Project description:All vertebrates have multiple genes encoding for different CASQ isoforms. Increasing interest has been focused on mammalian and human CASQ genes since mutations of both cardiac (CASQ2) and skeletal (CASQ1) isoforms cause different, and sometime severe, human pathologies Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work expression, biochemical properties and cellular and sub-cellular localization of Danio rerio native CASQ isoforms are investigated. By quantitative PCR three mRNAs were detected in skeletal muscle and one mRNA in heart. Three zebrafish CASQs were identified by mass spectrometry and they share properties with mammalian skeletal and cardiac CASQs. Skeletal calsequestrins were found primarily, but not exclusively, at the sarcomere Z-line level where Terminal Cisternae of Sarcoplasmic reticulum are located.
Project description:Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).