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bioRxiv · 10.64898/2026.01.23.701188

Supported membrane assay probes PLCγ1 activity in LAT condensates

DeGrandchamp, J. B.·Rajesh, S. V.·Lew, L. J. N.·Groves, J. T.
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Abstract

Phospholipase C-{gamma}1 (PLC{gamma}1) plays a critical role linking T cell receptor activation with downstream signaling pathways including calcium. PLC{gamma}1 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. PLC{gamma}1 in these LAT condensates becomes activated, leading to induction of extracellular calcium influx. While PLC{gamma}1-driven calcium signaling is clearly correlated with LAT condensation, it is less clear how--or if-- the LAT condensation state facilitates PLC{gamma}1 activity. Here we develop an image-based PLC{gamma}1 activity assay in supported bilayers that enables simultaneous measurement of both PLC{gamma}1 recruitment to phosphorylated LAT and PLC{gamma}1-catalyzed hydrolysis of PIP2 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 PKC{theta}. Assays are performed directly with mammalian cell lysate containing fluorescent PLC{gamma}1 fusion constructs. The results reveal that PLC{gamma}1 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 PLC{gamma}1 signal gating and LAT condensation, the physical environment of the condensate itself is not a key regulator of PLC{gamma}1 signaling. More broadly, this assay system offers a quantitative means of probing how PLC{gamma}1 activity is controlled at the membrane.

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DeGrandchamp, J. B., Rajesh, S. V., Lew, L. J. N., Groves, J. T.. 2026-01-25. Supported membrane assay probes PLCγ1 activity in LAT condensates. https://doi.org/10.64898/2026.01.23.701188

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