PLCγ1 promotes LAT cluster formation in vitro. (A) Domain structure of PLCγ1. (B) Schematics of the assay. (C) Top: TIRF microscopy revealed that both Grb2 and PLCγ1 (full-length) promote LAT microcluster formation. Alexa Fluor 488–labeled LAT at 300 molecules/µm2 was incubated with 125 nM Sos1 and 250 nM Grb2 or PLCγ1 for 0.5 h before imaging. Scale bar, 5 µm. Bottom: Quantification of Grb2- or PLCγ1-driven LAT microclusters. LAT clustering was quantified as normalized variance (Su et al., 2016). Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. *, P < 0.05; **, P < 0.01. (D) FRAP analysis revealed that PLCγ1-driven microclusters are less dynamic than Grb2-driven LAT microclusters. Shown are mean ± SD; n = 10 clusters.