Ankyrin-B is a PI3P effector that promotes polarized α5ß1-integrin recycling via recruiting RabGAP1L to early endosomes.

Endosomal membrane trafficking requires coordination between phosphoinositide lipids, Rab GTPases, and microtubule-based motors to dynamically determine endosome identity and promote long-range organelle transport. Here we report that ankyrin-B (AnkB), through integrating all three systems, functions as a critical node in the protein circuitry underlying polarized recycling of α5ß1-integrin in mouse embryonic fibroblasts, which enables persistent fibroblast migration along fibronectin gradients. AnkB associates with phosphatidylinositol 3-phosphate (PI3P)-positive organelles in fibroblasts and binds dynactin to promote their long-range motility. We demonstrate that AnkB binds to Rab GTPase Activating Protein 1-Like (RabGAP1L) and recruits it to PI3P-positive organelles, where RabGAP1L inactivates Rab22A, and promotes polarized trafficking to the leading edge of migrating fibroblasts. We further determine that α5ß1-integrin depends on an AnkB/RabGAP1L complex for polarized recycling. Our results reveal AnkB as an unexpected key element in coordinating polarized transport of α5ß1-integrin and likely of other specialized endocytic cargos.