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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.

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.

Skeletal muscle regeneration is a multistep process involving the activation, proliferation, differentiation, and fusion of muscle stem cells, known as satellite cells. Fusion of satellite cell-derived myoblasts (SCMs) is indispensable for generating the multinucleated, contractile myofibers during muscle repair. However, the molecular and cellular mechanisms underlying SCM fusion during muscle regeneration remain incompletely understood. Here, we reveal a critical role for branched actin polymerization in SCM fusion during mouse skeletal muscle regeneration. Using conditional knockouts of the Arp2/3 complex and its actin nucleation-promoting factors N-WASP and WAVE, we demonstrate that branched actin polymerization is specifically required for SCM fusion but dispensable for satellite cell proliferation, differentiation, and migration. We show that the N-WASP and WAVE complexes have partially redundant functions in regulating SCM fusion and that branched actin polymerization is essential for generating invasive protrusions at fusogenic synapses in SCMs. Together, our study identifies branched-actin regulators as key components of the myoblast fusion machinery and establishes invasive protrusion formation as a critical mechanism enabling myoblast fusion during skeletal muscle regeneration.

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.

The Wiskott–Aldrich syndrome protein (WASP) and the WASP family verprolin-homologous protein (WAVE) family are essential molecules that connect GTPases to the actin cytoskeleton, thereby controlling actin polymerisation through the actin-related protein 2/3 complex. This control is crucial for forming actin-based membrane protrusions necessary for cell migration and invasion. The elevated expression of WASP/WAVE proteins in invasive breast cancer cells highlights their significant role in promoting cell motility and invasion. This review summarises the discovery, structural properties, and activation mechanisms of WASP/WAVE proteins, focuses on the contribution of the WASP/WAVE family to breast cancer invasion and migration, particularly synthesises the results of nearly a decade of research in this field since 2015. By exploring promising therapeutic strategies for breast cancer, including small molecule inhibitors and biological agents, this review stresses the potential for developing anticancer drugs that target the WASP/WAVE family and associated pathways, intending to improve the prognosis for patients with metastatic breast cancer.

The ARP2/3 complex, which nucleates branched actin filament networks, is itself activated by nucleation-promoting factors (NPFs). New understanding is being gained into NPF control of ARP2/3 and the biological roles of ARP2/3 branched actin. The actin-related protein 2/3 (ARP2/3) complex nucleates branched actin filament networks, but requires nucleation promoting factors (NPFs) to stimulate this activity. NPFs include proteins such as Wiskott–Aldrich syndrome protein (WASP), neural WASP (NWASP), WASP family verprolin-homologous protein (WAVE; also known as SCAR) and the recently identified WASP and SCAR homologue (WASH) complex. The mechanisms underlying NPF-dependent regulation and the cellular functions of ARP2/3 are being unravelled using new chemical and genetic approaches. Of particular interest is the role of the ARP2/3 complex in vesicular trafficking and directional cell motility.

Key Points Wiskott–Aldrich Syndrome protein (WASP) is an important regulator of the actin cytoskeleton in haematopoietic cells. WASP-deficiency gives rise to the human disease Wiskott–Aldrich Syndrome (WAS), an X-linked primary immunodeficiency. Constitutively active mutations of WASP have recently been described to give rise to a distinct human disease: X-linked neutropenia. WASP activity is attenuated by multiple signalling pathways downstream of surface receptors. Conformational change, phosphorylation and degradation are important mechanisms. Although WASP does not seem to have a key role in haematopoiesis, WASP confers selective advantage for many mature haematopoietic cell types and is emerging as a key regulator of lymphocyte homeostasis. WASP is required for a diverse range of cell functions in innate and adaptive immune cells. These relate both to the role of WASP in cytoskelatal rearrangement and as an intrinsic signalling molecule. Autoimmunity is an important feature of WAS which is poorly understood. Recent studies suggest that defective regulatory T cell function is an important component. Insights into the basic mechanisms of WASP-associated disease are advancing our understanding of immune regulation with wider application, and this allows the potential for future therapeutic benefit. As a key regulator of the actin cytoskeleton, Wiskott–Aldrich syndrome protein (WASP) is involved in diverse immune responses, including leukocyte migration and activation. This Review describes how various mutations in mice and humans have led us to a greater appreciation of the many immunological functions of WASP. The Wiskott–Aldrich syndrome protein (WASP) is an important regulator of the actin cytoskeleton that is required for many haematopoietic and immune cell functions, including effective migration, phagocytosis and immune synapse formation. Loss of WASP activity leads to Wiskott–Aldrich syndrome, an X-linked disease that is associated with defects in a broad range of cellular processes, resulting in complex immunodeficiency, autoimmunity and microthrombocytopenia. Intriguingly, gain of function mutations cause a separate disease that is mainly characterized by neutropenia. Here, we describe recent insights into the cellular mechanisms of these two related, but distinct, human diseases and discuss their wider implications for haematopoiesis, immune function and autoimmunity.

Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.

Wiskott–Aldrich syndrome (WAS) is caused by mutations in the WAS gene and is characterized by immunodeficiency, eczema and microthrombocytopenia. The molecular link between WAS mutations and microthrombocytopenia is unknown. Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins, including the WAS protein (WASp)/WASp-interacting protein (WIP) complex. Here we report that mice with a megakaryocyte/platelet-specific Pfn1 deficiency display microthrombocytopenia due to accelerated turnover of platelets and premature platelet release into the bone marrow. Both Pfn1-null mouse platelets and platelets isolated from WAS patients contained abnormally organized and hyperstable microtubules. These results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients. Patients with mutations in the gene encoding the cytoskeleton regulator WAS have platelet defects. Here the authors show that the WAS-binding protein, Profilin1, is essential for platelet formation in mice, and that its deficiency reproduces the bleeding disorder of patients with WAS mutations.

Neural Wiskott–Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro . Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.