Endocytic dynamics and surface emergent property of leukocyte Integrins

Project Summary Regulation and sorting of leukocyte integrins are fundamental questions in cell adhesion and polarity that has great implication for various inflammatory and autoimmune diseases, but it has been poorly studied compared to cell adhesion receptors in fibroblast cells. We propose to investigate the sorting of leukocyte integrins and tetraspanin proteins that play critical role in immune cell adhesion and migration. We propose its dynamic sorting depends on a novel F-BAR-dependent mechanism that not only depends on curvature, but on a specific range of shallow curvature. We also propose that this mechanism is governing the mesoscale pattern these receptors are assembled, including propagating gradients and cortical oscillations, which link trafficking events to cell polarity. Specifically, in Aim 1, we plan to characterize surface expression and dynamics of these potential transmembrane protein cargos including integrin αMβ2 and CD81 after systematically identify and validate membrane cargos for the endocytic pathway mediated by F-BAR protein FBP17 and CIP4 using nonbiased proteomic approach. In Aim 2, we aim to isolate the factor of membrane curvature and tension using well-defined in vitro systems and to determine their effects on F- BAR membrane binding and tubulation. We will employ a nanobar-based supported bilayer system to critically evaluate if F-BAR protein senses curvature. We will also test the hypothesis that membrane tubulation may require lipid sorting and active membrane mechanics. These experiments will provide a quantitative biophysical understanding of how F-BAR proteins tubulate membranes without the hydrophobic insertion mechanisms commonly used by other BAR proteins. In Aim 3, we will dissect the functional consequences of the altered trafficking by examining spontaneous migration or migration under confinement. In particular, we will investigate the mechanism for polarized membrane receptor gradient formation. Collectively, combining advanced single cell imaging, genome-editing, proteomics, and in vitro reconstitution, the proposed research will shed key insights into the regulation and function of leukocyte integrins through sophisticated coordination and regulatory mechanisms operating at multiple spatial and temporal scales that have not yet been investigated.