Arp2/3-mediated turnover of large clathrin lattices is regulated through the tyrosine kinase ACK

Abstract

Clathrin mediated endocytosis (CME) is a vital cellular process that mediates cell signaling by controlling the internalization of extracellular cargo and activated receptors. Arp2/3-branched actin provides force to assist in membrane invagination and scission in CME. Loss of Arp2/3-branched actin in conditional Arpc2 KO fibroblasts results in an increase of large arrested clathrin lattices (LACLs) visualized by live-cell TIRF imaging. Additional structural details of LACLs were revealed using electron microscopy and include an increase in arrested clathrin lattices of various curvatures. Numerous CME proteins have heightened levels of tyrosine phosphorylation at these LACLs in Arpc2 KO cells. We identified the non-receptor tyrosine kinase ACK (Activated Cdc42-Associated Kinase) as a key upstream regulator of LACL turnover. CRISPR KO of ACK abrogates LACL tyrosine phosphorylation and impairs cells capacity to resolve LACLs. Our results support a model where ACK recruitment and activation at LACLs drives branched actin formation to help resolve large accumulations of arrested CME structures.