SecreTrap - a tool to measure peptide hormone secretion at the single cell level

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Project Summary The secretion of peptide hormones regulates many of the metabolic and behavioral functions of higher organisms. Therefore, monitoring of peptide hormones has been widely used in physiology and medicine, most prominently for measuring blood insulin levels. However, there is currently no method available to measure peptide hormone release continuously, neither in patients nor during experimental work. In this project, we propose to develop a fluorescent probe that monitors the secretion of peptides hormones such as glucagon and insulin on the microscope stage in real time. The method is based on fluorescent protein-peptide fusions that are co-secreted with the endogenous peptide hormones. While those fusions have been introduced earlier, a major problem is the rapid diffusion away from the secreting cell after the release happened. Here, we introduce a novel system called SecreTrap which is designed to catch the secreted peptide hormone construct and accumulate it on the surface of the secreting cell. SecreTrap will produce an accumulating signal that will overcome the above mentioned diffusion and the resulting very short-lived signal. SecreTrap further features an optogenetic component that clears the trap on the cell surface by a flash of light. This feature is required to prevent saturation of the trapping device on the cell surface. The trap and the optogenetic tool is based on the nanomolar interaction of the well-known LOV2 domain with a short designed peptide (Zdk1). Upon illumination, the LOV2-Zdk1 interaction is massively weakened thus releasing the accumulated peptide. The optogenetic part has two important roles to play. First, it helps freeing the trap of any accumulated peptide fusion before the start of the experiment. Second, at the end of the experiment, all secreted peptide fusion during the time of the experiment can be released within a few seconds giving quantitative data about the overall amount of peptide released during a given period. By Western blot this value can be correlated with secreted endogenous peptide levels. Initially, we will focus on glucagon and insulin secretion from pancreas cells. This will allow us to use established cell lines as well as primary mouse cells in intact islets. While cultured cells will be transfected by standard procedures, we will pack the constructs into an adenovirus for delivery into islet cells. Because in mouse islets, the α-cells reside predominantly on the perimeter of the islet, we expect excellent expression of the constructs and an accumulation of the glucagon fusion protein on the outside of the islet. We will expand the technique to neuropeptide Y (NPY) release from pancreatic β-cells to demonstrate general feasibility. NPY secretion is important in many tissues and a reporter system will have broad applications. This project is fully focusing on tool development and will leave major applications of the new technique to future studies. Obviously, a continuous imaging technique such as SecreTrap is well suited for cell biology and pharmacological studies as well as screening of drug candidates.