Drosophila integrin adhesion complexes are essential for hemocyte migration in vivo

Wiskott-Aldrich syndrome proteins (WASPs) are nucleationpromoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR (also known as WAVE), WASP and WASH (also known as CG13176), have been analyzed so far. Here, we characterized WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, we found that WHAMY specifically interacted with activated Rac1 through its two CRIB domains, which were sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promoted exceptionally fast actin filament elongation, although it did not activate the Arp2/3 complex. Loss-and gain-of-function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further implied synergistic functions between WHAMY and WASP during morphogenesis. Double mutants were late-embryonic lethal and showed severe defects in myoblast fusion. Trans-heterozygous mutant animals showed strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution.

“…As recently reported, wild-type macrophages started to initiate random single-cell migration 2 h after puparium formation (Moreira et al, 2013) (Fig. 5C; Movie 10).…”

Section: Whamy Function Is Important For Macrophage Cell Motility In supporting

confidence: 86%

WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development in Drosophila

Brinkmann

Winterhoff

Önel

et al. 2016

“…Previous data have revealed an important function of βPS-integrin-mediated cell adhesion in two-dimensional random cell migration in white prepupae, in vivo (Moreira et al, 2013). Sub- The originally published mutant fly stock with the deficiency in trans, the stock carrying the P-element as well as the viable washΔ185* stock that we established showed a complete loss of WASH expression in ovaries in western blot analysis (C,D).…”

Section: Wash Is Required For Cell Migration Of Prepupal Macrophagesmentioning

confidence: 84%

“…5A, quantification in Fig. 5D-F; Movie 7; Moreira et al, 2013;Brinkmann et al, 2016). Prepupal wash mutant cells also showed a reduced cell spread area in vivo (Fig.…”

Section: Wash Is Required For Cell Migration Of Prepupal Macrophagesmentioning

confidence: 90%

“…Because wash mutant cells spread properly on lectin-coated surfaces, we suggest that WASH plays a role in integrin-mediated cell spreading rather than in regulating lamellipodial cell protrusions. In Drosophila, the main β-integrin is βPS-integrin, also known as Myospheroid (Mys), which is required for cell spreading and cell migration of embryonic and pupal macrophages (Comber et al, 2013;Moreira et al, 2013). Structured illumination microscopy (SIM) revealed an overall punctate expression pattern of endogenous βPS-integrin in macrophages.…”

Section: Wash Regulates Integrin-mediated Cell Adhesionmentioning

confidence: 99%

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

Nagel

Bechtold

Rodríguez

et al. 2017

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.

“…The EGFP-Knittrig-FL transgene was expressed only weakly to allow live cell imaging on dorsal subepidermal macrophages of 2-to 4-hour pupae (Moreira et al, 2013). At this stage, wild-type macrophages are morphologically highly polarized with broad lamellipodial protrusions and migrate randomly beneath the epidermis or along tracheae (Moreira et al, 2013) (Fig.…”

Section: Knittrig Localizes At the Rear Of Migrating Macrophages In Vivomentioning

confidence: 99%

The Drosophila FHOD1-like formin Knittrig acts through Rok to promote stress fiber formation and directed macrophage migration during the cellular immune response

Lammel¹,

Bechtold²,

Risse³

et al. 2014

A tight spatiotemporal control of actin polymerization is important for many cellular processes that shape cells into a multicellular organism. The formation of unbranched F-actin is induced by several members of the formin family. Drosophila encodes six formin genes, representing six of the seven known mammalian subclasses. Knittrig, the Drosophila homolog of mammalian FHOD1, is specifically expressed in the developing central nervous system midline glia, the trachea, the wing and in macrophages. knittrig mutants exhibit mild tracheal defects but survive until late pupal stages and mainly die as pharate adult flies. knittrig mutant macrophages are smaller and show reduced cell spreading and cell migration in in vivo wounding experiments. Rescue experiments further demonstrate a cellautonomous function of Knittrig in regulating actin dynamics and cell migration. Knittrig localizes at the rear of migrating macrophages in vivo, suggesting a cellular requirement of Knittrig in the retraction of the trailing edge. Supporting this notion, we found that Knittrig is a target of the Rho-dependent kinase Rok. Co-expression with Rok or expression of an activated form of Knittrig induces actin stress fibers in macrophages and in epithelial tissues. Thus, we propose a model in which Rok-induced phosphorylation of residues within the basic region mediates the activation of Knittrig in controlling macrophage migration.