Biophysical and functional coupling of protein condensates to ordered lipid domains in T-cells

Project Summary T-cells integrate complex signals to make accurate decisions for activating immune responses, including cytokine secretion, degranulation, and proliferation. This integration requires precise regulation of signaling proteins at the cell membrane. Recently, we demonstrated that key membrane-associated T-cell signaling proteins are organized into condensed protein assemblies that regulate signal transduction. Namely, phosphorylation of the transmembrane adaptor protein Linker of Activation of T-cells (LAT) induces multivalent interactions with cytoplasmic adaptors that produce biomolecular condensates. This condensation is required for T-cell signal transduction. In parallel, we have shown that LAT, through its transmembrane domain, associates with cholesterol-driven lipid nanodomains called lipid rafts, which have also been heavily implicated in immune cell signaling. The involvement of LAT in both lipid nanodomains and cytoplasmic protein condensates suggests its potential to functionally couple these aspects of cellular organization. Combining expertise in lipid domains and protein condensates, our preliminary observations demonstrate that protein condensates can recruit membrane domains and promote their formation. Further, we find that this raft- condensate coupling is required for T-cell activation. These observations prompt fundamental questions about the biophysical mechanisms of coupled phase separation between the cytoplasm and membrane and its consequences in T-cell signaling. We will explore these questions with an integrated approach, spanning from biophysical characterization of purified systems to signaling and advanced microscopy in live T-cells. Aim 1 examines LAT-mediated coupling between protein condensates and membrane domains. In both model membranes and living cells, engineered protein probes and fluorescent lipid reporters are used to examine the biophysical properties of condensate-associated membranes. We also evaluate how condensates can change protein affinity for membrane domains. Aim 2 explores how LAT condensates regulate membrane organization. T-cell plasma membranes and cytoplasm both lie near phase coexistence boundaries and are thus poised for large-scale structural rearrangements. We investigate the hypothesis that protein condensates regulate lateral lipid organization. We report novel observations of micron-scale cholesterol-rich domains in activated T-cells and explore their basis and consequences. Further, we evaluate electrostatic interactions between condensates and lipids to reveal how condensates sort lipids and proteins in the membrane plane. Finally, preliminary data show that coupling of LAT condensation with membrane domains is essential for signal transduction leading to T-cell activation. Aim 3 will dissect the molecular mechanisms through which condensates and ordered domains cooperatively regulate signal propagation from the T-cell receptor. Together, these studies aim to reveal biophysical principles involved in membrane organization, signal transduction, and ultimate activation of T-cells.