PLCγ1 cooperates with Grb2 to regulate LAT microcluster formation. (A) TIRF microscopy revealed that PLCγ1 regulates LAT microcluster formation in a nonmonotonic manner. Physiologically relevant concentrations of proteins were used in the assay: LAT at 300 molecules/µm2, Grb2 at 3 µM, Sos1 at 0.3 µM, and PLCγ1 at 50 nM. LAT was labeled with Alexa Fluor 488, PLCγ1 (SH2-2-3 domains) was labeled with DY547, and Sos1 was labeled with Alexa Fluor 647. Scale bar, 5 µm. (B) Quantification of LAT clustering, membrane recruitment of Sos1. Shown are mean ± SD; n = 3 independent experiments. Unpaired two-tailed t test was used. **, P < 0.01. (C) PLCγ1 accelerates LAT cluster formation. TIRF microscopy revealed the time course of LAT microcluster formation in the presence or absence of PLCγ1. LAT–Alexa Fluor 488 at 1,000 molecules/µm2 was incubated with 1,000 nM Grb2 and 500 nM Sos1 and/or 50 nM PLCγ1 at time 0. Shown are mean ± SEM; n = 3 independent experiments.
