To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell\'s plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host\'s endocytic system for efficient internalization.

Neurite outgrowth is a critical step in neural development, leading to the generation of neurite branches that allow individual neurons to make contacts with multiple neurons within the target region. Polyglutamine-binding protein 1 (PQBP1) is a highly conserved protein with a key role in neural development. Our recent mass spectrometric analysis showed that PQBP1 associates with neural Wiskott-Aldrich syndrome protein (N-WASP), an important actin polymerization-promoting factor involved in neurite outgrowth. Here, we report that the WW domain of PQBP1 directly interacts with the proline-rich domain of N-WASP. The disruption of this interaction leads to impaired neurite outgrowth and growth cone size. Furthermore, we demonstrate that PQBP1/N-WASP interaction is critical for the recruitment of N-WASP to the growth cone, but does not affect N-WASP protein levels or N-WASP-induced actin polymerization. Our results indicated that PQBP1 regulates neurite outgrowth by recruiting N-WASP to the growth cone, thus representing an alternative molecular mechanism via which PQBP1-mediates neurite outgrowth.

The modulation of actin polymerization is a common theme among microbial pathogens. Even though microorganisms show a wide repertoire of strategies to subvert the activity of actin, most of them converge in the ones that activate nucleating factors, such as the Arp2/3 complex. Brucella spp. are intracellular pathogens capable of establishing chronic infections in their hosts. The ability to subvert the host cell response is dependent on the capacity of the bacterium to attach, invade, avoid degradation in the phagocytic compartment, replicate in an endoplasmic reticulum-derived compartment and egress. Even though a significant number of mechanisms deployed by Brucella in these different phases have been identified and characterized, none of them have been described to target actin as a cellular component. In this manuscript, we describe the identification of a novel virulence factor (NpeA) that promotes niche formation. NpeA harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP and stabilizes the autoinhibited state. Our results show that NpeA is secreted in a Type IV secretion system-dependent manner and that deletion of the gene diminishes the intracellular replication capacity of the bacterium. In vitro and ex vivo experiments demonstrate that NpeA binds N-WASP and that the short linear motif is required for the biological activity of the protein.IMPORTANCEThe modulation of actin-binding effectors that regulate the activity of this fundamental cellular protein is a common theme among bacterial pathogens. The neural Wiskott-Aldrich syndrome protein (N-WASP) is a protein that several pathogens target to hijack actin dynamics. The highly adapted intracellular bacterium Brucella has evolved a wide repertoire of virulence factors that modulate many activities of the host cell to establish successful intracellular replication niches, but, to date, no effector proteins have been implicated in the modulation of actin dynamics. We present here the identification of a virulence factor that harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP stabilizing its autoinhibited state. We demonstrate that this protein is a Type IV secretion effector that targets N-WASP-promoting intracellular survival and niche formation.

Filopodia are thin, actin-rich projection from the plasma membrane that promote cancer cell invasion and migration. Sex-determining region Y-related high-mobility group-box 4 (SOX4) is a crucial transcription factor that plays a role in the development and metastasis of colorectal cancer (CRC). However, the involvement of SOX4 in cytoskeleton remodeling in CRC remains unknown. For the first time, we demonstrate that SOX4 is a potent regulator of filopodia formation in CRC cells. Overexpression of SOX4 protein enhances both migration and invasion ability of HCT116, and CACO2 cells, which is relevant to the metastasis. Furthermore, through phalloidin staining, cytoskeleton re-assembly was observed in SOX4-modified cell lines. Enhanced expression of SOX4 increased the number and length of filopodia on cell surface. In contrast, silencing SOX4 in SW620 cells with higher endogenous expression of SOX4, impeded the filopodia formation. Moreover, SOX4 was found to be positively regulating the expression of central regulators of actin cytoskeleton - N-Wiskott-Aldrich syndrome protein (N-WASP); WAVE2; Actin related proteins, ARP2 and ARP3. Inhibiting the N-WASP/ARP2/3 pathway diminishes the filopodia formation and the migration of CRC cells. These results indicate the crucial role of SOX4 in the regulation of filopodia formation mediated by N-WASP/ARP2/3 pathway in CRC cells.

Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson\'s disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.

Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic outgrowth. Breast and prostate cancers are common malignancies in women and men, respectively. Here, we observe that β1-integrin is required for human prostate and breast cancer cell adhesion to ECs under shear-stress conditions in vitro and to lung blood vessel ECs in vivo. We identify IQGAP1 and neural Wiskott-Aldrich syndrome protein (NWASP) as regulators of β1-integrin transcription and protein expression in prostate and breast cancer cells. IQGAP1 and NWASP depletion in cancer cells decreases adhesion to ECs in vitro and retention in the lung vasculature and metastatic lung nodule formation in vivo. Mechanistically, NWASP and IQGAP1 act downstream of Cdc42 to increase β1-integrin expression both via extracellular signal-regulated kinase (ERK)/focal adhesion kinase signaling at the protein level and by myocardin-related transcription factor/serum response factor (SRF) transcriptionally. Our results identify IQGAP1 and NWASP as potential therapeutic targets to reduce early metastatic dissemination.

The higher-order assembly of Bin-amphiphysin-Rvs (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, into lattice on the membrane is essential for the formation of subcellular structures. However, the regulation of their ordered assembly has not been elucidated. Here, we show that the higher ordered assembly of growth-arrested specific 7 (GAS7), an F-BAR domain protein, is regulated by the multivalent scaffold proteins of Wiskott-Aldrich syndrome protein (WASP)/neural WASP, that commonly binds to the BAR domain superfamily proteins, together with WISH, Nck, the activated small guanosine triphosphatase Cdc42, and a membrane-anchored phagocytic receptor. The assembly kinetics by fluorescence resonance energy transfer monitoring indicated that the GAS7 assembly on liposomes started within seconds and was further increased by the presence of these proteins. The regulated GAS7 assembly was abolished by Wiskott-Aldrich syndrome mutations both in vitro and in cellular phagocytosis. Therefore, Cdc42 and the scaffold proteins that commonly bind to the BAR domain superfamily proteins promoted GAS7 assembly.

Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the N-terminal WH1 domain. The basic region also participates in Cdc42 binding, especially for WASP; consequently Cdc42 binding significantly compromises the ability of the basic region in WASP, but not N-WASP, to bind PIP2. PIP2 binding to the WASP basic region is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the activation of WASP and N-WASP may contribute to their different functional roles.

During neural development, the actin filament network must be precisely regulated to form elaborate neurite structures. N-WASP tightly controls actin polymerization dynamics by activating an actin nucleator Arp2/3. However, the importance of N-WASP-Arp2/3 signaling in the assembly of neurite architecture in vivo has not been clarified. Here, we demonstrate that N-WASP-Arp2/3 signaling plays a crucial role in the maturation of cerebellar Purkinje cell (PC) dendrites in vivo in mice. N-WASP was expressed and activated in developing PCs. Inhibition of Arp2/3 and N-WASP from the beginning of dendrite formation severely disrupted the establishment of a single stem dendrite, which is a characteristic basic structure of PC dendrites. Inhibition of Arp2/3 after stem dendrite formation resulted in hypoplasia of the PC dendritic tree. Cdc42, an upstream activator of N-WASP, is required for N-WASP-Arp2/3 signaling-mediated PC dendrite maturation. In addition, overactivation of N-WASP is also detrimental to dendrite formation in PCs. These findings reveal that proper activation of N-WASP-Arp2/3 signaling is crucial for multiple steps of PC dendrite maturation in vivo.

A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition1-3. Here we report on the discovery that, in Caenorhabditis elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP and the ARP2/3 complex4-8 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow and then transition to disassembly before dissolving completely. We find that, in contrast to condensate growth through diffusion9, the growth dynamics of cortical condensates are chemically driven. Notably, the associated chemical reactions obey mass action kinetics that govern both composition and size. We suggest that the resultant condensate dynamic instability10 suppresses coarsening of the active micro-emulsion11, ensures reaction kinetics that are independent of condensate size and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.