Mutual regulation between membrane rafts and protein condensates in T cell activation.

Abstract

T cells are central effectors of adaptive immunity, mediating critical defenses against infections and cancer. T cell activation must be tightly regulated to integrate and transduce various signals detected at the plasma membrane and elicit proper immune responses. Despite decades of research, the regulatory mechanisms of normal and aberrant T cell activation are not fully understood. Recently, we reported that biomolecular condensates form at the T cell immunological synapse via liquid-liquid phase separation driven by multivalent macromolecular interactions. Specifically, the transmembrane protein Linker for Activation of T cells (LAT) is phosphorylated upon antigenic stimulation to multivalently interact with proteins called Grb2 and Sos1, forming condensates with fluid properties in vitro and in living cells. Notably, LAT is recruited to the ordered, cholesterol-rich membrane domains known as lipid rafts, and such recruitment has been implicated in T cell activation. Thus, we hypothesized that LAT may mediate interplay between protein and membrane lipid phase separation in T-cell signaling. Here, we report physical and functional coupling between membrane rafts and protein condensates via LAT using both in vitro reconstituted membrane systems and live cells. To study how LAT condensates and membrane rafts cooperatively regulate T-cell signaling, we perturbed membrane raft domains and observed a decrease in condensate density and a dampening of downstream signaling. Moreover, using fluorescent probes that are sensitive to local membrane lipid packing in living cells, we quantitatively measure changes in membrane biophysical properties during condensate assembly and see an increase in lipid packing in the local membrane environment surrounding LAT condensates. By evaluating effects of lipid remodeling on LAT condensate properties, and effect of condensate formation on membrane properties, we provide evidence that membrane rafts and LAT condensates mutually regulate T cell activation to tune immune signaling.