Filopodia In Vitro and In Vivo.

Filopodia are dynamic cell surface protrusions used for cell motility, pathogen infection, and tissue development. The molecular mechanisms determining how and where filopodia grow and retract need to integrate mechanical forces and membrane curvature with extracellular signaling and the broader state of the cytoskeleton. The involved actin regulatory machinery nucleates, elongates, and bundles actin filaments separately from the underlying actin cortex. The refined membrane and actin geometry of filopodia, importance of tissue context, high spatiotemporal resolution required, and high degree of redundancy all limit current models. New technologies are improving opportunities for functional insight, with reconstitution of filopodia in vitro from purified components, endogenous genetic modification, inducible perturbation systems, and the study of filopodia in multicellular environments. In this review, we explore recent advances in conceptual models of how filopodia form, the molecules involved in this process, and our latest understanding of filopodia in vitro and in vivo.

OMD Curation Toolkit: a workflow for in-house curation of public omics datasets.

BACKGROUND: Major advances in sequencing technologies and the sharing of data and metadata in science have resulted in a wealth of publicly available datasets. However, working with and especially curating public omics datasets remains challenging despite these efforts. While a growing number of initiatives aim to re-use previous results, these present limitations that often lead to the need for further in-house curation and processing. RESULTS: Here, we present the Omics Dataset Curation Toolkit (OMD Curation Toolkit), a python3 package designed to accompany and guide the researcher during the curation process of metadata and fastq files of public omics datasets. This workflow provides a standardized framework with multiple capabilities (collection, control check, treatment and integration) to facilitate the arduous task of curating public sequencing data projects. While centered on the European Nucleotide Archive (ENA), the majority of the provided tools are generic and can be used to curate datasets from different sources. CONCLUSIONS: Thus, it offers valuable tools for the in-house curation previously needed to re-use public omics data. Due to its workflow structure and capabilities, it can be easily used and benefit investigators in developing novel omics meta-analyses based on sequencing data.

Ena/VASP proteins in cell edge protrusion, migration and adhesion.

The tightly coordinated, spatiotemporal control of actin filament remodeling provides the basis of fundamental cellular processes, such as cell migration and adhesion. Specific protein assemblies, composed of various actin-binding proteins, are thought to operate in these processes to nucleate and elongate new filaments, arrange them into complex three-dimensional (3D) arrays and recycle them to replenish the actin monomer pool. Actin filament assembly is not only necessary to generate pushing forces against the leading edge membrane or to propel pathogens through the cytoplasm, but also coincides with the generation of stress fibers (SFs) and focal adhesions (FAs) that generate, transmit and sense mechanical tension. The only protein families known to date that directly enhance the elongation of actin filaments are formins and the family of Ena/VASP proteins. Their mechanisms of action, however, in enhancing processive filament elongation are distinct. The aim of this Review is to summarize our current knowledge on the molecular mechanisms of Ena/VASP-mediated actin filament assembly, and to discuss recent insights into the cell biological functions of Ena/VASP proteins in cell edge protrusion, migration and adhesion.

Meeting the transfusion needs of a patient with anti-Ena requires an international effort.

This extraordinary case showcases the identification of a rare anti-Ena specificity that was assisted by DNA-based red blood cell antigen typing and collaboration between the hospital blood bank in the United States, the home blood center in Qatar, the blood center Immunohematology Reference Laboratory, as well as the American Rare Donor Program (ARDP) and the International Society for Blood Transfusion (ISBT) International Rare Donor Panel. Ena is a high-prevalence antigen, and blood samples from over 200 individuals of the extended family in Qatar were crossmatched against the patient's plasma with one compatible En(a-) individual identified. The ISBT International Rare Donor Panel identified an additional donor in Canada, resulting in a total of two En(a-) individuals available to donate blood for the patient.

Utility of repeat extractable nuclear antigen antibody testing: a retrospective audit.

OBJECTIVES: Autoantibodies to ENA are frequently ordered during the workup of suspected autoimmune connective tissue diseases. There are no current guidelines for repeat test ordering. The objective of this study was to assess the utility of repeat ENA testing after an initial negative result. METHODS: A retrospective study was conducted in a single, multicentre tertiary health network in Melbourne, Australia. Results of all ENA tests were extracted from the hospital laboratory information system. For patients who had a change in ENA result from negative to positive, clinical information was obtained from the hospital records regarding new diagnosis of an ANA-associated rheumatic disease (AARD). RESULTS: A total of 23 438 ENA tests were performed in 19 603 patients from 29 July 2013 to 28 September 2020. In total, 20 918 (89.2%) were negative with 215 (0.9%) being equivocal. Of the 2305 positive tests, the most common ENA auto-antibody specificity detected was anti-Ro52 (1185, 51.4%). A total of 2636 of 19 603 patients (13.4%) had more than one ENA test performed during the study period. Of these, most (2523, 95.7%) had stable ENA results with no change compared with the first test. Only 53 patients (2.2%) had an ENA result that changed from negative to positive. Excluding patients with pre-existing rheumatic conditions and those under 18, there were five new AARDs found in the remaining 34 patients. CONCLUSION: Repeat ENA test results rarely change or result in a new diagnosis of an AARD, with repeated testing only warranted if there is a change in clinical manifestations.

EVL regulates VEGF receptor-2 internalization and signaling in developmental angiogenesis.

Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.

Environmental RNA as a Noninvasive Tool for Assessing Toxic Effects in Fish: A Proof-of-concept Study Using Japanese Medaka Exposed to Pyrene.

Although environmental RNA (eRNA) is emerging as a noninvasive tool to assess the health status of aquatic macroorganisms, the potential of eRNA in assessing chemical hazards remain largely untested. In this study, we investigated the ability of eRNA to detect changes in gene expression in Japanese medaka fish (Oryzias latipes) in response to sublethal pyrene exposure, as a model toxic chemical. We performed standardized acute toxicity tests and collected eRNA from tank water and RNA from fish tissue after 96 h of exposure. Our results showed that over 1000 genes were detected in eRNA and the sequenced read counts of these genes correlated with those in fish tissue (r = 0.50). Moreover, eRNA detected 86 differentially expressed genes in response to pyrene, some of which were shared by fish RNA, including the suppression of collagen fiber genes. These results suggest that eRNA has the potential to detect changes in gene expression in fish in response to environmental stressors without the need for sacrificing or causing pain to fish. However, we also found that the majority of sequenced reads of eRNA (>99%) were not mapped to the reference medaka genome and they originated from bacteria and fungi, resulting in low sequencing depth. In addition, eRNA, in particular nuclear genes, was highly degraded with a median transcript integrity number (TIN) of <20. These limitations highlight the need for future studies to improve the analytical methods of eRNA application.

Effects of acetaminophen exposure on behavior and erythrocyte nuclear morphology of juvenile Oreochromis niloticus.

The occurrence of pharmaceuticals in the environment can have undesirable effects on nontarget animals, including fish. The present experiment assessed the effects of subchronic exposure to waterborne acetaminophen (N-acetyl-p-aminophenol) (APAP) on selected behavioral aspects (physical avoidance response, ventilation rate, and food detection rate) and erythrocyte nuclear abnormality (ENA) in juvenile Oreochromis niloticus. Two groups of fish were exposed to APAP dissolved in aged municipal water (T1: 2 mg/L and T2: 10 mg/L) for 8 weeks in three replicates (n = 7 fish per tank), alongside a control group (C) without APAP. APAP-exposed fish spent significantly (p < .05) longer time to detect food (T1: 32.6 ± 4.55 s and T2: 39.6 ± 4.66 s) compared to the control group (19.9 ± 2.46 s). Both APAP-exposed groups exhibited attenuated physical avoidance responses (76.7%, 68.7%, and 87.3% in T1, T2, and C, respectively) and a lower mean ventilation rate compared to the control group (194.5 ± 15.5, 179.1 ± 11.6, and 233.2 ± 19.0 per min in T1, T2, and C, respectively). The frequency occurrence of ENA types such as bi-nucleated, notched nuclei, lobed nuclei, and blebbed nuclei (except micronuclei) was significantly higher (p < .05) in APAP-exposed groups compared to the control, with more pronounced effects in the T2 group. The study concludes that APAP exposure prompts significant alterations in behavior and erythrocyte nuclear morphology, emphasizing the value of monitoring and regulating the entry of pharmaceuticals, including APAP, into aquatic environments to prevent unintended effects on non-target organisms like Oreochromis niloticus.

Phylogenetic and Structure-Function Analyses of ENA ATPases: A Case Study of the ENA1 Protein from the Fungus Neurospora crassa.

ENA transporters are a group of P-type ATPases that are characterized by actively moving Na+ or K+ out of the cell against their concentration gradient. The existence of these transporters was initially attributed to some fungi, although more recently they have also been identified in mosses, liverworts, and some protozoa. Given the current increase in the number of organisms whose genomes are completely sequenced, we set out to expand our knowledge about the existence of ENA in organisms belonging to other phylogenetic groups. For that, a hidden Markov model profile was constructed to identify homologous sequences to ENA proteins in protein databases. This analysis allowed us to identify the existence of ENA-type ATPases in the most primitive groups of fungi, as well as in other eukaryotic organisms not described so far. In addition, this study has allowed the identification of a possible new group of P-ATPases, initially proposed as ENA but which maintain phylogenetic distances with these proteins. Finally, this work has also addressed this study of the structure of ENA proteins, which remained unknown due to the lack of crystallographic data. For this purpose, a 3D structure prediction of the NcENA1 protein of the fungus Neurospora crassa was performed using AlphaFold2 software v2.3.1. From this structure, the electrostatic potential of the protein was analyzed. With all these data, the protein regions and the amino acids involved in the transport of Na+ or K+ ions across the membrane were proposed for the first time. Targeted mutagenesis of some of these residues has confirmed their relevant participation in the transport function of ENA proteins.

The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalization.

It is well known that in addition to its classical role in protein turnover, ubiquitylation is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Our results indicate that the yeast Na+ pump Ena1 is an epsin (Ent1 and Ent2 in yeast)-specific cargo and that its internalization requires K1090, which likely undergoes Art3-dependent ubiquitylation. In addition, an Ena1 serine and threonine (ST)-rich patch, proposed to be targeted for phosphorylation by casein kinases, was also required for its uptake. Interestingly, our data suggest that this phosphorylation was not needed for cargo ubiquitylation. Furthermore, epsin-mediated internalization of Ena1 required a specific spatial organization of the ST patch with respect to K1090 within the cytoplasmic tail of the pump. We hypothesize that ubiquitylation and phosphorylation of Ena1 are required for epsin-mediated internalization.

An Actin-, Cortactin- and Ena-VASP-Linked Complex Contributes to Endothelial Cell Focal Adhesion and Vascular Barrier Regulation.

BACKGROUND/AIMS: Increase in vascular permeability is a cardinal feature of all inflammatory diseases and represents an imbalance in vascular contractile forces and barrier-restorative forces, both of which are highly dependent on actin cytoskeletal dynamics. In addition to the involvement of key vascular barrier-regulatory, actin-binding proteins, such as nmMLCK and cortactin, we recently demonstrated a role for a member of the Ena-VASP family known as Ena-VASP-like (EVL) in promoting vascular focal adhesion (FA) remodeling and endothelial cell (EC) barrier restoration/preservation. METHODS: To further understand the role of EVL in EC barrier-regulatory processes, we examined EVL-cytoskeletal protein interactions in FA dynamics in vitro utilizing lung EC and in vivo murine models of acute inflammatory lung injury. Deletion mapping studies and immunoprecipitation assays were performed to detail the interaction between EVL and cortactin, and further evaluated by assessment of changes in vascular EC permeability following disruption of EVL-cortactin interaction. RESULTS: Initial studies focusing on the actin-binding proteins, nmMLCK and cortactin, utilized deletion mapping of the cortactin gene (CTTN) to identify cortactin domains critical for EVL-cortactin interaction and verified the role of actin in promoting EVL-cortactin interaction. A role for profilins, actin-binding proteins that regulate actin polymerization, was established in facilitating EVL-FA binding. CONCLUSION: In summary, these studies further substantiate EVL participation in regulation of vascular barrier integrity and in the highly choreographed cytoskeletal interactions between key FA and cytoskeletal partners.

Fatal haemolytic transfusion reaction due to anti-Ena and identification of a novel GYPA c.295delG variant in a Thai family.

BACKGROUND AND OBJECTIVES: High-frequency antigen Ena (MNS 28) is expressed on glycophorin A (GPA). En(a-) individuals can form anti-Ena when exposed to GPA. A Thai patient formed an antibody that reacted against all reagent red blood cells (RBCs). The patient received incompatible blood resulting in a fatal haemolytic transfusion reaction (HTR). This study aimed to characterize the antibody detected in the patient and investigate the cause of HTR. MATERIALS AND METHODS: Blood samples from the patient and three of his family members were investigated. Massively parallel sequencing (MPS) and DNA-microarray were used for genotyping. Standard haemagglutination techniques were used for phenotyping and antibody investigations. RESULTS: DNA sequencing showed the patient was homozygous for GYPA*M c.295delG (p.Val99Ter) predicting En(a-). Three family members were heterozygous for GYPA c.295delG. MPS and DNA-microarray predicted the patient was N- discordant with the N+ RBC phenotype. The patient's plasma was positive with enzyme/chemical-treated reagent RBCs but failed to react with En(a-) and Mk Mk RBCs. CONCLUSION: The GYPA c.295delG variant prevented GPA expression on RBCs resulting in En(a-) phenotype. The N+ phenotype result was probably due to the anti-N typing reagent detecting 'N' (MNS30) on GPB. The patient's alloantibody has anti-Ena specificity.

Determination of pediatric reference limits for 10 commonly measured autoantibodies.

OBJECTIVES: The objective of this study was to establish pediatric reference limits for autoimmune disease markers in the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) cohort of healthy children and adolescents to support their interpretation and clinical decision making. The CALIPER is a national study of healthy children aiming to close gaps in pediatric laboratory medicine by establishing a robust database of pediatric reference intervals for pediatric disease biomarkers (caliperdatabase.org). METHODS: Healthy children and adolescents (n=123, aged 1-19) were recruited to CALIPER with informed consent. Serum autoantibody testing conducted on the BIO-FLASH analyzer (Werfen, Barcelona, Spain) included anti-dsDNA IgG, anti-Sm IgG, anti-RNP IgG, anti-SSB/La IgG, anti-Ro60 IgG, anti-Ro52 IgG, anti-cardiolipin IgG, anti-MPO IgG, anti-PR3 IgG, and anti-tTG IgA. Pediatric reference limits representing 95th, 97.5th, and 99th percentiles were calculated using the non-parametric rank method according to Clinical Laboratory Standards Institute C28-A3 guidelines. RESULTS: The proportion of samples with results above the lower limit of the analytical measuring range were: anti-cardiolipin IgG 90%, anti-dsDNA 22%, anti-Sm 13%, anti-RNP 0.8%, anti-SSB/La 0%, anti-Ro60 0%, anti-Ro52 0%, anti-MPO 25%, anti-PR3 9%, and anti-tTG IgA 28%. Pediatric reference limits and associated 90% confidence intervals were established for all 10 markers. All autoantibodies could be described by one age range except for anti-cardiolipin IgG and anti-MPO. A sex-specific difference was identified for anti-tTG IgA. CONCLUSIONS: Robust pediatric reference limits for 10 commonly clinically utilized autoimmune markers established herein will allow for improved laboratory assessment and clinical decision making in pediatric patients using the BIO-FLASH assay platform worldwide.

Capping protein is dispensable for polarized actin network growth and actin-based motility.

Heterodimeric capping protein (CP) binds the rapidly growing barbed ends of actin filaments and prevents the addition (or loss) of subunits. Capping activity is generally considered to be essential for actin-based motility induced by Arp2/3 complex nucleation. By stopping barbed end growth, CP favors nucleation of daughter filaments at the functionalized surface where the Arp2/3 complex is activated, thus creating polarized network growth, which is necessary for movement. However, here using an in vitro assay where Arp2/3 complex-based actin polymerization is induced on bead surfaces in the absence of CP, we produce robust polarized actin growth and motility. This is achieved either by adding the actin polymerase Ena/VASP or by boosting Arp2/3 complex activity at the surface. Another actin polymerase, the formin FMNL2, cannot substitute for CP, showing that polymerase activity alone is not enough to override the need for CP. Interfering with the polymerase activity of Ena/VASP, its surface recruitment or its bundling activity all reduce Ena/VASP's ability to maintain polarized network growth in the absence of CP. Taken together, our findings show that CP is dispensable for polarized actin growth and motility in situations where surface-directed polymerization is favored by whatever means over the growth of barbed ends in the network.

Renadirsen, a Novel 2'OMeRNA/ENA® Chimera Antisense Oligonucleotide, Induces Robust Exon 45 Skipping for Dystrophin In Vivo.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients' DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2'-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2'OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.

Ena orchestrates remodelling within the actin cytoskeleton to drive robust Drosophila macrophage chemotaxis.

The actin cytoskeleton is the engine that powers the inflammatory chemotaxis of immune cells to sites of tissue damage or infection. Here, we combine genetics with live in vivo imaging to investigate how cytoskeletal rearrangements drive macrophage recruitment to wounds in Drosophila We find that the actin-regulatory protein Ena is a master regulator of lamellipodial dynamics in migrating macrophages, where it remodels the cytoskeleton to form linear filaments that can then be bundled together by the cross-linker Fascin (also known as Singed in flies). In contrast, the formin Dia generates rare, probing filopods for specialised functions that are not required for migration. The role of Ena in lamellipodial bundling is so fundamental that its overexpression increases bundling even in the absence of Fascin by marshalling the remaining cross-linking proteins to compensate. This reorganisation of the lamellipod generates cytoskeletal struts that push against the membrane to drive leading edge advancement and boost cell speed. Thus, Ena-mediated remodelling extracts the most from the cytoskeleton to power robust macrophage chemotaxis during their inflammatory recruitment to wounds.

Non-organ-specific autoimmunity in adult 47,XXY Klinefelter patients and higher-grade X-chromosome aneuploidies.

Current literature regarding systemic autoimmune diseases in X-chromosome aneuploidies is scarce and limited to case reports. Our aim was to evaluate the frequency of anti-nuclear (ANAs), extractable nuclear (ENA), anti-double-stranded DNA (dsDNAs), anti-smooth muscle (ASMAs) and anti-mitochondrial (AMAs) antibodies in a large cohort of adults with Klinefelter's syndrome (KS, 47,XXY) and rare higher-grade sex chromosome aneuploidies (HGAs) for the first time. Sera from 138 X-chromosome aneuploid patients [124 adult patients with 47,XXY KS and 14 patients with HGA (six children, eight adults)] and 50 age-matched 46,XY controls were recruited from the Sapienza University of Rome (2007-17) and tested for ANAs, ENAs, anti-dsDNAs, ASMAs and AMAs. Non-organ-specific immunoreactivity was found to be significantly higher in patients with 47,XXY KS (14%) than in the controls (2%, p = 0.002). Among all the antibodies investigated, only ANAs were observed significantly more frequently in patients with 47,XXY KS (12.1%) than in the controls (2%, p = 0.004). No anti-dsDNA immunoreactivity was found. Stratifying by testosterone replacement therapy (TRT), non-organ-specific autoantibody frequencies were higher in TRT-naive (p = 0.01) and TRT-treated groups than in controls. No patients with HGA were found positive for the various autoantibodies. Non-organ-specific autoantibodies were significantly present in 47,XXY adult patients. Conversely, HGAs did not appear to be target of non-organ-specific immunoreactivity, suggesting that KS and HGAs should be considered as two distinct conditions. The classification and diagnosis of systemic autoimmune diseases is frequently difficult. To support a correct clinical evaluation of KS disease and to prevent eventual secondary irreversible immune-mediated damages, we highlight the importance of screening for non-organ-specific autoimmunity in Klinefelter's syndrome.

Enigmatic Pilus-Like Endospore Appendages of Bacillus cereus Group Species.

The endospores (spores) of many Bacillus cereus sensu lato species are decorated with multiple hair/pilus-like appendages. Although they have been observed for more than 50 years, all efforts to characterize these fibers in detail have failed until now, largely due to their extraordinary resilience to proteolytic digestion and chemical solubilization. A recent structural analysis of B. cereus endospore appendages (Enas) using cryo-electron microscopy has revealed the structure of two distinct fiber morphologies: the longer and more abundant "Staggered-type" (S-Ena) and the shorter "Ladder-like" type (L-Ena), which further enabled the identification of the genes encoding the S-Ena. Ena homologs are widely and uniquely distributed among B. cereus sensu lato species, suggesting that appendages play important functional roles in these species. The discovery of ena genes is expected to facilitate functional studies involving Ena-depleted mutant spores to explore the role of Enas in the interaction between spores and their environment. Given the importance of B. cereus spores for the food industry and in medicine, there is a need for a better understanding of their biological functions and physicochemical properties. In this review, we discuss the current understanding of the Ena structure and the potential roles these remarkable fibers may play in the adhesion of spores to biotic and abiotic surfaces, aggregation, and biofilm formation.

Rounding Out the Understanding of ACD Toxicity with the Discovery of Cyclic Forms of Actin Oligomers.

Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays.

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASP-mediated actin assembly in clustered arrays.