<br/>Liquid-liquid phase separation of proteins have been broadly observed in many cellular machineries, but the biological roles of many of these protein condensates remains ambiguous. Condensates of the membrane protein LAT with Grb2+Sos1 have been implicated in T cell activation, possibly by excluding inhibitory phosphatase CD45 through electrostatic repulsion and/or increasing the dwell time of effector protein Sos1 near the plasma membrane (PM). Intriguingly, LAT has been widely shown to reside in ordered PM domains known as lipid rafts. Here, we provide evidence that LAT condensates can induce and stabilize raft domains in the PM, and in turn that raft domains can nucleate and stabilize cytoplasmic protein condensates. Coupling between condensates and ordered domains were studied in both lipid model membranes and live T cells. First, reconstituted LAT condensates induced phase separation in biomimetic giant unilamellar vesicle membranes, with liquid ordered (Lo) domains being recruited to condensates. Lo domain recruitment by condensates were also observed in supported lipid bilayers. Importantly, phase separated membranes could concentrate LAT molecules in Lo regions, with this effect facilitating condensate formation. Concordantly, in activated Jurkat T cells, LAT condensates induced microscopic cholesterol-rich raft domains, evidenced by enrichment of raft markers, including endogenous GPI-anchored protein CD90, and exclusion of non-raft constructs, including transmembrane domain of CD45. Moreover, stabilizing raft domains by clustering the GPI-anchored protein CD90 not only potentiated the formation of LAT condensates, but also increased their size, reinforcing the effect of condensates for cell activation. The coupling of raft domains with the condensates also explains the long-lingering mysterious observation that crosslinking raft components can activate T cells. Altogether, we conclude that protein condensates are thermodynamically and mechanistically coupled to ordered membrane domains to regulate the functional organization underlying immune cell signal transduction.

Coupling between protein condensates and ordered membrane domains facilitates T cell activation

ACS Spring 2022

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