UC Davis

The ability of immune cells to detect, process, and quickly respond to environmental cues is critical for pathogen clearance and mounting inflammatory responses. For instance, neutrophils use chemotaxis- the migration of cells along chemical gradients- to rapidly migrate toward pathogen-derived cues and act as a first line of defense against infection. To do so, neutrophils translate extracellular spatial information into polarized subcellular domains of signaling activity, cytoskeletal rearrangements, and adhesion dynamics. Mounting precise responses to complex inflammatory cues relies upon information processing steps within the chemotaxis signaling cascade. Much of this processing is known to occur through members of the Rho family of GTPases, whose activity is focused by autonomous feedback circuits and crosstalk connections, to activate the necessary signaling pathways at distinct times and subcellular locations. However, limitations in our ability to both measure GTPase activities and perturb signaling inputs with high spatial and temporal resolution have impeded our understanding of neutrophil information processing. In this work, we present new light-based molecular tools and automated imaging assays to simultaneously perturb and measure Rho GTPase activities in space and time. First, we outline a high-resolution chemotaxis assay that allows precise patterning of chemoattractant gradients while simultaneously measuring Rho GTPase activities using FRET-based biosensors. Second, we present an optimized optogenetic strategy for light-based control of protein recruitment to the plasma membrane, which can be used to activate Rho GTPases with precise spatial control. Finally, we pair this optogenetic system with novel red-shifted Rho GTPase FRET biosensors to directly measure crosstalk signaling within the Rho GTPase network. Together, these powerful new tools and experimental approaches provide the capability to dissect information processing by Rho family GTPases with unprecedented spatial and temporal resolution.