Activators of the Arp2/3 complex, termed nucleation-promoting factors (NPFs), are required for the proper spatial and temporal control of actin assembly in cells. Mammalian cells express several NPFs, each of which functions in a distinct cellular process, including WASP and N- WASP in phagocytosis and endocytosis, WAVE and JMY in cell migration, and WHAMM in ER- to-Golgi transport. Although another NPF called WASH was recently identified, the cellular localization and function of this protein were unclear. Here we demonstrated that human WASH alone potently activated the Arp2/3 complex in vitro and in cells, suggesting that the protein is not autoinhibited like N-WASP, but is likely regulated by interacting proteins. In cells, WASH was associated with Rab5-positive early endosomes and Rab11-positive recycling endosomes that were enriched for actin filaments. Silencing of WASH or Arp2/3 complex expression by RNAi, or disruption of actin function by drug treatments, caused enlargement and elongation of endosomes. Intriguingly, WASH silencing, as well as actin disruption, delayed EGF transport to LAMP1- positive late endosomes. These observations indicate that actin polymerization by WASH influences the shape and maturation of endosomes, and highlight a previously unrecognized role for WASH and the Arp2/3 complex in the degradative steps of endocytic trafficking.
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