LAT condensation gates PLCγ1 activation via bimodal LAT phosphorylation.
T cells can respond to even a single molecular binding event of antigen to a TCR. A key step in the TCR signaling pathway that definitively exhibits this single-molecule response is the initiation of calcium influx by activation of PLCγ1 in the LAT protein condensate. Here, we describe detailed kinetic measurements examining how protein condensation of LAT regulates activation of PLCγ1 using a reconstituted membrane system. The results reveal that membrane recruitment of PLCγ1
T cells can respond to even a single molecular binding event of antigen to a TCR. A key step in the TCR signaling pathway that definitively exhibits this single-molecule response is the initiation of calcium influx by activation of PLCγ1 in the LAT protein condensate. Here, we describe detailed kinetic measurements examining how protein condensation of LAT regulates activation of PLCγ1 using a reconstituted membrane system. The results reveal that membrane recruitment of PLCγ1 is tightly controlled by the LAT phosphorylation state, with no measurable independent recruitment to PIP 2 or PIP 3 lipids via the PLCγ1 PH domains. We further observe PLCγ1 is rapidly activated by membrane-associated kinase upon recruitment, irrespective of the LAT condensation state. These studies also reveal a crosstalk mechanism in which the TEC family kinases responsible for PLCγ1 activation also phosphorylate LAT. This interaction establishes a positive feedback loop in LAT phosphorylation, mediated through LAT condensation, which drives a bimodal LAT phosphorylation response to TCR activation. Kinetic modeling reveals how this LAT phosphorylation response can cooperatively gate PLCγ1 activation from a single TCR. These results suggest the LAT condensate facilitates both signal amplification and noise suppression in PLCγ1 activation through a bimodal switch affecting LAT phosphorylation.
