Oscillatory signals regulate a wide variety of integral physiological and cellular processes, from G- protein coupled receptor (GPCR) signaling to circadian rhythms. Although an actively studied area, even the most well-known and commonly studied pathways can have controversy and lack of clarity on circuit architecture. This is because pathway perturbation studies using conventional molecular or genetic tools only provide limited information resulting in multiple plausible mechanisms. This proposal will develop tools and methods based on non-linear frequency and waveform response analysis to dissect such oscillatory pathways in ways that are not possible with conventional molecular or genetic perturbations alone. Specifically, we will use microfluidics to apply a periodic chemical input to cells and observed phase-locked cellular responses using real-time fluorescent readouts of intracellular signaling. The observed frequency response characteristics will be evaluated using computer models of the signaling pathway. Signaling circuit architecture as well as modes of action and mechanisms of inhibitors, agonists, and modulators will be dissected. Although the method should be applicable to any oscillatory signaling pathway, we will first focus on two GPCR signaling pathways (M3 muscarinic acetylcholine receptor and type 5 metabotropic glutamate receptor) that have very different proposed mechanisms of oscillation and that are physiologically and pharmacologically important (diabetes and schizophrenia).
Aim 1. Analyze Phase Locking Response of Cells Under Base Conditions: Perform microfluidic pulsed stimulation of live cells with receptor ligands. Obtain high time resolution real-time imaging of intracellular signals using genetically encoded fluorescent indicators of calcium and IP3.
Aim 2. Construct Mathematical Models of Signaling Circuitry: First construct plausible mathematical models based on published data. Then refine the circuit architecture and parameters to match observations in Aim 1, guided by results of uncertainty and sensitivity analyses.
Aim 3. Delineate Mechanisms of Action of Modulators Through Phase Locking Analysis: Study how phase locking responses of cells change in the presence of inhibitors, agonists, and modulators. Use the experimental observations with mathematical models to delineate mechanisms of action.
Aim 4. Disseminate self-regulating chips that make microfluidic phase-locking studies accessible to anyone.
Oscillatory signaling pathways play critical roles in biological function, including mediating signals from G- protein coupled receptors, which are targeted by the majority of clinically used pharmaceuticals. This proposal will develop novel ways to dissect such pathways and to identify molecules to modulate such pathways. This will in turn impact public health by enhancing basic biological knowledge and accelerating drug development.
|Sumit, M; Takayama, S; Linderman, J J (2017) New insights into mammalian signaling pathways using microfluidic pulsatile inputs and mathematical modeling. Integr Biol (Camb) 9:6-21|
|Kim, Sejoong; LesherPerez, Sasha Cai; Kim, Byoung Choul C et al. (2016) Pharmacokinetic profile that reduces nephrotoxicity of gentamicin in a perfused kidney-on-a-chip. Biofabrication 8:015021|
|Chang, S Laura; Cavnar, Stephen P; Takayama, Shuichi et al. (2015) Cell, isoform, and environment factors shape gradients and modulate chemotaxis. PLoS One 10:e0123450|
|Kim, Sung-Jin; Yokokawa, Ryuji; Lesher-Perez, Sasha Cai et al. (2015) Multiple independent autonomous hydraulic oscillators driven by a common gravity head. Nat Commun 6:7301|
|Sumit, M; Neubig, R R; Takayama, S et al. (2015) Band-pass processing in a GPCR signaling pathway selects for NFAT transcription factor activation. Integr Biol (Camb) 7:1378-86|
|Kirschner, Denise E; Hunt, C Anthony; Marino, Simeone et al. (2014) Tuneable resolution as a systems biology approach for multi-scale, multi-compartment computational models. Wiley Interdiscip Rev Syst Biol Med 6:289-309|
|Cavnar, S P; Ray, P; Moudgil, P et al. (2014) Microfluidic source-sink model reveals effects of biophysically distinct CXCL12 isoforms in breast cancer chemotaxis. Integr Biol (Camb) 6:564-76|
|Mosadegh, Bobak; Mazzeo, Aaron D; Shepherd, Robert F et al. (2014) Control of soft machines using actuators operated by a Braille display. Lab Chip 14:189-99|
|Zhao, Shuang; Chang, S Laura; Linderman, Jennifer J et al. (2014) A Comprehensive Analysis of CXCL12 Isoforms in Breast Cancer(1,2.) Transl Oncol :|
|Lai, David; Frampton, John P; Tsuei, Michael et al. (2014) Label-free direct visual analysis of hydrolytic enzyme activity using aqueous two-phase system droplet phase transitions. Anal Chem 86:4052-7|
Showing the most recent 10 out of 22 publications