Subcellular Localization Analysis of Sigma 1 Receptor (S1R) and Binding Immunoglobulin Protein (BiP) for Identification of Antagonists versus Agonists

Abstract

<b>Abstract ID 17604</b> <b>Poster Board 124</b> Sigma receptors are attractive therapeutics targets in numerous neurological diseases and cancers. Sigma 1 receptors (S1Rs) are localized in the endoplasmic reticulum (ER) membranes associated with mitochondria where they may participate in cellular stress signaling. During ER stress or stimulation by ligands, S1Rs dissociate from binding immunoglobulin protein (BiP) and exert chaperone activity. S1R agonists promote dissociation while antagonists maintain the complex with BiP. As S1R and BiP are expressed throughout the cell, fluorescence microscopy techniques might not be sensitive to localization and co-localization changes upon ER stress and ligand binding. Therefore, this study aims to perform a quantitative image analysis of S1R and BiP subcellular localization and co-localization during ER stress, and ER stress with S1R ligand treatment. Chinese hamster ovary (CHO) cells were maintained in adherent culture and a suspension was prepared by trypsinizing and adjusting to 200 cells/μL. After CHO cell attachment to fibronectin-coated coverslips, one group was treated with 3 μM thapsigargin to cause ER stress, and another group was treated with 1 μM BD-1047 following 3 μM thapsigargin treatment. Hoechst and phalloidin were multiplexed with secondary antibodies for S1R and BiP primary antibodies. The acquired images were subjected to multistep processing to enable analysis of S1R and BiP localization using MetaMorph software. Background and intensity unevenness were corrected with an object size estimation of 3 pixels, and then high frequencies were suppressed by replacing each pixel intensity with the local average intensity. 90% low threshold values were subtracted for each image and the resulting images were auto-scaled within 95% low and 4% high range. A sample size of 25 cells per treatment group was analyzed. All the quantifications were done at three different cellular regions: whole cell, perinuclear and nuclear. The differences among groups were analyzed by a one-way ANOVA followed by Tukey9s multiple comparison test in GraphPad Prism. ER stress increased the average area of discrete objects of both S1R and BiP, increased total S1R and BiP area in whole cell measurements, and increased S1R area in nuclear and perinuclear regions. Co-localization of S1R and BiP upon ER stress is observed throughout the cell, nuclear and perinuclear regions. The co-localization is lowered by BD-1047 in perinuclear and nuclear regions where S1R and BiP participate in stress signaling activity. We conclude this image analysis approach is useful for potential classification of S1R ligands as either agonists or antagonists and predicting ligand efficiency in particular disease states. This work was supported by SIUE School of Pharmacy and SIUE Graduate School.