The role of receptor dynamics in eukaryotic chemotaxis

University Of Arizona, Tucson AZ

Investigators

Abstract

This project will provide unprecedented insight into how different types of motile cells are able to detect gradients of chemicals (chemoattractants) and migrate towards the source, which is how many microorganisms find food and what guides immune cells to sites of inflammation or infection during the immune response in mammals. This important cellular behavior is termed chemotaxis and is not understood. Through an unknown mechanism, highly motile cells such as the amoeba Dictyostelium discoideum and mammalian white blood cells can detect a chemoattractant gradient that is as small as a 2% difference between the front and back of the cells, but how cells achieve such gradient sensing is not known. Using Dictyostelium as an experimental model, this project will uncover an important aspect of the gradient sensing machinery and how it functions to control the migration of cells. In addition, the project will strongly emphasize providing multidisciplinary research training, laboratory experiences, and mentorship to diverse high school and undergraduate students in both California and Arizona. This will be achieved through partnerships with local schools as well as the development of an undergraduate colloquium course aimed at preparing and engaging students in scientific research early in their undergraduate career. In Dictyostelium, the cAMP chemoattractant receptor cAR1 couples with heterotrimeric Gα2βγ protein (G2) to drive chemotaxis in response to cAMP. Evidence suggests the presence of G2 signaling asymmetry during chemotaxis, but the role of cAR1 in creating this signaling asymmetry and promoting gradient sensing is unclear. The proposed work addresses this important question, with the hypothesis that distinct receptor phosphorylation events modulate gradient sensing and subsequent activation of the polarized Ras chemotactic pathway by spatiotemporally altering cAR1-G2 interaction and G2 activation dynamics in a cAMP concentration-dependent manner. This hypothesis will be tested in two Specific Aims. Aim 1 will determine the role of cAR1 in gradient sensing and the chemotactic response through a combination of site-directed mutagenesis, biochemical assays, and quantitative imaging of live cells migrating in precise gradients produced by microfluidic devices. The resulting data will form the basis for creating mathematical models of gradient sensing and its modulation by cAR1 phosphorylation to identify underlying mechanisms. Aim 2 will define the cAR1-G2 interaction dynamics and role in chemotaxis using Bioluminescence Resonance Energy Transfer (BRET) methods in live Dictyostelium cells coupled to mathematical modeling, which will allow integrating the data in a comprehensive chemotaxis model combining results of both Aim 1 and Aim 2. Together, these studies will reveal how the chemoattractant receptor functions and how it is regulated to promote gradient sensing and dictate the downstream polarized chemotactic pathways that control the directed migration of cells. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.