Single Cell Analysis of MAPK Signaling Dynamics in Multicellularity

Johns Hopkins University, Baltimore MD

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Abstract

Project Summary Cells have a remarkable ability to reproducibly respond to a changing environment even with promiscuous protein-protein interactions, stochastic gene expression, and macromolecular crowding. Our goal is to identify and characterize the mechanisms that robustly amplify signaling events over noise to govern cell biology and ultimately coordinate collective cell behavior. We use as a model the Mitogen Activated Protein Kinase (MAPK) pathway, a conserved signaling network involved in virtually all aspects of cell biology. Our focus on quantitatively understanding the timing of signaling events at the single cell and single molecule levels in the context of physiologically relevant multicellular systems allows us to dissect how signal and noise relate to functional outcome. We have developed biosensors and fully automated imaging platforms to simultaneously quantify multiple kinase activities in thousands of live single cells. More recently, we have generated transgenic mouse models to monitor signaling events in unperturbed mouse tissues and developing embryos. Using these approaches we have uncovered spatial and temporal patterns of signaling events that filter biological noise to effect deterministic outcomes. Our unique methodology features the high temporal resolution, sensor multiplexing capabilities, and single cell resolution essential for studying signaling network dynamics in single cells of a multicellular system. My research program is divided in two areas: (A) connecting single molecule behaviors to changes in cell state and (B) connecting single cell signaling activities to collective cell behaviors. This grant cycle, we propose to focus in four main questions: 1- What are the most upstream molecular events that engage the MAPK signaling network? 2- How do cells monitor and adjust the biophysical properties of the cytoplasm? 3- How are the collective properties of cytoskeleton driving cell-cell communication in tissues?, and 4- What are the mechanisms driving the unicellular to multicellular transition in mammalian embryos? Through these fundamental questions, our research program has expanded into several exciting new areas of modern biology including mechanobiology, phase transitions, and the regulation of macromolecular crowding. Using genetic and biochemical approaches coupled with powerful fluorescent biosensor and live cell imaging, we will define new biological mechanisms that control cell biology.