcium, fluorescence resonance energy transfer (FRET), lysosome, sensor, signalling, targeting