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.