Dynamic actin polymerization on endosomes regulates integrin trafficking, cell adhesion and cell migration

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 serve distinct functions in specific cellular processes, including N-WASP in phagocytosis and endocytosis, WAVE and JMY in cell migration, and WHAMM in ER-to-Golgi transport. Although another NPF termed WASH was recently identified, the cellular function and activity of this protein are not well defined. We demonstrate that human WASH potently activates the Arp2/3 complex in vitro and in cells. Furthermore, we show that WASH localizes to early/sorting endosomes and recycling endosomes. These WASH-positive compartments are often associated with actin networks. Silencing of WASH or Arp2/3 complex expression by RNAi, or disruption of actin function by drug treatments, leads to enlargement and elongation of endocytic compartments. Moreover, disruption of actin dynamics or WASH depletion delays EGF transport to LAMP1-positive late endosomes. These observations indicate that actin polymerization downstream of WASH influences the shape and maturation of endosomes, and shed light on a previously unrecognized role for WASH and the Arp2/3 complex in endocytic trafficking./nWASH and actin dynamics have now been implicated in multiple endocytic trafficking pathways including receptor recycling, cargo degradation, and retromer-mediated receptor retrieval. All of these trafficking pathways converge at the early/sorting endosome, raising the question whether WASH plays a general role in sorting and trafficking cargo through this compartment or if there is a requirement for WASH in trafficking specific cargos. To answer this question, we must characterize the influence of WASH on trafficking cellular cargos that utilize distinct routes through endocytic pathway. Here, we investigate the influence of WASH and Arp2/3 complex activity during integrin recycling, cell adhesion and migration. We observed that subdomains of early/sorting endosomes associate with dynamic WASH and filamentous actin (F-actin), and that alpha5-integrins traffic through this population of endosomes. Depletion of WASH causes accumulation of alpha5-integrins in intracellular compartments, reduction of alpha5-integrin localization at adhesive structures, and reduction in focal adhesion number. Recycling of alpha5-integrins from internal endocytic structures to focal adhesions is disrupted upon WASH depletion or Arp2/3 complex inhibition. Furthermore, WASH-depleted cells display greatly reduced affinity for specific ECM proteins including fibronectin. Interestingly, the reduced adhesion capacity of WASH-depleted cells results in more rapid migration of these cells in wound healing assays. These results implicate WASH, the Arp2/3 complex, and actin in the specialized trafficking of integrins. Our findings highlight a role for actin dynamics 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.

Advisor: Welch, Matthew W