Figure 8.

PI 3-kinase inhibitors decrease the number of actin comet tails in OCRL-deficient cells and overall model. (A) Purified mCherry-2×FYVE domain used to stain for PI(3)P in cells fixed after treatment with DMSO control, 2 µM wortmannin, or 10 µM Vps34-IN1 in serum-free media for 1 h before imaging. (B) Representative images from time-lapse videos of OCRL-deficient cells expressing F-tractin and treatment with DMSO or the inhibitors, as shown in Video 6. Actin comets were reduced on PI 3-kinase inhibitor treatment. Bars, 10 µm. (C) Quantification of actin comets from n = 30 cell regions for each treatment. Bars indicate the mean ± SEM for each condition. Difference assessed by ordinary one-way ANOVA with Dunnett’s multiple comparisons test, overall ANOVA, and comparing each inhibitor treatment to DMSO control cells. ***, P ≤ 0.0001. (D) Pathway of curvature signaling to actin polymerization via PI(4,5)P₂/PI(3)P/SNX9 during CME. High curvature activates a cascade of phosphoinositol metabolism to change membrane identity and trigger actin polymerization at PI(4,5)P₂/PI(3)P-enriched and highly curved sites via the oligomerization of SNX9 during endocytosis. In Lowe syndrome, OCRL deficiency increases PI(4,5)P₂ levels on PI(3)P intermediates, triggering SNX9 assembly. PI(4,5)P₂ also activates Cdc42 (independently of the curvature) for the relief of N-WASP inhibition. N-WASP oligomerization (via SNX9 binding) finally drives superactivation of the Arp2/3 complex and, thus, actin polymerization.