Novel Step Detection Algorithms for Photobleaching Analysis of Protein Complexes with Many Subunits

Abstract

Pancreatic islets secrete multiple hormones, including insulin, that are required to maintain euglycemia while meeting the energy demand of the body during everyday activities. We are interested in understanding the interplay between molecular mechanisms that precisely regulate these secretions. One specific paracrine modulator, dopamine, functions as a negative regulator of insulin secretion in the context of the pancreatic islet. It is secreted by the insulinproducing b-cells, activates an autocrine negative feedback that decreases the frequency of glucose-stimulated [Ca 2þ ] i oscillations, and in turn, inhibits insulin secretion. The G-protein coupled dopamine receptors are present in islet cells, but it is not clear how activation of the these receptors results in the observed changes in [Ca 2þ ] i in intact pancreatic islet cells. We are using an mTurquoise-Based cAMP biosensor with an improved dynamic range (Klarenbeek, J.B., et al., PLoS One, 2011), along with organic and genetically encoded Ca 2þ -indicator dyes. Labeled cells are studied by live imaging using perfused pancreatic islet. Spectral unmixing is used to extract the fluorescence emissions. This experimental setup allows us to monitor the effect of specific dopamine receptor agonists and antagonists on the two main cellular second messengers. Also, islets from mice with a genetic target mutation of the DRD3 (D3-KO) are used to measure how the deletion of the dopaminergic feedback changes the second messenger dynamics. With the same approach we are measuring the effects of the overexpression of dopamine receptor D3 in wild-type and D3-KO islets. The information from these experiments will help elucidate the mechanism of dopamine signaling in the pancreatic islet.