In vitro LC3 enhances intrinsic nucleation and Arp2/3 activation by JMY. (A) FRET assay for LC3 binding to JMY. The curves are fluorescence intensity as a function of emission wavelength of Alexa Fluor 546–labeled JMY (green) as donor, Alexa Fluor 647–labeled LC3 (pink) as acceptor, and mixture of both fluorescently labeled proteins (red). (B) Binding isotherm derived from FRET assay for the interaction of LC3 with the N-terminal region (residues 1–314) of JMY (Kd = ∼50 nM). We added various concentrations of Alexa Fluor 546–labeled NT JMY to 200 nM Alexa Fluor 647–labeled LC3 in 100 mM KCl with 20 mM Hepes buffer, pH 7.4. (C) Dose dependence of LC3 stimulation of JMY’s intrinsic nucleation activity. LC3 promotes JMY’s intrinsic nucleation activity in pyrene-actin polymerization assay (left). The initial slope (first 250 s) of each curve is normalized and plotted as a proxy for nucleation rate (right). (D) Dose dependence of LC3 enhancement of Arp2/3 complex activation by JMY. Pyrene-actin polymerization assays were performed in 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM Imidazole, pH 7.0, with 1 µM actin, 200 nM JMY, 25 nM Arp2/3 as noted, 16 mM NaCl, and LC3 as indicated.
