Nucleation-promoting factors (NPFs) activate the ARP2/3 complex, leading to actin polymerization, cytoskeletal reorganization and subsequent changes in cell morphology. Although WASP and SCAR homologue (WASH) acts as an NPF in vitro, its role in vivo was unclear. Now, two studies published in Developmental Cell show that WASH has a role in endosomal sorting.

Gomez and Billadeau and Derivery et al. first investigated the intracellular localization of WASH. Using immunofluorescence staining and size-exclusion chromatography, both groups found that WASH localizes at early endosomes and interacts with tubulin. Gomez and Billadeau found that in immortalized T cells WASH localizes with endosomes polarizing towards the immunological synapse during T cell activation, suggesting a role in T cell receptor trafficking. Consistent with this, Derivery et al. observed that depletion of WASH leads to impaired recycling of transferrin, confirming a role for WASH in this process. Furthermore, they showed that WASH preferentially associates with early and recycling endosomes (expressing RAB4 or RAB11) over endosomes of the degradative pathway (expressing RAB7). Specifically, Gomez and Billadeau identified a requirement for WASH in retrograde transport (from the endosomes to the Golgi), as depletion of WASH resulted in inhibition of retrograde transport of cation-independent mannose-6-phosphate receptor (CI-MPR).

So what is the function of WASH in endosomes? As ARP2/3 and filamentous actin (F-actin) accumulate on endosomes, Derivery et al. examined the role of WASH in actin polymerization in this compartment. WASH depletion markedly inhibited the recruitment of ARP2/3 to endosomes, which suggests that WASH is a key regulator of F-actin polymerization in this compartment. However, this is not consistent with the findings of Gomez and Billadeau, who observed that WASH depletion did not affect the recruitment of F-actin and ARP2/3 to endosomes. Further work will be required to clarify this discrepancy. Nevertheless, both studies reveal an important role for WASH in actin polymerization in endosomes, with depletion resulting in the formation of thin and long endosomal tubules. This was shown by Derivery et al. to be due to the loss of ARP2/3 activation, as targeting the WASH VCA domain (known to bind ARP2/3 and actin) with a blocking antibody had the same effect as WASH depletion.

NPFs normally function as part of multiprotein complexes, so both groups sought to identify the other proteins in the complex. Gomez and Billadeau isolated the previously uncharacterized FAM21, which constitutively associated with WASH through the WASH domain that mediates endosomal localization. FAM21 (or KIAA0592 in Derivery et al.) was also among the seven WASH multiprotein subunits identified by Derivery et al.; these included capping protein, which inhibits actin dynamics at the barbed end.

Taken together, these data identify an integral role for WASH in the endosomal sorting pathway (recycling and retrograde transport), which it achieves by binding tubulin and activating ARP2/3. The authors propose a model in which WASH regulates endosomal trafficking and fission by linking endosomes to the microtubule network and by generating force through actin polymerization.

Author: Rachel David - Copyright © 2010 Nature Publishing Group, a division of MacMillan Publishers Limited; used with permission