title: Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP.
abstract: The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Henderson
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health.
authors.forename: Emily S
authors.initials: ES
authors.lastname: Wires
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health.
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Trychta
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health.
authors.forename: Xiaokang
authors.initials: X
authors.lastname: Yan
authors.affiliation: National Institute on Drug Abuse, National Institutes of Health; bharvey@mail.nih.gov.
authors.forename: Brandon K
authors.initials: BK
authors.lastname: Harvey
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D033342:Copepoda / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D059865:Endoplasmic Reticulum Stress; D006706:Homeostasis; D006801:Humans; D008156:Luciferases / Q000032:analysis* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D017510:Protein Folding; D021381:Protein Transport; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000032:analysis / Q000378:metabolism
chemical_list: D008156:Luciferases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium