Domains required for PLCγ1-driven LAT clustering. (A) TIRF microscopy revealed LAT microcluster formation with a high concentration of PLCγ1 fragments. Alexa Fluor 488 LAT at 300 molecules/µm² was incubated with 125 nM Sos1 and 500 nM of indicated PLCγ1 fragments. Scale bar, 5 µm. (B) Quantification of LAT clustering in A. Shown are mean ± SD; n = 3 independent experiments. (C) TIRF microscopy revealed LAT microcluster formation in the presence or absence of PLCγ1. Alexa Fluor 488 LAT at 1,000 molecules/µm² was incubated with 500 nM Sos1 and 1,000 nM Grb2 with or without 100 nM full-length PLCγ1. Scale bar, 5 µm. (D) Quantification of LAT clustering. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test. ***, P < 0.001. (E) FRAP analysis revealed that PLCγ1 decreases the recovery of LAT signal in clusters after photobleaching. Shown are mean ± SD; n = 10 clusters.