The plasma membrane serves as an effective platform for signal transduction of membrane receptor pathways. Activation of the T-cell receptor (TCR) triggers the formation of membrane-associated condensates that are formed through liquid-liquid phase separation. These condensates are assembled by multivalent interactions between the tyrosine-phosphorylated receptor/adaptor and the SH2 domain-containing protein at membrane-proximal milieu. Here, we describe a biochemical reconstitution system that has been implemented to decipher the mechanisms of phospholipase PLCγ1-mediated LAT condensate formation. To characterize the interaction between specific phosphotyrosine-SH2 pair, we developed a total internal reflection fluorescence (TIRF) microscopy-based system to quantify the binding preference of each SH2 domain to specific tyrosine in the context of membranes. An assay to determine the condensate-mediated protection of phosphotyrosines from being dephosphorylated by phosphatase is also elaborated. These assays could be applied to study other transmembrane receptor pathway as well as condensates formed on endomembrane systems including the endoplasmic reticulum, mitochondrion, and Golgi apparatus.
Keywords: Biochemical reconstitution; CD45; Condensate; PLCγ1; Phase separation; Plasma membrane; TIRF.
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