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Mycolactone is a diffusible lipid secreted by the human pathogen Mycobacterium ulcerans, which induces the formation of open skin lesions referred to as Buruli ulcers. Here, we show that mycolactone operates by hijacking the Wiskott-Aldrich syndrome protein (WASP) family of actin-nucleating factors. By disrupting WASP autoinhibition, mycolactone leads to uncontrolled activation of ARP2/3-mediated assembly of actin in the cytoplasm. In epithelial cells, mycolactone-induced stimulation of ARP2/3 concentrated in the perinuclear region, resulting in defective cell adhesion and directional migration. In vivo injection of mycolactone into mouse ears consistently altered the junctional organization and stratification of keratinocytes, leading to epidermal thinning, followed by rupture. This degradation process was efficiently suppressed by coadministration of the N-WASP inhibitor wiskostatin. These results elucidate the molecular basis of mycolactone activity and provide a mechanism for Buruli ulcer pathogenesis. Our findings should allow for the rationale design of competitive inhibitors of mycolactone binding to N-WASP, with anti-Buruli ulcer therapeutic potential.

Key Points Wiskott–Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins activate the ARP2/3 complex to promote reorganization of the actin cytoskeleton. The N-terminal domain of the WASP and WAVE proteins contributes to stable protein–protein interactions, thereby contributing to the formation of the protein complex. Intramolecular interaction suppresses the ARP2/3-complex-activating capability of WASP and neural (N-)WASP. The role of intramolecular or intermolecular interactions in the regulation of WAVE proteins is still unclear. Phosphatidylinositol phosphates bind to WASP and WAVE proteins and contribute to the localization of these proteins, and they also regulate their capability to activate the ARP2/3 complex. Many proteins with Src-homology-3 (SH3) domains bind to the proline-rich region of WASP and WAVE proteins, and enhance the ARP2/3 activation of WASP and WAVE. Most of the proteins with SH3 domains are adaptors that link WASP and WAVE proteins to other proteins or the cell membrane. The Bin, amphiphysin, Rvs167 (BAR) and extended Fer-CIP4 homology (EFC) domains are membrane-binding and membrane-deforming domains. Most BAR- or EFC-domain-containing proteins have SH3 domains that bind to N-WASP, and are involved in endocytosis. The Rac binding (RCB, also known as the IRSp53-Mim-homology domain (IMD)) domain of IRSp53 shares structural similarity to the BAR domain and binds to the membrane. IRSp53 binds to WAVE2 for lamellipodium formation. WASP and N-WASP are involved in cell-shape changes that occur inwardly (endocytosis) and outwardly (filopodia and podosomes). WAVE proteins are involved in outward (lamellipodia) cell-shape changes only. Membrane deformation might be coupled to WASP- and WAVE-mediated cytoskeletal changes. Membrane-binding and membrane-deforming proteins have emerged as binding partners of the Wiskott–Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins that regulate the actin cytoskeleton. Membrane deformation and cytoskeletal reorganization might be coupled in processes that require alteration of membrane shapes, including endocytosis and membrane protrusion. Wiskott–Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins are scaffolds that link upstream signals to the activation of the ARP2/3 complex, leading to a burst of actin polymerization. ARP2/3-complex-mediated actin polymerization is crucial for the reorganization of the actin cytoskeleton at the cell cortex for processes such as cell movement, vesicular trafficking and pathogen infection. Large families of membrane-binding proteins were recently found to interact with WASP and WAVE family proteins, therefore providing a new layer of membrane-dependent regulation of actin polymerization.

Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ∼1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors.

To maintain tissue integrity during epithelial morphogenesis, adherens junctions (AJs) must resist the mechanical stresses exerted by dynamic tissue movements. Junctional stability is dependent on actomyosin contractility within the actin ring. Here we describe a novel function for the axon guidance receptor, Neogenin, as a key component of the actin nucleation machinery governing junctional stability. Loss of Neogenin perturbs AJs and attenuates junctional tension. Neogenin promotes actin nucleation at AJs by recruiting the Wave regulatory complex (WRC) and Arp2/3. A direct interaction between the Neogenin WIRS domain and the WRC is crucial for the spatially restricted recruitment of the WRC to the junction. Thus, we provide the first example of a functional WIRS–WRC interaction in epithelia. We further show that Neogenin regulates cadherin recycling at the AJ. In summary, we identify Neogenin as a pivotal component of the AJ, where it influences both cadherin dynamics and junctional tension. The stability of epithelial adherens junctions depends on tension generated by actomyosin contractility. Here Lee et al . describe a novel role for the axon guidance receptor Neogenin in maintaining junctional stability by recruiting actin nucleation machinery to adherens junctions.

Subtelomeres are duplication-rich, structurally variable regions of the human genome situated just proximal of telomeres. We report here that the most terminally located human subtelomeric genes encode a previously unrecognized third subclass of the Wiskott-Aldrich Syndrome Protein family, whose known members reorganize the actin cytoskeleton in response to extracellular stimuli. This new subclass, which we call WASH, is evolutionarily conserved in species as diverged as Entamoeba. We demonstrate that WASH is essential in Drosophila. WASH is widely expressed in human tissues, and human WASH protein colocalizes with actin in filopodia and lamellipodia. The VCA domain of human WASH promotes actin polymerization by the Arp2/3 complex in vitro. WASH duplicated to multiple chromosomal ends during primate evolution, with highest copy number reached in humans, whose WASH repertoires vary. Thus, human subtelomeres are not genetic junkyards, and WASH's location in these dynamic regions could have advantageous as well as pathologic consequences.

Proteins containing repeats of the WASP homology 2 (WH2) actin-binding module are multifunctional regulators of actin nucleation and assembly. Now biochemical analyses of VopF from Vibrio cholerae reveal a new regulatory mechanism of actin-filament barbed-end dynamics including enhanced nucleation, uncapping and assisted elongation. Proteins containing repeats of the WASP homology 2 (WH2) actin-binding module are multifunctional regulators of actin nucleation and assembly. The bacterial effector VopF in Vibrio cholerae , like VopL in Vibrio parahaemolyticus , is a unique homodimer of three WH2 motifs linked by a C-terminal dimerization domain. We show that only the first and third WH2 domains of VopF bind G-actin in a non-nucleating, sequestered conformation. Moreover, dimeric WH2 domains in VopF give rise to unprecedented regulation of actin assembly. Specifically, two WH2 domains on opposite protomers of VopF direct filament assembly from actin or profilin–actin by binding terminal subunits and uncapping capping protein from barbed ends by a new mechanism. Thus, VopF does not nucleate filaments by capping a pointed-end F-actin hexamer. These properties may contribute to VopF pathogenicity, and they show how dimeric WH2 peptides may mediate processive filament growth.

Key Points Lamellipodial protrusion depends on the force generated by actin polymerization. Actin polymerization is the sum of the activities of nucleators — for example, the actin-related protein 2/3 (ARP2/3) complex — and elongators — formins and ENA/VASP proteins. Small GTPases, such as RAC and CDC42, control both actin nucleators and actin elongators; RAC activates the WASP family verprolin-homologous protein (WAVE) complex upstream of the ARP2/3 complex independently of the activation of the formin FMNL2 by CDC42, but RAC may coordinate ARP2/3 with ENA/VASP proteins by inducing a complex between WAVE and lamellipodin. The speed of cell migration depends on the turnover of actin branched junctions and on the elongation of actin networks. An intrinsic instability of lamellipodia is due to ARP2/3 inhibitory proteins, such as Arpin, which is also activated downstream of RAC. The persistence of lamellipodia is the major controller of cell directionality. Directional persistence (that is, the characteristic time during which a cell sustains its migration in the same direction) is the combinatory result of several intertwined positive- and negative-feedback loops that sustain or stop actin polymerization at the leading edge. Lamellipodial protrusion is powered by actin polymerization that is mediated through the actin-related protein 2/3 (ARP2/3)-induced nucleation of branched actin networks and the elongation of actin filaments. These processes are regulated by positive and negative feedback loops centred around the GTPase RAC, and the balance between them determines lamellipodial and directional persistence during cell migration. Membrane protrusions at the leading edge of cells, known as lamellipodia, drive cell migration in many normal and pathological situations. Lamellipodial protrusion is powered by actin polymerization, which is mediated by the actin-related protein 2/3 (ARP2/3)-induced nucleation of branched actin networks and the elongation of actin filaments. Recently, advances have been made in our understanding of positive and negative ARP2/3 regulators (such as the SCAR/WAVE (SCAR/WASP family verprolin-homologous protein) complex and Arpin, respectively) and of proteins that control actin branch stability (such as glial maturation factor (GMF)) or actin filament elongation (such as ENA/VASP proteins) in lamellipodium dynamics and cell migration. This Review highlights how the balance between actin filament branching and elongation, and between the positive and negative feedback loops that regulate these activities, determines lamellipodial persistence. Importantly, directional persistence, which results from lamellipodial persistence, emerges as a critical factor in steering cell migration.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency associated with an increased susceptibility to herpesvirus infection and hematologic malignancy as well as a deficiency of NK cell function. It is caused by defective WAS protein (WASp). WASp facilitates filamentous actin (F-actin) branching and is required for F-actin accumulation at the NK cell immunological synapse and NK cell cytotoxicity ex vivo. Importantly, the function of WASp-deficient NK cells can be restored in vitro after exposure to IL-2, but the mechanisms underlying this remain unknown. Using a WASp inhibitor as well as cells from patients with WAS, we have defined a direct effect of IL-2 signaling upon F-actin that is independent of WASp function. We found that IL-2 treatment of a patient with WAS enhanced the cytotoxicity of their NK cells and the F-actin content at the immunological synapses formed by their NK cells. IL-2 stimulation of NK cells in vitro activated the WASp homolog WAVE2, which was required for inducing WASp-independent NK cell function, but not for baseline activity. Thus, WAVE2 and WASp define parallel pathways to F-actin reorganization and function in human NK cells; although WAVE2 was not required for NK cell innate function, it was accessible through adaptive immunity via IL-2. These results demonstrate how overlapping cytoskeletal activities can utilize immunologically distinct pathways to achieve synonymous immune function.

WHAMM, a member of the Wiskott-Aldrich syndrome protein (WASP) family, is an actin nucleation promoting factor (NPF) that also associates with membranes and microtubules. Here we report that WHAMM is required for autophagic lysosome reformation (ALR). WHAMM knockout causes impairment of autolysosome tubulation, which results in accumulation of enlarged autolysosomes during prolonged starvation. Mechanistically, WHAMM is recruited to the autolysosome membrane through its specific interaction with PI(4,5)P 2 . WHAMM then works as an NPF which promotes assembly of an actin scaffold on the surface of the autolysosome to promote autolysosome tubulation. Our study demonstrates an unexpected role of the actin scaffold in regulating autophagic lysosome reformation. After autophagic cargo degradation, autolysosomes undergo a reformation process to recycle lysosomal membrane components. Here, Dai et al. demonstrate that the actin nucleation promoting factor WHAMM is required for autolysosome reformation by providing an actin scaffold to drive tubulation.

Human papillomavirus type 16 (HPV16) is an etiological agent of human cancers that requires endocytosis to initiate infection. HPV16 entry into epithelial cells occurs through a non-canonical endocytic pathway that is actin-driven, but it is not well understood how HPV16–cell surface interactions trigger actin reorganization in a way that facilitates entry. This study provides evidence that Wiskott–Aldrich syndrome protein family verprolin-homologous proteins 1 and 2 (WAVE1 and WAVE2) are molecular mediators of actin protrusions that occur at the cellular surface upon HPV addition to cells, and that this stimulation is a key step prior to endocytosis and intracellular trafficking. We demonstrate through post-transcriptional gene silencing and genome editing that WAVE1 and WAVE2 are critical for efficient HPV16 infection, and that restoration of each in knockout cells rescues HPV16 infection. Cells lacking WAVE1, WAVE2, or both internalize HPV16 at a significantly reduced rate. Microscopic analysis of fluorescently labeled cells revealed that HPV16, WAVE1, WAVE2, and actin are all colocalized at the cellular dorsal surface within a timeframe that precedes endocytosis. Within that same timeframe, we also found that HPV16-treated cells express cellular dorsal surface filopodia, which does not occur in cells lacking WAVE1 and WAVE2. Taken together, this study provides evidence that WAVE1 and WAVE2 mediate a key step prior to HPV entry into cells that involves actin reorganization in the form of cellular dorsal surface protrusions.