Understanding robust cellular information processing in complex environments and development of enabling single-cell analysis technologies - The overarching goal of our laboratory is to understand how cells process signals and communicate robustly in complex environments. We combine experiments, modeling, and development of technologies for high- throughput single-cell analysis. Here, we build upon two grants that proposed fundamental (R01GM128042) and technological (R01GM127527) studies of immune signaling. Our goals are integrated around two key questions: How do cells encode information into signaling molecules? Environmental inputs are detected by sentinel cells, which in response produce cytokines. Recent studies led to the hypothesis that cytokine dynamics encode information from the environment. Determining how pathogen and stress inputs are encoded is crucial for understanding infection, autoimmunity, sepsis and cancer. We will use single-cell protein production/secretion assays and live-cell analysis to answer key questions including: How do cells exposed to multiple sequential stimuli encode the memory of prior stimuli? How does cellular density and coordination influence cytokine responses? What are the mechanisms of cytokine production variability by myeloid or epithelial cells, and what causes excessive cytokine production/secretion? Technologies to analyze single-cell protein production/secretion: We will realize a method for simultaneous measurement of single-cell expressed proteins, protein complexes and mRNA using single-cell sequencing readout. High-throughput microfluidic technologies for time-dependent measurement of proteins secreted by single live-cells will be developed. We will also develop microfluidic co-culture systems for creating controlled dynamic microenvironments. How do cells process combinatorial/dynamic signals? Signals generated by sentinel cells are processed by transcriptional pathways. NF-κB, an inflammatory pathway that controls responses to many signals, is a prime example of a system that creates fine-tuned responses. We will study NF-κB as a model system to answer key questions in signal processing in single-cells: How does NF-κB process combinatorial signals? During infection, immunity and stress, cells are exposed to combinations and temporal sequences of multiple cytokines. How NF- κB processes such inputs is not understood. How does NF-κB dynamics regulate gene expression in space and time? While much attention has been given to temporal characteristics of signaling, little is known about how NF- κB decodes signals propagating over different spatial scales. How do single cells/populations respond to dynamic (increasing, decreasing, oscillating) inputs? High-throughput live-cell analysis technologies: To better answer these and other general questions in signaling, we will develop broadly applicable live-cell analysis technologies: We will develop microfluidic systems that culture, track and analyze single-cells and populations under predetermined dynamic/combinatorial signals. We will develop microfluidic technologies for spatial analysis of live cells and cell signaling events. We will develop computational/statistical methods for automated analysis for image segmentation, cell/organoid tracking, and prediction of cellular outcomes.
