Manipulation and Elucidation of Intracellular Signaling Mechanisms through Periodic Stimulation Using Microfluidics.

Abstract

Orchestration of cellular operations often requires faithful conversion of chemical signals from the environment into intracellular messages that cells must decipher with their internal protein machinery. Intracellular messages are conveyed by chemical messengers, such as calcium. Signals from the environment and chemical messengers are regularly frequency-encoded: biological information is stored in the periodicity, not just the amplitude, of signals. Despite the wealth of mathematical models available for predicting and interpreting the mechanisms mediating the conversion of extracellular signals into messenger signals, there is a paucity of experimental setups enabling manipulation and further elucidation of this crucial conversion process. These limitations were overcome by developing a microfluidic platform able to deliver periodic extracellular chemical signals to mammalian cells and amenable to real-time imaging of messenger signal dynamics. While microfluidic-mediated periodic chemical stimulation afforded greater control over the timing of calcium messenger signals, compared to continuous chemical stimulation, fidelity was compromised; however, this deficiency was surmounted to a degree by modulating periodic stimulation parameters. These results provided concrete strategies for effectively manipulating intracellular calcium signals, using physiologically-relevant stimulant concentrations and periodicities. Our theoretical results predicted that small changes in cellular components could yield precipitous changes in calcium response fidelity, showing that fidelity can be highly sensitive to both stimulation and intrinsic parameters. By demonstrating experimentally that these cellular components can dramatically modulate the fidelity of intracellular signals, these studies provide insight into how the body achieves high fidelity control of signaling. Compromised fidelity of intracellular signals, while potentially harmful, provided valuable insight into the chemical mechanisms mediating the conversion of extracellular

signals into calcium signals. limitations, nor predict the effects of altering periodic stimulation parameters on the calcium response fidelity. Simple revisions to model mechanisms were able to account for all our experimental results, demonstrating that this approach is powerful for evaluating models and elucidating signaling mechanisms. Collectively, this thesis research delineated that by theoretically and experimentally analyzing cells’ abilities to convert periodic chemical signals into intracellular chemical messengers, manipulation and elucidation of cellular signaling mechanisms was achieved.