Figure 7.

The effect of LC3 on actin nucleation and Arp2/3 activation by JMY reveals a cryptic actin regulatory sequence in the N-terminal region of JMY. (A and B) NT JMY (residues 1–314) promotes concentration-dependent actin nucleation in the absence (A) and presence (B) of the Arp2/3 complex. Actin assembly was monitored by the fluorescence of pyrene-labeled actin (left). The initial slope of each curve is normalized and plotted as a proxy for nucleation rate (right). Pyrene-actin polymerization assays are performed at 23°C in 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, and 10 mM Imidazole, pH 7.0, with 2 µM actin, 25 nM Arp2/3, and NT JMY as noted. (C) LC3 enhances NT JMY (residues 1–314) intrinsic actin nucleation activity. Actin assembly in the presence of various concentrations of LC3 was monitored by the fluorescence of pyrene-labeled actin (left). The initial slope (first 250 s) of each curve is normalized and plotted as a proxy for nucleation rate (right). Buffer conditions: same as in A with 1 µM actin, 200 nM NT JMY, 16 mM NaCl, and LC3 as indicated.