Analysis of neutrophil chemotaxis spatial signal processing using optogenetic receptor control
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Analysis of neutrophil chemotaxis spatial signal processing using optogenetic receptor control

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Neutrophils are early responders of the innate immune system that use chemotaxis, the directed migration of cells along a chemical gradient, to reach sites of infection and inflammation. Deciphering the spatial patterns of chemoattractant signals is a fundamental challenge at the cellular level due to diffusion and inherent stochasticity of signaling molecules. Despite these sources of noise, neutrophils respond to changing chemoattractant gradients with high spatial and temporal precision by integrating information from receptors with a cell-autonomous polarity and motility signaling program1–4. The wiring of the chemotaxis signaling network is complex with multiple layers of feedbacks and potentially feedforward connections. Understanding how cell polarity programs interpret and respond to chemotaxis receptor signals remains a key challenge in the field, as deciphering the interconnections of the network are not trivial. To better understand how cell polarity regulators in the Rho GTPase family signal within chemotaxing cells, I developed a high-magnification chemotaxis assay that enables measurement of Rho GTPase activity biosensors downstream of photo-activation of chemoattractants (Chapter 2). Chemoattractant gradients are inherently spatial, and it remains unclear how cells encode this spatially relevant information from the receptors in the downstream chemotaxis signaling programs. Experiments using diffusible chemoattractants are limiting, as achieving well-defined and highly local receptor stimulation is unfeasible. To overcome this challenge, I developed a new molecular tool kit that uses light-driven chemotaxis signaling to improve the spatial and temporal control of receptor activation. I paired this receptor with a red-shifted biosensor for a cell front polarity regulator, enabling stimulation and response measurements in the same cells. Using this system, I explored how cell polarity signals are propagated in space and time downstream of receptor stimulation. Additionally, I investigated how negative signals shape the polarity response (Chapter 3). Through collaboration, the light-activated chemotaxis receptor was also used to unlock another experimental question that was limited by chemoattractant diffusion. Specifically, we investigated whether existing cell front/back polarity is resistant to receptor activation at the cell rear (Chapter 4). Collectively, the methodologies developed, and information gleaned from this work lay the foundation for systematic interrogation of spatial signal transduction that occurs during neutrophil chemotaxis.

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