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. V C 2010 Wiley-Liss, Inc.

WASH is a nucleation-promoting factor for the Arp2/3 complex that is implicated in multiple endocytic trafficking pathways including receptor recycling, cargo degradation, and retromer-mediated receptor retrieval. We sought to examine whether WASH plays an important role in trafficking of specialized cargo molecules such as integrins, for which trafficking is highly regulated during cell migration. We observed that subdomains of early/sorting endosomes associated with dynamic WASH and filamentous actin, and α5-integrins trafficked through this population of endosomes. Depletion of WASH caused accumulation of α5-integrins in intracellular compartments, reduction of α5-integrin localization at focal adhesions, and reduction in focal adhesion number. Transport of α5-integrins from internal endocytic structures to focal adhesions was disrupted upon WASH depletion or Arp2/3 complex inhibition. Furthermore, WASH-depleted cells displayed greatly reduced affinity for specific ECM proteins including fibronectin, and impaired cell spreading ability. Interestingly, the reduced adhesion capacity of WASH-depleted cells resulted in their migrating more rapidly than control cells in wound healing assays. Our results define a requirement for WASH, Arp2/3 complex, and actin in specialized trafficking of integrins. These findings highlight a role for actin dynamics in influencing cell adhesion and migration via endocytic trafficking of integrins, in addition to the well-established role of actin in plasma membrane dynamics and contractility.

Rac1B, a small GTP-binding protein in Dictyostelium discoideum, is involved in regulation of the actin cytoskeleton. Scanning electron microscopy revealed distinctive phenotypes for the wild-type, constitutively active, constitutively inactive and overexpressing cell lines. Immunofluorescence showed constitutively active Rac1B localized to lamellipodia and sites of cell-to-cell contact. In contrast, constitutively inactive Rac1B was homogeneously distributed throughout the cell. Phalloidin staining demonstrated that active Rac1B co-localizes with F-actin. Amoebae expressing mutant Rac1B exhibited defects in endocytosis, cytokinesis and multicellular development. Overexpression of wild-type Rac1B positively affected fluid-phase endocytosis, whereas expression of either constitutively active or inactive forms of Rac1B inhibited endocytic rates. The greatest defects in cytokinesis were observed in amoebae producing constitutively active Rac1B or overexpressing wild-type Rac1B. These cells were severely multinucleated and divided by traction-mediated cytofission when placed onto a solid surface. Cells expressing mutant Rac1B were unable to form viable fruiting bodies. Elucidating the role of Rac1B in filamentous actin dynamics will lead to a better understanding of cell adhesion, development and cell motility.