Mutual regulation between coupled protein condensates and ordered membrane domains.

Abstract

Downstream of T-cell receptor ligation, biomolecular condensates are produced via the multivalent binding between Linker for Activation of T-cells (LAT) and its effectors (Grb2 and Sos1) to facilitate T-cell activation. LAT is a transmembrane protein known to be recruited to ordered lipid domains, suggesting potential coupling between protein cytoplasmic condensates and lateral lipid-based phase-separation and organization. We report evidence demonstrating thermodynamic coupling between lipid- and protein-based phase separation. In model membranes, condensates formed on phase-separating membranes template ordered lipid domains and induce phase separation. Conversely, lipid phase separation stabilizes protein condensate formation suggesting strong coupling between lipid- and protein-based phase separation. Furthermore, experiments in T-cells show that LAT/Grb2/Sos1 condensates recruit ordered domain lipids and proteins in living cells. We observed that crosslinking of a membrane component that prefers ordered domains (i.e. the endogenous GPI-anchored protein Thy1) stabilizes lipid phase separation and templates condensate formation. Finally, we tested whether condensate-domain coupling is necessary for T-cell activation by decoupling raft domains from condensates through either mutation of the transmembrane domain to modulate its raft affinity or mutation of residues necessary for condensate formation. We found that the coupling of cytoplasmic condensate formation to lipid rafts is essential for T-cell activation. Thus, we conclude that the thermodynamic coupling between LAT/Grb2/Sos1 cytoplasmic condensates and lipid rafts can cooperatively and functionally reorganize the membrane to facilitate T-cell activation.