Apollo-IRE1: A genetically encoded sensor for live cell and multiplexed imaging of ER stress.

Abstract

Several metabolic diseases, such as Alzheimer's, diabetes, and arthritis, are caused in part by the cellular accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER). In response to protein misfolding, a transmembrane stress-sensor called IRE1α activates the Unfolded Protein Response (UPR) to maintain protein folding homeostasis. To measure ER stress response in living beta-cells with spatial and temporal resolution, we developed a genetically encoded sensor based on fluorescent protein-tagged IRE1α. This sensor homo-oligomerizes to dimeric and tetrameric conformations under early and chronic ER stress, respectively, resulting in homoFRET and an associated decrease in steady-state fluorescence anisotropy (i.e., decreased IRE1 anisotropy/polarisation of fluorescence due oligomerization (Apollo-IRE1)). Apollo-IRE1 responds to chemical and physiological (high glucose) stress in a rat beta-cell line (INS-1E) and mouse islets. Preliminary data suggest that UPR-associated XBP1 mRNA splicing is highly correlated with sensor response. Furthermore, individual cells with the highest sensor response (correlating to late-stage UPR) show elevated levels of pro-apoptotic protein TXNIP. The single colour design of Apollo sensors also makes them ideal for multiplexing, offering an exciting opportunity to explore multiparametric analysis of cellular metabolic characteristics. To demonstrate the multiplexing capability of Apollo-IRE1, we will explore simultaneously imaging of a sensor for NADPH (Apollo-NADP+) and by immunofluorescence with other ER stress markers. These data validate and demonstrate the utility of Apollo-IRE1 in sensing early and chronic ER stress through live cell imaging and multiplexed analysis.