Phosphatidylinositol 3,4-bisphosphate regulates neurite initiation and dendrite morphogenesis via actin aggregation

Neurite initiation is critical for neuronal morphogenesis and early neural circuit development. Recent studies showed that local actin aggregation underneath the cell membrane determined the site of neurite initiation. An immediately arising question is what signaling mechanism initiated actin aggregation. Here we demonstrate that local clustering of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P₂), a phospholipid with relatively few known signaling functions, is necessary and sufficient for aggregating actin and promoting neuritogenesis. In contrast, the related and more extensively studied phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol (3,4,5)-trisphosphate (PIP₃) molecules did not have such functions. Specifically, we showed that beads coated with PI(3,4)P₂ promoted actin aggregation and neurite initiation, while pharmacological interference with PI(3,4)P₂ synthesis inhibited both processes. PI(3,4)P₂ clustering occurred even when actin aggregation was pharmacologically blocked, demonstrating that PI(3,4)P₂ functioned as the upstream signaling molecule. Two enzymes critical for PI(3,4)P₂ generation, namely, SH2 domain-containing inositol 5-phosphatase and class II phosphoinositide 3-kinase α, were complementarily and non-redundantly required for actin aggregation and neuritogenesis, as well as for subsequent dendritogenesis. Finally, we demonstrate that neural Wiskott-Aldrich syndrome protein and the Arp2/3 complex functioned downstream of PI(3,4)P₂ to mediate neuritogenesis and dendritogenesis. Together, our results identify PI(3,4)P₂ as an important signaling molecule during early development and demonstrate its critical role in regulating actin aggregation and neuritogenesis.