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Multiplexed biochemical imaging reveals the extent and complexity of non-genetic heterogeneity in DNA damage-induced caspase dynamics. 10.17863/CAM.20943 Fries, Maximilian Werner Venkitaraman, Ashok R. Genetically identical cells show a heterogeneous response to a multitude of signals such as growth factors and DNA damage. While this heterogeneity has been shown to be a major determinant of treatment success in several diseases including cancer, little is known about how differences in biochemical signalling networks underlie such heterogeneity. State-of-the-art methodologies to study biochemical networks are often invasive and enable to quantify biochemical events only on cell populations or at a single point in time for a single cell, and therefore, cannot adequately quantify the fast, asynchronous and heterogeneous responses. In order to address these limitations, we have developed a unique sensing platform based on fluorescence lifetime imaging microscopy (FLIM) capable to multiplex at least three biosensors by utilizing Förster Resonance Energy Transfer (FRET) efficiently. After an overall introduction in Chapter 1, I describe the rational design and characterization of novel FRET pairs aiming to utilize the visible spectrum efficiently in combination with FLIM in Chapter 2. We combined blue, green and red donor fluorescent proteins that are excited at the same wavelength (840 nm for two-photon excitation) with genetically encoded quenchers, i.e. non-fluorescent chromoproteins as acceptors. This sensing platform enables the simultaneous detection of three biochemical reactions within single living cells providing new opportunities to characterize and understand non-genetic heterogeneity. In Chapter 3, I will demonstrate the first application of this novel platform by studying the activity of three key enzymes in DNA damage-induced cell death, caspase-2, -3, and -9. We confirm the heterogeneous nature of Cisplatin-induced cell death in genetically identical cells but reveal the existence of at least three subpopulations of cells characterized by distinct caspase dynamics. By combining biochemical and morphological information we infer the existence of different biochemical network topologies that are associated with alternative death phenotypes each cell adopts, such as apoptosis and programmed necrosis. Finally, deconvolution of cellular populations and direct measurement of a three-node caspase network - formerly impossible - permitted us to design perturbations of cell fate choices utilizing clinically relevant inhibitors. These perturbations resulted in changes in cell fate in response to Cisplatin, a clinically desirable outcome that suggests new avenues for combinatorial drugging and a new strategy to reveal cancer vulnerabilities that may be otherwise confounded by typical genetic and non-genetic heterogeneity. University of Cambridge 2018-04-28 Thesis Doctoral Doctor of Philosophy (PhD) en Gates Cambridge Trust https://www.repository.cam.ac.uk/handle/1810/273868 https://www.repository.cam.ac.uk/bitstreams/e6550d42-aa3b-4865-8af8-e31c8a67ee41/download 87eda9de84448d1f82354d60eee3eb5f https://www.repository.cam.ac.uk/bitstreams/f2e3dcf7-fdd6-41e2-b3ec-b027257d78a9/download 15b8f4fb65933cde78283e8f5a33aa15 https://www.rioxx.net/licenses/all-rights-reserved/ Apoptosis Caspase FLIM MICROSCOPY FRET Multiplexing DNA Damage Fluorescent proteins