Phase-Locked Signals Elucidate Circuit Architecture of an Oscillatory Pathway

This paper introduces the concept of phase-locking analysis of oscillatory cellular signaling systems to elucidate biochemical circuit architecture. Phase-locking is a physical phenomenon that refers to a response mode in which system output is synchronized to a periodic stimulus; in some instances, the number of responses can be fewer than the number of inputs, indicative of skipped beats. While the observation of phase-locking alone is largely independent of detailed mechanism, we find that the properties of phase-locking are useful for discriminating circuit architectures because they reflect not only the activation but also the recovery characteristics of biochemical circuits. Here, this principle is demonstrated for analysis of a G-protein coupled receptor system, the M3 muscarinic receptor-calcium signaling pathway, using microfluidic-mediated periodic chemical stimulation of the M3 receptor with carbachol and real-time imaging of resulting calcium transients. Using this approach we uncovered the potential importance of basal IP3 production, a finding that has important implications on calcium response fidelity to periodic stimulation. Based upon our analysis, we also negated the notion that the Gq-PLC interaction is switch-like, which has a strong influence upon how extracellular signals are filtered and interpreted downstream. Phase-locking analysis is a new and useful tool for model revision and mechanism elucidation; the method complements conventional genetic and chemical tools for analysis of cellular signaling circuitry and should be broadly applicable to other oscillatory pathways.Author Summary: Key to robust discernment of cell circuit architecture is to have as many distinct response features as possible for comparison and evaluation. One under-appreciated characteristic of oscillatory circuits is that under periodic stimulation, these systems will exhibit responses synchronized to this stimulatory input, a phenomenon termed phase-locking. We demonstrate that phase-locked response characteristics vary noticeably depending on circuit activation and recovery properties; these response characteristics thereby provide a unique set of criteria for oscillatory circuit architecture analysis. The concept is validated through experiments on an oscillatory calcium pathway in mammalian cells; the experimental setup allowed us to explore, for the first time, the properties of chemically induced phase-locking of intracellular signals. Observations of this phenomenon were then used to test the predictions of several existing mathematical models of calcium signaling. Most of the models we evaluated were unable to match all our experimental observations, suggesting that current models are missing mechanistic elements in the context of calcium signaling for the cell type and receptor/stimulant tested. The observations of phase-locking further led us to identify one simple mechanistic modification that would account for all the experimental observations. The techniques and methodology presented should be broadly applicable to a variety of biological oscillators.