Supported membrane assay probes PLCγ1 activity in LAT condensates

Abstract Phospholipase C-γ1 (PLCγ1) plays a critical role linking T cell receptor activation with downstream signaling pathways including calcium. PLCγ1 activation in T Cells relies on phosphotyrosine-mediated recruitment to the membrane-bound scaffold LAT, which becomes crosslinked through a bond percolation network with Grb2 and other scaffold and signaling molecules to form a signaling condensate. PLCγ1 in these LAT condensates becomes activated, leading to induction of extracellular calcium influx. While PLCγ1-driven calcium signaling is clearly correlated with LAT condensation, it is less clear how—or if— the LAT condensation state facilitates PLCγ1 activity. Here we develop an image-based PLCγ1 activity assay in supported bilayers that enables simultaneous measurement of both PLCγ1 recruitment to phosphorylated LAT and PLCγ1-catalyzed hydrolysis of PIP 2 in the membrane. The condensation state of LAT is independently controlled by adjusting levels of co-condensation proteins such as Grb2, SOS, GADS, and SLP76. The hydrolysis product, diacylglycerol (DAG), remains in the membrane and is monitored as a readout of catalytic activity using a DAG sensor based on the C1b (DAG binding) domain of PKCθ. Assays are performed directly with mammalian cell lysate containing fluorescent PLCγ1 fusion constructs. The results reveal that PLCγ1 is highly active when recruited to dispersed LAT and that the condensed state does not promote activity. Overall, this assay platform reveals that despite the correlation between PLCγ1 signal gating and LAT condensation, the physical environment of the condensate itself is not a key regulator of PLCγ1 signaling. More broadly, this assay system offers a quantitative means of probing how PLCγ1 activity is controlled at the membrane.