University of Minnesota
School of Physics & Astronomy

Biophysics Seminar

Thursday, September 22nd 2016
11:00 am:
Biophysics Seminar in 120 PAN
Speaker: Prof. David Piston, Washington University School of Medicine
Subject: Imaging the Molecular Mechanisms of Pancreatic Hormone Secretion
Note change of room from previous announcement.

The release of insulin from pancreatic β-cells plays a central role in maintaining normal blood glucose levels. Regulated trans-membrane gradients of calcium and potassium ion channels signal to increase or decrease insulin release. This is exemplified with the stimulation of dopamine, a neurotransmitter, which reduces intracellular calcium oscillations by activation of the dopamine receptor D3 (DRD3) and therefore inhibits secretion of insulin (Ustione & Piston, Mol. Endocrinol. 26, 1928 (2012)). We seek to better understand the physiological and cellular functions of dopamine signaling as it could potentially lead to a novel treatment of diabetes. DRD3 is a G protein-coupled receptor that we propose might interact with cellular ion channels through the Gβγ complex. Towards understanding which channels and pathways are involved, we have turned to quantitative imaging assays, the first of which is based on Förster resonance energy transfer (FRET), a technique widely used to study biomolecular dynamics and protein interactions in live cells. Limitations due to brightness differences, donor:acceptor stoichiometry, and cross-talk between the donor and acceptor can lead to misleading or even meaningless results. To help alleviate these problems with cellular FRET measurements, we have developed a new approach for absolute and high precision measurements of FRET efficiency is based upon the use of an optical switching acceptor. By employing a defined train of optical perturbations to control the on and off states of the acceptor, it is possible to modulate the fluorescence intensity of the donor, and this can be analyzed using a lock-in detection approach. Secondly, we have leveraged two-color Fluorescence Fluctuation Spectroscopy (FFS), which can be used to directly measure diffusion and binding rates of proteins within the cell. Using FFS to measure protein binding and diffusion allows quantification at the molecular level of protein interactions. In addition, protein molecular concentration can also be determined. Quantifying the molecular interactions within the proposed dopaminergic feedback pathway illuminates the signaling pathway, and thus provides essential information for developing a therapeutic treatment of non-insulin dependent diabetes.

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