Chad He, Linda Kippner
Collaborators: Hang Lu (Georgia Tech)
Funding Source: NIAID R01
Reactive oxygen species (ROS) are produced in distinct cellular locations – by the organelle location of oxidases and mitochondria – and exert their effects only nanometers from the site of production. Little is known about how cells use and discriminate between plasma membrane generated, mitochondrial, or extracellular sources of reactive oxygen species to control signal transduction. The objective of this project is to investigate ROS spatiotemporal dynamics during T cell signaling through the development of site-specific ROS dyes, high-throughput microfluidic systems, and computational models. We hypothesize that the subcellular sources of ROS create a tightly connected network between mitochondria, endoplasmic reticulum and plasma membrane oxidases to regulate T cell signaling. The rationale for this research is that by understanding when and where ROS is used to target protein oxidation during antigen recognition, cellular oxidation can move from phenomenological observation to a relevant diagnostic biomarker for disease state. The outcomes of this work are expected to fundamentally advance our understanding of how cells use spatially distinct ROS sources to regulate receptor-initiated signaling. This knowledge will have large impact in ultimately redefining intracellular oxidation by more biologically relevant metrics for diagnosis and treatment of diseases.
Kniss-James, A.S., Rivet, C.A., Chingozha, L., Lu, H., Kemp, M.L. “Single-cell resolution of intracellular T cell Ca2+ dynamics in response to frequency-based stimulation”. Integrative Biology, DOI: 10.1039/C6IB00186F, 2017.
*Rivet, C.A., *Kniss, A., Gran, M.A., Potnis, A., Hill, A., Lu, H., Kemp, M.L. “Calcium dynamics of ex vivo long-term cultured CD8+ T cells are regulated by age related changes in redox metabolism”. PLoS One,11(8):e0159248, 2016.
*Warren, E.A.K., *Netterfield, T.S., Sarkar, S., Kemp, ML, Payne, C.K.. “Spatially-resolved Intracellular Sensing of Hydrogen Peroxide in Living Cells”. Scientific Reports 5:16929, DOI: 10.1038/srep16929, 2015.
*He L, *Kniss A, San-Miguel A, Rouse T, Kemp ML, Lu H. An automated platform enabling dynamic stimuli delivery and cellular response readout for high-throughput single-cell signaling studies. Lab on a Chip, 15(6):1497-507, 2015.
*He, L, *Kniss, A, Kemp, ML, Lu, H. Automated high-throughput microsystem for tunable temporal stimulation and analysis of non-adherent cells”. International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS) extended abstract, 2013.
Kniss, A., Lu, H., Jones, D.P., Kemp, M.L. “A microfluidic systems biology approach for live single-cell mitochondrial ROS imaging”. Methods in Enzymology, 526: 219-30, 2013.
Reddie, K.G., Humphries, W.H., Bain C.P., Payne, C.K., Kemp, M.L., Murthy, N. “Fluorescent coumarin thiol probes measure the thiol-disulfide redox couple”. Organic Letters, 14(3): 680, 2012.
Chung, K., Rivet, C.A., Kemp, M.L. Lu, H. “Imaging single-cell signaling dynamics with a deterministic high-density single-cell trap array”. Analytical Chemistry, 83(18):7044-52, 2011.
Rivet, C.A., Hill, A.S., Lu, H., Kemp, M.L. “Predicting cytotoxic T cell age from multivariate analysis of static and dynamic biomarkers” Molecular and Cellular Proteomics, 10(3), 2011.
Hirsch, A., Rivet, C., Zhang, B., Kemp, M.L., Lu, H. “Parallel multi-time point cell stimulation and lysis on-chip for studying early signaling events in T-cell activation”, Lab on a Chip, 9:536-544, 2009.