Single-cell NF-kappaB signaling in macrophages

The transcription factor NF-kappaB is heavily involved in innate immunity, and shows complex dynamic patterns of nuclear translocation in response to many stimuli. These dynamics are hypothesized to contain information about a particular stimulus. Single cell studies of NF-kappaB have shown high variability across cells, raising questions about the reliability of biochemical information encoding. Additionally, the majority of work to date has only measured NF-kappaB activity in non-immune cell types, leaving the question of how dynamics function in native contexts, like macrophage activation by Toll-like receptor signaling. To answer these questions, I developed an automated image processing algorithm to accurately track individual macrophages over 12-24 hours. I use this algorithm to measure multiple dimensions of macrophage activation, including NF-kappaB activity, in fluorescently-tagged cells.;Thousands of NF-kappaB responses to lipopolysaccharide in macrophages were generated with this automated process, then applied in the development of an algorithm that estimates the information transduction capacities of biochemical networks. I show that NF-kappaB dynamics, as well as response dynamics in the ERK and Ca2+ systems, all demonstrate enhanced information transmission compared to nondynamic responses. Theoretical analysis demonstrates that dynamics play a key role in overcoming extrinsic noise. Experimental measurements of information transmission in the ERK network under varying signal-to-noise levels confirm these predictions and show that signaling dynamics mitigate extrinsic noise-induced information loss. By reducing information loss from cell-to-cell variability, dynamic responses improve the accuracy of signaling networks.;Finally, I present a study that couples single cell measurements with an iteratively-developed computational model to examine the respective roles of the MyD88 and TRIF pathways in determining dynamic responses TLR4 stimulation. I show how each pathway encodes distinct features in NF-kappaB dynamics, and contributes uniquely to the high variability observed in single-cell measurements. In one pathway, the assembly of a macromolecular signaling platform dictates initial response timing and provides for a reliable NF-kappaB signal. In the other, ligand-induced receptor translocation and endosomal maturation combine to produce noisy yet sustained NF-kappaB signals via TRIF. Thus, I arrive at a predictive understanding of how these molecular mechanisms provide for ligand-dose and pathogen-specific signaling dynamics and information transduction capacities.