title: Generation and characterization of a lysosomally targeted, genetically encoded Ca(2+)-sensor.
abstract: 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.
authors.affiliation: The Physiological Laboratory, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown St, Liverpool L69 3BX, UK.
authors.forename: Hannah V
authors.initials: HV
authors.lastname: McCue
authors.forename: Joanna D
authors.initials: JD
authors.lastname: Wardyn
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burgoyne
authors.forename: Lee P
authors.initials: LP
authors.lastname: Haynes
mesh_terms: D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1 / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D060149:Tetraspanin 30 / Q000378:metabolism; D014162:Transfection
chemical_list: D002147:Calmodulin; D008164:Luminescent Proteins; D052118:Lysosomal-Associated Membrane Protein 1; D018942:Macrolides; D011993:Recombinant Fusion Proteins; D060149:Tetraspanin 30; C579281:YCaM3.6 protein; D006632:Histamine; C040929:bafilomycin A1; D002118:Calcium