Drosophila WASH is required for integrin-mediated cell adhesion, cell motility and lysosomal neutralization

Abstract

The Wiskott-Aldrich syndrome protein and SCAR homolog (WASH; also known as Washout in flies) is a conserved actin-nucleationpromoting factor controlling Arp2/3 complex activity in endosomal sorting and recycling. Previous studies have identified WASH as an essential regulator in Drosophila development. Here, we show that homozygous wash mutant flies are viable and fertile. We demonstrate that Drosophila WASH has conserved functions in integrin receptor recycling and lysosome neutralization. WASH generates actin patches on endosomes and lysosomes, thereby mediating both aforementioned functions. Consistently, loss of WASH function results in cell spreading and cell migration defects of macrophages, and an increased lysosomal acidification that affects efficient phagocytic and autophagic clearance. WASH physically interacts with the vacuolar (V)-ATPase subunit Vha55 that is crucial to establish and maintain lysosome acidification. As a consequence, starved flies that lack WASH function show a dramatic increase in acidic autolysosomes, causing a reduced lifespan. Thus, our data highlight a conserved role for WASH in the endocytic sorting and recycling of membrane proteins, such as integrins and the V-ATPase, that increase the likelihood of survival under nutrient deprivation.

Cellular functions of WASP family proteins at a glance

“…Interestingly, a mutation in VPS35 (D620N) (Vilariño-Güell et al, 2011;Zimprich et al, 2011) that is associated with earlyonset Parkinson's disease has been shown to diminish the interaction of the SHRC with VPS35 and impair vesicular trafficking from the late endosome (Follett et al, 2013;McGough et al, 2014;Zavodszky et al, 2014). Studies in Drosophila have recently shown important roles for WASH in regulating integrin receptor trafficking and lysosome acidification (Nagel et al, 2017). A similar role for WASH in the recycling of integrins has been seen in mammalian cells .…”

“…This is mediated by a TRIM27-containing ubiquitin ligase complex that also contains MAGEL2 and UBE2O, and is regulated by the deubiquitinase USP7 (Hao et al, 2015). Finally, although deletion of WASH in Drosophila was initially found to impair oogenesis and larval development (Linardopoulou et al, 2007;Liu et al, 2009), a recent study using a different WASH mutant showed that homozygous wash mutant flies are viable and fertile (Nagel et al, 2017). This discrepancy was likely caused by a secondsite lethal mutation in the Drosophila strain used in the former studies.…”

“…These results implicate WASH as a positive regulator of autophagy. It is unclear how to reconcile these two disparate findings, but it was recently shown that genetic deletion of WASH in Drosophila also leads to excessive autophagy (Nagel et al, 2017). Thus, the conservation of WASH as an inhibitor of autophagy in invertebrates and vertebrates is suggestive of an evolutionary conserved function.…”

Mroh1, a lysosomal regulator localized by WASH-generated actin

“…Disruption of the WASH complex halts the maturation and trafficking of lysosomes in Dictyostelium (Carnell et al, 2011; Park et al, 2013) and phagolysosomes in Drosophila macrophages (Nagel et al, 2017). In Dictyostelium , instead of undergoing their normal transition into neutral post-lysosomes from wash -knockout (KO) cells remain acidic and fail to undergo exocytosis (Carnell et al, 2011).…”

“…As the V-ATPase has F-actin-binding domains (Holliday et al, 2000; Huss et al, 2011), there could be direct coupling between WASH-generated F-actin and the V-ATPase that coordinates its trafficking. Recent work has also found that WASH and V-ATPase can be co-immunoprecipitated from Drosophila cells, suggesting a possible direct interaction between the complexes (Nagel et al, 2017). …”