Longitudinal monitoring of Gaussia and Nano luciferase activities to concurrently assess ER calcium homeostasis and ER stress in vivo

• Published in: PloS one Vol. 12; no. 4; p. e0175481
• Main Authors: Wires, Emily S, Henderson, Mark J, Yan, Xiaokang, Bäck, Susanne, Trychta, Kathleen A, Lutrey, Molly H, Harvey, Brandon K
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 12.04.2017; Public Library of Science (PLoS)
• Subjects: Amino acid sequence; Animal models; Animals; Asparagine; Aspartate-ammonia ligase; ATP; Biological Assay; Biological samples; Biology and Life Sciences; Calcium; Calcium - metabolism; Calcium Signaling; Cell death; Cell Line, Tumor; Computer applications; Copepoda - enzymology; Deep sea; Degradation; Depletion; Disease; Drug abuse; Emission measurements; Emissions; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum Stress; Enzymes; Foot & mouth disease; Foot-and-mouth disease; Gene expression; Glucose; Heart diseases; Homeostasis; Humans; In vivo methods and tests; Inositols; Kinases; Lipids; Luciferase; Luciferases - metabolism; Luminescence; Medicine and Health Sciences; Membrane proteins; Metabolism; Monitoring; Movement disorders; Neurodegenerative diseases; Neuronal-glial interactions; Oxidation; Parkinson's disease; Physical Sciences; Physiological aspects; Protein synthesis; Proteins; Residues; Ribonucleic acid; RNA; Rodents; Splicing; Stress; Stress (Physiology); Substrates; T-cell receptor; Temperature effects; Toxicity; Translocation; Tropism; Unfolded Protein Response; Viruses; United States; United States > US; Maryland; Baltimore Maryland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0175481

The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism and calcium storage. The ability to longitudinally monitor ER homeostasis in the same organism would offer insight into progressive molecular and cellular adaptations to physiologic or pathologic states, but has been challenging. We recently described the creation of a Gaussia luciferase (GLuc)-based secreted ER calcium-modulated protein (SERCaMP or GLuc-SERCaMP) to longitudinally monitor ER calcium homeostasis. Here we describe a complementary tool to measure the unfolded protein response (UPR), utilizing a UPRE-driven secreted Nano luciferase (UPRE-secNLuc) to examine the activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1) pathways of the UPR. We observed an upregulation of endogenous ATF6- and XBP1-regulated genes following pharmacologically-induced ER stress that was consistent with responsiveness of the UPRE sensor. Both GLuc and NLuc-based reporters have favorable properties for in vivo studies, however, they are not easily used in combination due to overlapping substrate activities. We describe a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. This study is the first to describe the in vivo measurement of UPRE activation by sampling blood, using an approach that allows concurrent interrogation of two components of ER homeostasis.