Branched actin segregates endocytic cargo to control sorting and fission

At the early endosome, cargos are sorted into subdomains; receptors destined for recycling to the plasma membrane are sorted into tubulovesicular structures that undergo fission and release cargo-laden vesicles that traffic along microtubules. Although branched actin has been implicated in the establishment/maintenance of endosomal membrane subdomains, its role in cargo segregation, fission, and recycling has not been extensively studied. Using chemical inhibitors and siRNA knockdown to impede formin- and ARP2/3-mediated actin assembly, we show that branched actin, but not linear actin, is required for endosome fission and receptor recycling. To examine the spatial relationship between actin and cargo, we transfected cells with constitutively active RAB5 Q79L to generate enlarged endosomes and demonstrated that internalized transferrin localized to discrete endosomal regions adjacent to branched actin. ARP2/3 inhibition disrupted this organization, maintaining receptors at the endosome and resulting in broader cargo distribution on the endosomal membrane and coalescence of clathrin-dependent degradative cargo with clathrin-independent recycling cargo. Our findings identify ARP2/3-mediated branched actin as a key regulator of cargo segregation, subdomain maintenance, and fission at the early endosome.