Investigating insulin vesicle maturation pathways using soft X-ray tomography.

Abstract

Insulin secretion is a well-studied phenomenon consisting of an interplay between various cellular signaling pathways. Despite the importance of insulin in regulating blood glucose, little is known about the pathways governing insulin vesicle acidification and maturation. Proper maturation of vesicles is imperative for the processing of the active form of insulin and vesicle trafficking. Here, we use Soft X-ray Tomography (SXT) to investigate the effect of a variety of insulin secretagogues and chemical probes to determine pathways leading to maturation of insulin vesicles. SXT is an ideal approach to this problem as it enables high-throughput, three-dimensional organelle mapping of intact cells. This enables us to map and quantify the trafficking of vesicles, informing us about subcellular rearrangements and organelle contacts during the secretory process. In addition, SXT allows us to detect changes in molecular densities and in turn, changes in biochemical density and packaging efficiency of insulin vesicles. Our results indicate varied levels of insulin vesicle density and differences in organelle contacts, specifically insulin vesicle-mitochondria contacts, under the influence of compounds acting via orthogonal signaling pathways. Overall, our screening pipeline will be useful to allow for rapid and high throughput identification of cellular pathways involved in enhancing insulin packaging and maturation.