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ARPC2 is a subunit of the Arp2/3 complex, which is essential for lamellipodia, invadopodia and filopodia, and ARPC2 has been identified as a migrastatic target molecule. To identify ARPC2 inhibitors, we generated an ARPC2 knockout DLD‐1 human colon cancer cell line using the clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) system and explored gene signature‐based strategies, such as a connectivity map (CMap) using the gene expression profiling data of ARPC2 knockout and knockdown cells. From the CMap‐based drug discovery strategy, we identified pimozide (a clinically used antipsychotic drug) as a migrastatic drug and ARPC2 inhibitor. Pimozide inhibited the migration and invasion of various cancer cells. Through drug affinity responsive target stability (DARTS) analysis and cellular thermal shift assay (CETSA), it was confirmed that pimozide directly binds to ARPC2. Pimozide increased the lag phase of Arp2/3 complex‐dependent actin polymerization and inhibited the vinculin‐mediated recruitment of ARPC2 to focal adhesions in cancer cells. To validate the likely binding of pimozide to ARPC2, mutant cells, including ARPC2F225A, ARPC2F247A and ARPC2Y250F cells, were prepared using ARPC2 knockout cells prepared by gene‐editing technology. Pimozide strongly inhibited the migration of mutant cells because the mutated ARPC2 likely has a larger binding pocket than the wild‐type ARPC2. Therefore, pimozide is a potential ARPC2 inhibitor, and ARPC2 is a new molecular target. Taken together, the results of the present study provide new insights into the molecular mechanism and target that are responsible for the antitumor and antimetastatic activity of pimozide. Pimozide is identified as a migrastatic drug and ARPC2 inhibitor from connectivity map‐based drug discovery strategy. Pimozide inhibits migration and invasion in various cancer cell lines, and suppresses metastasis in an in vivo antimetastatic assay. Through drug affinity responsive target stability (DARTS) analysis and cellular thermal shift assay (CETSA), it was confirmed that pimozide directly binds to ARPC2.

New Arp2/3 complex inhibitors The actin cytoskeleton is dynamic and plays a critical role in several cell biological processes such as cell adhesion, migration and endocytosis. In addition, several pathogens exploit this system to invade and survive within host cells. The Arp2/3 complex nucleates actin filaments and has a unique property to form branched actin networks. In this study, Nolen et al . describe the generation of two types of small molecules that bind to different sites on the Arp2/3 complex and inhibit its actin nucleating activity in vitro and in vivo . These inhibitors provide a powerful approach to study Arp2/3 complex-mediated actin reorganization events in living cells. The actin cytoskeleton plays a critical role in cell biological processes such as cell adhesion, migration and endocytosis. Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movement. Two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments are now described; these inhibitors provide a powerful approach for studying the Arp2/3 complex in living cells. Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements 1 . However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.

Fusarium oxysporum is a devastating plant-pathogenic fungus that causes vascular wilt disease in many economically important crops, including watermelon, worldwide. F. oxysporum f. sp. nievum ( Fon ) causes serious yield loss in watermelon production. However, the molecular mechanism of Fusarium wilt development by Fon remains largely unknown. Fusarium oxysporum f. sp. niveum ( Fon ), a soilborne phytopathogenic fungus, causes watermelon Fusarium wilt, resulting in serious yield losses worldwide. However, the underlying molecular mechanism of Fon virulence is largely unknown. The present study investigated the biological functions of six FonPUFs , encoding RNA binding Pumilio proteins, and especially explored the molecular mechanism of FonPUF1 in Fon virulence. A series of phenotypic analyses indicated that FonPUFs have distinct but diverse functions in vegetative growth, asexual reproduction, macroconidia morphology, spore germination, cell wall, or abiotic stress response of Fon . Notably, the deletion of FonPUF1 attenuates Fon virulence by impairing the invasive growth and colonization ability inside the watermelon plants. FonPUF1 possesses RNA binding activity, and its biochemical activity and virulence function depend on the RNA recognition motif or Pumilio domains. FonPUF1 associates with the actin-related protein 2/3 (ARP2/3) complex by interacting with FonARC18, which is also required for Fon virulence and plays an important role in regulating mitochondrial functions, such as ATP generation and reactive oxygen species production. Transcriptomic profiling of ΔFonPUF1 identified a set of putative FonPUF1-dependent virulence-related genes in Fon , possessing a novel A-rich binding motif in the 3′ untranslated region (UTR), indicating that FonPUF1 participates in additional mechanisms critical for Fon virulence. These findings highlight the functions and molecular mechanism of FonPUFs in Fon virulence. IMPORTANCE Fusarium oxysporum is a devastating plant-pathogenic fungus that causes vascular wilt disease in many economically important crops, including watermelon, worldwide. F. oxysporum f. sp. nievum ( Fon ) causes serious yield loss in watermelon production. However, the molecular mechanism of Fusarium wilt development by Fon remains largely unknown. Here, we demonstrate that six putative Pumilio proteins-encoding genes ( FonPUFs ) differentially operate diverse basic biological processes, including stress response, and that FonPUF1 is required for Fon virulence. Notably, FonPUF1 possesses RNA binding activity and associates with the actin-related protein 2/3 complex to control mitochondrial functions. Furthermore, FonPUF1 coordinates the expression of a set of putative virulence-related genes in Fon by binding to a novel A-rich motif present in the 3′ UTR of a diverse set of target mRNAs. Our study disentangles the previously unexplored molecular mechanism involved in regulating Fon virulence, providing a possibility for the development of novel strategies for disease management.

Mammalian oocyte maturation involves two asymmetric meiotic divisions that require the positioning of the meiotic spindle near the cortical area from which the extrusion of the polar bodies occurs. Li and colleagues show that the nucleating activity of the Arp2/3 complex, localized at the cortical actin cap, induces actin-filament flow away from the complex, creating a cytoplasmic streaming that pushes the spindle towards the cortex. Mature mammalian oocytes are poised for completing meiosis II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap 1 , 2 , 3 . Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.

Directed movement of normal cells occurs when actin‐related protein 2 and 3 complex (Arp2/3 complex) triggers the actin polymerization that forms lamellipodia immediately after binding to WAVE2. In order to determine whether the same mechanism correlates with liver metastasis from colorectal cancer, paired mirror sections of 154 cancer specimens (29 cases with liver metastasis and 125 cases without liver metastasis in which T factor, gender, primary tumor site, and age at operation were matched) were examined immunohistochemically for the localization of Arp2 and WAVE2. Expression of both Arp2 and WAVE2 was detected in the same cancer cells in 55 (35.7%) of the 154 cases, but not detected in the normal colonic epithelial cells. Univariate analysis showed that the colocalization was significantly predictive of liver metastasis (risk ratio [RR] 8.760. Likewise, histological grade (RR 2.46), lymphatic invasion (RR 9.95), and tumor budding (RR 4.00) were significant predictors. Among these, colocalization and lymphatic invasion were shown to be independent risk factors by multivariate analysis. Another 59 colorectal specimens were examined for mRNA expression of Arp2 by real time polymerase chain reaction. High mRNA levels of Arp2, that in situ hybridization revealed to be expressed by the cancer cells, were significantly associated with liver metastasis. However, its effect was absorbed by the influence of risk of the colocalization that is closely related to high expression of Arp2. These results indicate that the colocalization of Arp2 and WAVE2 is an independent risk factor for liver metastasis of colorectal carcinoma. (Cancer Sci 2007; 98: 992–999)

Actin filament assembly by the actin-related protein (Arp) 2/3 complex is necessary to build many cellular structures, including lamellipodia at the leading edge of motile cells and phagocytic cups, and to move endosomes and intracellular pathogens. The crucial role of the Arp2/3 complex in cellular processes requires precise spatiotemporal regulation of its activity. While binding of nucleation-promoting factors (NPFs) has long been considered essential to Arp2/3 complex activity, we recently showed that phosphorylation of the Arp2 subunit is also necessary for Arp2/3 complex activation. Using molecular dynamics simulations and biochemical assays with recombinant Arp2/3 complex, we now show how phosphorylation of Arp2 induces conformational changes permitting activation. The simulations suggest that phosphorylation causes reorientation of Arp2 relative to Arp3 by destabilizing a network of salt-bridge interactions at the interface of the Arp2, Arp3, and ARPC4 subunits. Simulations also suggest a gain-of-function ARPC4 mutant that we show experimentally to have substantial activity in the absence of NPFs. We propose a model in which a network of auto-inhibitory salt-bridge interactions holds the Arp2 subunit in an inactive orientation. These auto-inhibitory interactions are destabilized upon phosphorylation of Arp2, allowing Arp2 to reorient to an activation-competent state.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by the pathological accumulation of collagen-rich extracellular matrix, resulting in irreversible lung remodeling and respiratory failure. The incomplete understanding of IPF pathogenesis has hindered the development of effective therapeutics. Here, we investigate the mechanism by which the actin-related protein 2/3 complex subunit 2 (ARPC2) contributes to the fibrotic response in lung fibroblasts. Modulating of ARPC2 expression levels altered the expression of profibrotic genes, including α-smooth muscle actin (ACTA2), in TGF-β1-treated MRC-5 cells at the transcriptional level. We further show that ARPC2 regulates the TGF-β1-mediated nuclear translocation of myocardin-related transcription factor-A (MRTFA), a central driver of fibrotic gene induction. Our data indicate that ARPC2 plays a distinct role in profibrotic gene expression and MRTFA nuclear localization, distinguishing its function from other components of the actin-related protein 2/3 (ARP2/3) complex. Furthermore, ARPC2 appears to modulate the TGF-β1-dependent formation of MRTFA/G-actin complexes. Finally, transcriptomic analysis of cells depleted of ARPC2, ACTR2, or MRTFA revealed that ARPC2 and MRTFA co-regulate a specific repertoire of fibrotic genes. These observations support a profibrotic function for ARPC2 during fibroblast-to-myofibroblast transition (FMT), highlighting it as a potential therapeutic target for IPF.

Microfilament and Ca 2+ dynamics play important roles in stress signaling in plants. Through genetic screening of Arabidopsis thaliana mutants that are defective in stress-induced increases in cytosolic Ca 2+ ([Ca 2+ ] cyt ), we identified Actin-Related Protein2 (Arp2) as a regulator of [Ca 2+ ] cyt in response to salt stress. Plants lacking Arp2 or other proteins in the Arp2/3 complex exhibited enhanced salt-induced increases in [Ca 2+ ] cyt , decreased mitochondria movement, and hypersensitivity to salt. In addition, mitochondria aggregated, the mitochondrial permeability transition pore opened, and mitochondrial membrane potential ψm was impaired in the arp2 mutant, and these changes were associated with salt-induced cell death. When opening of the enhanced mitochondrial permeability transition pore was blocked or increases in [Ca 2+ ] cyt were prevented, the salt-sensitive phenotype of the arp2 mutant was partially rescued. These results indicate that the Arp2/3 complex regulates mitochondrial-dependent Ca 2+ signaling in response to salt stress.

The increasing genetic tractability of Chlamydia trachomatis is accelerating the ability to characterize the unique infection biology of this obligate intracellular parasite. These efforts are leading to a greater understanding of the molecular events associated with key virulence requirements. Chlamydia trachomatis is a medically significant human pathogen and is an epithelial-tropic obligate intracellular parasite. Invasion of nonprofessional phagocytes represents a crucial step in the infection process and has likely promoted the evolution of a redundant mechanism and routes of entry. Like many other viral and invasive bacterial pathogens, manipulation of the host cell cytoskeleton represents a focal point in Chlamydia entry. The advent of genetic techniques in C. trachomatis , such as creation of complete gene deletions via fluorescence-reported allelic exchange mutagenesis (FRAEM), is providing important tools to unravel the contributions of bacterial factors in these complex pathways. The type III secretion chaperone Slc1 directs delivery of at least four effectors during the invasion process. Two of these, TarP and TmeA, have been associated with manipulation of actin networks and are essential for normal levels of invasion. The functions of TarP are well established, whereas TmeA is less well characterized. We leverage chlamydial genetics and proximity labeling here to provide evidence that TmeA directly targets host N-WASP to promote Arp2/3-dependent actin polymerization. Our work also shows that TmeA and TarP influence separate, yet synergistic pathways to accomplish chlamydial entry. These data further support an appreciation that a pathogen, confined by a reductionist genome, retains the ability to commit considerable resources to accomplish bottle-neck steps during the infection process. IMPORTANCE The increasing genetic tractability of Chlamydia trachomatis is accelerating the ability to characterize the unique infection biology of this obligate intracellular parasite. These efforts are leading to a greater understanding of the molecular events associated with key virulence requirements. Manipulation of the host actin cytoskeleton plays a pivotal role throughout Chlamydia infection, yet a thorough understanding of the molecular mechanisms initiating and orchestrating actin rearrangements has lagged. Our work highlights the application of genetic manipulation to address open questions regarding chlamydial invasion, a process essential to survival. We provide definitive insight regarding the role of the type III secreted effector TmeA and how that activity relates to another prominent effector, TarP. In addition, our data implicate at least one source that contributes to the functional divergence of entry mechanisms among chlamydial species.

Actin-related protein (Arp) 2/3 complex mediates the formation of actin filament branches during endocytosis and at the leading edge of motile cells. The pathway of branch formation is ambiguous owing to uncertainty regarding the stoichiometry and location of VCA binding sites on Arp2/3 complex. Isothermal titration calorimetry showed that the CA motif from the C terminus of fission yeast WASP (Wsp1p) bound to fission yeast and bovine Arp2/3 complex with a stoichiometry of 2 to 1 and very different affinities for the two sites (Kds of 0.13 and 1.6 µM for fission yeast Arp2/3 complex). Equilibrium binding, kinetic, and cross-linking experiments showed that (i) CA at high-affinity site 1 inhibited Arp2/3 complex binding to actin filaments, (ii) low-affinity site 2 had a higher affinity for CA when Arp2/3 complex was bound to actin filaments, and (iii) Arp2/3 complex had a much higher affinity for free CA than VCA cross-linked to an actin monomer. Crystal structures showed the C terminus of CA bound to the low-affinity site 2 on Arp3 of bovine Arp2/3 complex. The C helix is likely to bind to the barbed end groove of Arp3 in a position for VCA to deliver the first actin subunit to the daughter filament.