Cervero, P.; Hey, S.; Weber, K.; Barcelona, B.; Herzog, R.; Linder, S.

Using primary human macrophages, we analyze the molecular machinery that enables direct contact of microtubule +tips with podosomes, and its connection to clathrin-based endocytosis. We show that the podosome cap protein drebrin, together with its +tip-localized binding partner EB3, regulates podosome-microtubule contact. Importantly, drebrin depletion leads to reduced endocytosis, particularly of {beta}2 integrin at podosomes. This is based on drebrin's interaction with {beta}2 integrin, and its direct binding of clathrin heavy chain via a canonical clathrin box and a novel box, which we identify also in other mammalian proteins. Our data enable a model of the molecular machinery that regulates direct contact between podosomes and microtubule +tips. Moreover, we provide evidence for spatial selectivity in endocytosis of {beta} integrins, while also revealing a more general principle of endocytic trafficking regulation, through drebrin working as a multivalent, actin-based hub that links the clathrin system with microtubule-based transport.