University of Minnesota
School of Physics & Astronomy

Biophysics Seminar

Thursday, March 8th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Elizabeth Smith (Elias Puchner lab), School of Physics and Astronomy, University of Minnesota
Subject: Characterization of Ire1 interactions and dynamics with quantitative super-resolution microscopy

Quantitative Super-Resolution Microscopy is a powerful technique to study biological processes below the diffraction limit. In this work, we employ our intracellular calibrated Photoactivated Localization Microscopy (PALM) technique to perform quantitative molecular counting of proteins involved in the unfolded protein response (UPR). The UPR is a signaling pathway which dynamically regulates endoplasmic reticulum (ER) protein folding capacity in response to cellular stress. As is true with many signaling pathways, the spatiotemporal organization of the UPR-specific biomolecules is an inherent feature of the pathway activation and downstream response. Specifically, in response to stress, Ire1 (a bifunctional transmembrane kinase/endoribonuclease) oligomerizes and forms discrete signaling clusters which recruit and splice an mRNA encoding a transcription activator. Using PALM in conjunction with traditional fluorescence microscopy we characterize the interactions and dynamics of Ire1 at wild type expression levels in yeast cells. Specifically, we quantify the oligomeric state, of Ire1 under stressed and unstressed conditions, track the motion of Ire1 during signaling activity, and determine the sensitivity and resolution of spatial cross-correlation in a model system combining traditional and super-resolution fluorescencemicroscoy in the same protein construct (Ire1_yeGFP_mEos2). Finally we perform colocalization experiments with downstream UPR biomolecules to further characterize the role of Ire1 signaling centers in control of gene expression. This study provides insight into the spatiotemporal organization of Ire1 and its downstream partners in the signaling response of the UPR.

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