Correlating Rat Basophil Leukemia Cell Activation with Interleukin 4 RNA Production using Single Molecule Fluorescence In-Situ Hybridization, Automated Super-Resolution Microscopy, and GPU-Enabled Image Analysis

Abstract

Single-molecule, single-cell studies of genetic expression have provided key insights into how cells respond to external stimuli. Direct single-cell measurements of individual bio-molecules, such as small RNA (sRNA) or messenger RNA (mRNA) transcripts, make it possible to quantify the heterogeneity in spatiotemporal responses of key signaling and regulatory processes. Critically, these investigations provide information on cellular fluctuations-information that is hidden by typical biochemical ensemble measurement approaches. Recent work has suggested that macroscopic cell properties, such as cell morphology, are correlated with gene expression. Here we present single-cell studies of a signal-activated gene network: Interleukin 4 (IL4) RNA production in rat basophil leukemia (RBL) cells during the allergic response. IL4 mRNA production has been closely linked to histamine release by RBL cells in ensemble measurements. To further investigate this regulatory network, we fluorescently label individual IL4 RNA transcripts in populations of RBL cells, subject to varying external stimuli. A custom super-resolution microscope and GPU-accelerated data analysis package are used to measure the number of fluorescently labeled IL4 transcripts in populations of RBL cells on a cell-by-cell basis. To test the hypothesis that RBL cell morphology may connected to IL4 production and histamine release, we analyze white light images of RBL cells and cross-reference cell morphology with IL4 RNA levels. We find that the activation of RBL cells, determined by white-light imaging, is well correlated with IL4 mRNA expression yet highly heterogeneous for certain stimuli.