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Numerous studies using a combination of confocal microscopic- and pharmacological-based approaches have demonstrated that the actin cytoskeleton dynamically responds to pathogen infection. Here, we observed that phalloidin treatment induced actin nucleation, resulting in enhanced resistance of wheat against the stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst). To define the mechanism underpinning this process, we characterized a family of conserved actin-binding proteins, the actin related protein (ARP) family, which controls actin polymerization. Specifically, we identified and characterized a wheat ARPC gene (TaARPC5), which encodes a 136-amino acid protein containing a P16-Arc domain, the smallest subunit of the ARP2/3 complex. TaARPC5 mRNA accumulation was induced following the infection of plants with the avirulent Pst strain, and following the elicitation with flagellin (e.g., flg22) as well. Subcellular localization analysis revealed that TaARPC5 is primarily localized to the cortical actin cytoskeleton, and its precise cellular localizations suggest the proximity to processes correlated with the actin-organelle interface. Upon treatment with virulent Pst, TaARPC5-knockdown plants exhibited a significant reduction in the expression of PTI-specific mRNAs. Conversely, we observed enhanced induction of reactive oxygen species (ROS) accumulation and a decrease in TaCAT1 expression following infection with an incompatible Pst isolate. Together with yeast complementation assays, the current study demonstrates a role for TaARPC5 in resistance signaling in wheat against Pst infection by regulating the host actin cytoskeleton.

The assembly of macromolecules on the plasma membrane concentrates cell surface biomolecules into nanometer- to micrometer-scale clusters (nano- or microdomains) that help the cell initiate or respond to signals. In plant-microbe interactions, the actin cytoskeleton undergoes rapid remodeling during pathogen-associated molecular pattern-triggered immunity (PTI). The nanoclustering of formin-actin nucleator proteins at the cell surface has been identified as underlying actin nucleation during plant innate immune responses. Here, we show that the condensation of nanodomain constituents and the self-assembly of remorin proteins enables this mechanism of controlling formin condensation and activity during innate immunity in Arabidopsis thaliana. Through intrinsically disordered region-mediated remorin oligomerization and formin interaction, remorin gradually recruits and condenses formins upon PTI activation in lipid bilayers, consequently increasing actin nucleation in a time-dependent manner postinfection. Such nanodomain- and remorin-mediated regulation of plant surface biomolecules is expected to be a general feature of plant innate immune responses that creates spatially separated biochemical compartments and fine tunes membrane physicochemical properties for transduction of immune signals in the host. Ministry of Education (MOE) Nanyang Technological University This study was supported by the National Science Foundation of China (Grants 31870170) to Xu Chen, the Nanyang Technological University (NTU) Singapore startup grant (M4081533) and Singapore Ministry of Education (MOE) Tier 1 (RG32/20) and Tier 3 (MOE2019-T3-1-012) funding awarded to Yansong Miao.

C1q tumor necrosis factor‐related peptide 8 (CTRP8) is the least studied member of the C1Q‐TNF‐related peptide family. We identified CTRP8 as a ligand of the G protein‐coupled receptor relaxin family peptide receptor 1 (RXFP1) in glioblastoma multiforme (GBM). The CTRP8‐RXFP1 ligand–receptor system protects human GBM cells against the DNA‐alkylating damage‐inducing temozolomide (TMZ), the drug of choice for the treatment of patients with GBM. The DNA protective role of CTRP8 was dependent on a functional RXFP1‐STAT3 signaling cascade and targeted the monofunctional glycosylase N‐methylpurine DNA glycosylase (MPG) for more efficient base excision repair of TMZ‐induced DNA‐damaged sites. CTRP8 also improved the survival of GBM cells by upregulating anti‐apoptotic BCl‐2 and BCL‐XL. Here, we have identified Janus‐activated kinase 3 (JAK3) as a novel member of a novel CTRP8‐RXFP1‐JAK3‐STAT3 signaling cascade that caused an increase in cellular protein content and activity of the small Rho GTPase Cdc42. This is associated with significant F‐actin remodeling and increased GBM motility. Cdc42 was critically important for the upregulation of the actin nucleation complex N‐Wiskott–Aldrich syndrome protein/Arp3/4 and actin elongation factor profilin‐1. The activation of the RXFP1‐JAK3‐STAT3‐Cdc42 axis by both RXFP1 agonists, CTRP8 and relaxin‐2, caused extensive filopodia formation. This coincided with enhanced activity of ezrin, a key factor in tethering F‐actin to the plasma membrane, and inhibition of the actin filament severing activity of cofilin. The F‐actin remodeling and pro‐migratory activities promoted by the novel RXFP1‐JAK3‐STAT3‐Cdc42 axis were blocked by JAK3 inhibitor tofacitinib and STAT3 inhibitor STAT3 inhibitor VI. This provides a new rationale for the design of JAK3 and STAT3 inhibitors with better brain permeability for clinical treatment of the pervasive brain invasiveness of GBM., The C1Q‐TNF‐related peptide family member C1q tumor necrosis factor‐related peptide 8 (CTRP8) and its G protein‐coupled receptor relaxin family peptide receptor 1 (RXFP1) are emerging as a novel ligand–receptor system that can protect glioblastoma cells against DNA‐alkylating damage and apoptosis. We describe a novel CTRP8‐RXFP1‐Janus‐activated kinase 3‐STAT3 signaling cascade that enhances cellular protein content and activity of small Rho GTPase Cdc42, which directs F‐actin cytoskeletal remodeling, filopodia formation, and increased glioblastoma multiforme migration.

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. WH2 domains are short sequence motifs found in many different actin binding proteins including WASP family proteins which regulate the actin nucleating complex Arp2/3. In this study we reveal a phosphorylation site, Serine 554, within the WH2 domain of the yeast WASP homologue Las17. Both phosphorylation and a phospho-mimetic mutation reduce actin monomer binding affinity while an alanine mutation, generated to mimic the non-phosphorylated state, increases actin binding affinity. The effect of these mutations on the Las17-dependent process of endocytosis in vivo was analysed and leads us to propose that switching of Las17 phosphorylation states may allow progression through distinct phases of endocytosis from site assembly through to the final scission stage. While the study is focused on Las17, the sole WASP family protein in yeast, our results have broad implications for our understanding of how a key residue in this conserved motif can underpin the many different actin regulatory roles with which WH2 domains have been associated.

Proteinaceous aggregates containing α-synuclein protein called Lewy bodies in the substantia nigra is a hallmark of Parkinson's disease. The molecular mechanisms of Lewy body formation and associated neuronal loss remain largely unknown. To gain insights into proteins and pathways associated with Lewy body pathology, we performed quantitative profiling of the proteome. We analyzed substantia nigra tissue from 51 subjects arranged into three groups: cases with Lewy body pathology, Lewy body-negative controls with matching neuronal loss, and controls with no neuronal loss. Using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we characterized the proteome both in terms of protein abundances and peptide modifications. Statistical testing for differential abundance of the most abundant 2963 proteins, followed by pathway enrichment and Bayesian learning of the causal network structure, was performed to identify likely drivers of Lewy body formation and dopaminergic neuronal loss. The identified pathways include (1) Arp2/3 complex-mediated actin nucleation; (2) synaptic function; (3) poly(A) RNA binding; (4) basement membrane and endothelium; and (5) hydrogen peroxide metabolic process. According to the data, the endothelial/basement membrane pathway is tightly connected with both pathologies and likely to be one of the drivers of neuronal loss. The poly(A) RNA-binding proteins, including the ones relevant to other neurodegenerative disorders (e.g., TDP-43 and FUS), have a strong inverse correlation with Lewy bodies and may reflect an alternative mechanism of nigral neurodegeneration.

Corneal wound healing depends on extracellular matrix (ECM) and topographical cues that modulate migration and proliferation of regenerating cells. In our study, silk films with either flat or nanotopography patterned parallel ridge widths of 2000, 1000, 800 nm surfaces were combined with ECMs which include collagen type I (collagen I), fibronectin, laminin, and Poly-d-Lysine to accelerate corneal wound healing. Silk films with 800 nm ridge width provided better cell spreading and wound recovery than other size topographies. Coating 800 nm patterned silk films with collagen I proves to optimally further increased mouse and rabbit corneal epithelial cells growth and wound recovery. This enhanced cellular response correlated with redistribution and increase in size and total amount of focal adhesion. Transcriptomics and signaling pathway analysis suggested that silk topography regulates cell behaviors via actin nucleation ARP-WASP complex pathway, which regulate filopodia formation. This mechanism was further explored and inhibition of Cdc42, a key protein in this pathway, delayed wound healing and decreased the length, density, and alignment of filopodia. Inhibition of Cdc42 in vivo resulted in delayed re-epithelization of injured corneas. We conclude that silk film nanotopography in combination with collagen I constitutes a better substrate for corneal wound repair than either nanotopography or ECM alone.

SummaryMicroglia actively promote the growth of high-grade gliomas. Within the glioma microenvironment an activated (amoeboid) microglial morphology has been observed, however the underlying causes and the related impact on microglia functions and their tumour promoting activities is unclear. Using the advantages of the larval zebrafish model, we demonstrate that pre-neoplastic glioma cells have an immediate impact on microglial morphology and functions. Overexpression of human HRasV12 in proliferating domains of the larval brain induces an amoeboid morphology of microglia, increases microglial numbers and decreases their motility and phagocytic activity. RNA sequencing analysis revealed lower expression levels of the actin nucleation promoting factor wasla in microglia. Importantly, a microglia specific rescue of wasla expression restores microglial morphology and functions. This results in increased phagocytosis of pre-neoplastic cells and slows down tumour progression. In conclusion, we identified a mechanism that de-activates core microglial functions within the emerging glioma microenvironment.

WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is involved in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation-promoting factor, which activates the actin-related protein 2/3 complex (the Arp2/3 complex). However, the role of WHAMM in mammalian oocyte maturation is poorly understood. The presence of WHAMM mRNA and protein during all stages of mouse oocyte maturation has been verified. It is mainly co-localized with the actin cage permeating the spindle during mouse oocyte maturation. Through the knockdown of WHAMM, we confirmed that it regulates spindle formation and affects the localization of the microtubule-organizing center (MTOC) during the early stages of spindle formation. Moreover, depletion of WHAMM impaired the formation of the spindle actin and chromosome alignment, which might be the cause of chromosomal aneuploidy and abnormal, asymmetric division. Treatment with brefeldin A (BFA), an inhibitor of vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus, induced abnormal and dispersed localization of WHAMM. Taken together, these findings show that WHAMM is an essential component of the actin cytoskeleton machinery and plays a crucial role in oocyte maturation, presumably by controlling the formation of spindles with normal length by activating the formation of the spindle actin via the Arp2/3 complex.

Candida albicans is a dimorphic fungus that converts from a yeast form to a hyphae form during infection. This switch requires the formation of actin cable to coordinate polarized cell growth. It's known that nucleation of this cable requires a multiprotein complex localized at the tip called the polarisome, but the mechanisms underpinning this process were unclear. Here, we found that C. albicans Aip5, a homolog of polarisome component ScAip5 in Saccharomyces cerevisiae that nucleates actin polymerization and synergizes with the formin ScBni1, regulates actin assembly and hyphae growth synergistically with other polarisome proteins Bni1, Bud6, and Spa2. The C terminus of Aip5 binds directly to G-actin, Bni1, and the C-terminal of Bud6, which form the core of the nucleation complex to polymerize F-actin. Based on insights from structural biology and molecular dynamic simulations, we propose a possible complex conformation of the actin nucleation core, which provides cooperative positioning and supports the synergistic actin nucleation activity of a tri-protein complex Bni1-Bud6-Aip5. Together with known interactions of Bni1 with Bud6 and Aip5 in S. cerevisiae, our findings unravel molecular mechanisms of C. albicans by which the tri-protein complex coordinates the actin nucleation in actin cable assembly and hyphal growth, which is likely a conserved mechanism in different filamentous fungi and yeast.

Weibel–Palade bodies (WPB) are endothelial cell-specific storage granules that regulate vascular hemostasis by releasing the platelet adhesion receptor von Willebrand factor (VWF) following stimulation. Fusion of WPB with the plasma membrane is accompanied by the formation of actin rings or coats that support the expulsion of large multimeric VWF fibers. However, factor(s) organizing these actin ring structures have remained elusive. We now identify the actin-binding proteins Spire1 and Myosin Vc (MyoVc) as cytosolic factors that associate with WPB and are involved in actin ring formation at WPB-plasma membrane fusion sites. We show that both, Spire1 and MyoVc localize only to mature WPB and that upon Ca2+ evoked exocytosis of WPB, Spire1 and MyoVc together with F-actin concentrate in ring-like structures at the fusion sites. Depletion of Spire1 or MyoVc reduces the number of these actin rings and decreases the amount of VWF externalized to the cell surface after histamine stimulation. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-04108-x.

Actin nucleation is achieved by collaborative teamwork of actin nucleator factors (NFs) and nucleation-promoting factors (NPFs) into functional protein complexes. Selective inter- and intramolecular interactions between the nucleation complex constituents enable diverse modes of complex assembly in initiating actin polymerization on demand. Budding yeast has two formins, Bni1 and Bnr1, which are teamed up with different NPFs. However, the selective pairing between formin NFs and NPFs into the nucleation core for actin polymerization is not completely understood. By examining the functions and interactions of NPFs and NFs via biochemistry, genetics, and mathematical modeling approaches, we found that two NPFs, Aip5 and Bud6, showed joint teamwork effort with Bni1 and Bnr1, respectively, by interacting with the C-terminal intrinsically disordered region (IDR) of formin, in which two NPFs work together to promote formin-mediated actin nucleation. Although the C-terminal IDRs of Bni1 and Bnr1 are distinct in length, each formin IDR orchestrates the recruitment of Bud6 and Aip5 cooperatively by different positioning strategies to form a functional complex. Our study demonstrated the dynamic assembly of the actin nucleation complex by recruiting multiple partners in budding yeast, which may be a general feature for effective actin nucleation by formins. Ministry of Education (MOE) Nanyang Technological University Skin Research Institute of Singapore (SRIS) Published version This study was supported by a NTU startup grant (M4081533), Skin Research Institute of Singapore (SRIS, SIG18002), and MOE Tier 2 (MOE2016-T2-1-005), MOE Tier 3 (MOE2019-T3-1-012) to Y.M., and MOE Tier 1 (2018-T1-001-096) to L.L. in Singapore.

Introduction: The Spire protein is an actin nucleator protein that drives actin nucleation by its WH-2 domains, it is present as two isoforms in mammals. Since actin cytoskeletal function is determined by the mechanism of action nucleation, we aimed to determine the immunolocalization and protein expression of Spire-2 protein in different stages of folliculogenesis within bovine ovaries. Methods: Four bovine ovaries were obtained from slaughterhouse. Histological sections were obtained through formalin fixation and paraffin embedding. The tissue architecture was determined by the hematoxylin and eosin staining. Immunolocalization of Spire-2 protein in different ovarian follicular stages was evaluated. The fluorescent images were analyzed via ImageJ software. Results: Spire-2 immunostaining was intense in oocytes compared to granulosa of the ovarian follicles, and ImageJ analysis revealed the dominance of Spire-2 within the oocytes. And within different stages of ovaries, Spire-2 level was found to be the highest in Graafian follicles when compared to primary and secondary follicles, which was also revealed using ImageJ analysis. Conclusion: This study demonstrates that Spire-2 protein is expressed within many components of bovine ovaries, particularly within the oocyte and granulosa cells of the follicle. Therefore, we suggest that Spire-2 protein might have unique functions in bovine ovaries related to folliculogenesis, asymmetric oocyte division, microtubule organization, and steroidogenesis.

FoxM1 activates genes that regulate S-G2-M cell-cycle progression and, when overexpressed, is associated with poor clinical outcome in multiple cancers. Here we identify FoxM1 as a tumor suppressor in mice that, through its N-terminal domain, binds to and inhibits Ect2 to limit the activity of RhoA GTPase and its effector mDia1, a catalyst of cortical actin nucleation. FoxM1 insufficiency impedes centrosome movement through excessive cortical actin polymerization, thereby causing the formation of non-perpendicular mitotic spindles that missegregate chromosomes and drive tumorigenesis in mice. Importantly, low FOXM1 expression correlates with RhoA GTPase hyperactivity in multiple human cancer types, indicating that suppression of the newly discovered Ect2-RhoAmDia1 oncogenic axis by FoxM1 is clinically relevant. Furthermore, by dissecting the domain requirements through which FoxM1 inhibits Ect2 GEF activity, we provide mechanistic insight for the development of pharmacological approaches that target protumorigenic RhoA activity.

SummaryDNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2–Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P2 containing membranes and dimerize in a protein density dependent manner. Although dispensable for PI(4,5)P2 binding and lipid kinase activity, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P2 and membrane bound kinase.

Cancer cells depend on actin cytoskeleton rearrangement to carry out hallmark malignant functions including activation, proliferation, migration and invasiveness. Wiskott–Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor and is a key regulator of actin polymerization in hematopoietic cells. The involvement of WASp in malignancies is incompletely understood. Since WASp is exclusively expressed in hematopoietic cells, we performed in silico screening to identify small molecule compounds (SMCs) that bind WASp and promote its degradation. We describe here one such identified molecule; this WASp-targeting SMC inhibits key WASp-dependent actin processes in several types of hematopoietic malignancies in vitro and in vivo without affecting naïve healthy cells. This small molecule demonstrates limited toxicity and immunogenic effects, and thus, might serve as an effective strategy to treat specific hematopoietic malignancies in a safe and precisely targeted manner., Cancer cells proliferate and invade via cytoskeletal proteins such as WASp, exclusively expressed in hematopoietic cells. Here the authors show a specific small molecule compound inhibiting cancer cell activity by WASp degradation and demonstrating its therapeutic potential in vitro and in vivo.

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress and protect stalled replication forks. All such responses rely on the formation of Replication Protein A (RPA)-ssDNA complexes, yet supra-physiological binding of RPA to ssDNA is toxic. How cells regulate RPA availability to promote fork protection and genome stability is largely unknown. Here we establish that during replication excess RPA is sequestered by monomeric actin and released upon replicative stress through transition to polymeric actin state. Impairment in actin nucleation leads to RPA sequestration, deprotection of ssDNA generated at the stressed forks and consequently, catastrophic fork collapse and hypersensitivity to replication inhibitors. In line with this, we show that increasing RPA load is sufficient to restore efficient fork protection in actin polymerization mutants. Collectively, this work identifies a simple yet robust RPA-buffering mechanism regulating its availability to bind ssDNA and protect replication forks against nucleolytic degradation. Inhibition of this pathway could be of therapeutic interest in treatment of cancers.

BackgroundCytoskeletal remodelling plays a pivotal role in the establishment of neuronal connectivity during development and in plasticity in adults. Mutations in the cytoskeleton regulatory protein Formin-2 (Fmn2) are associated with neurodevelopmental disorders like intellectual disability, though its function in neuronal morphogenesis has not been characterised in vivo.ResultsHere we develop a loss-of-function model for fmn2b, the zebrafish orthologue of Fmn2, using CRISPR/Cas9-mediated gene editing. fmn2b mutants display motor deficits starting from the earliest motor responses in the embryo. We find that fmn2b is expressed in spinal motor neurons and its loss reduces motor neuron innervation of the axial muscles without affecting myotome integrity. The translocation of caudal primary (CaP) motor neuron outgrowth is compromised in fmn2b mutants, while rostral primary (RoP) motor neurons have missing soma or stall at the horizontal myoseptum. Strikingly, axon collateral branching of the motor neurons is severely compromised and results in reduced synaptic coverage of the myotome. Rescue experiments identify the requirement for Fmn2-mediated actin nucleation for motor neuron outgrowth and arborisation.ConclusionsThe zebrafish loss-of-function model of Fmn2 reveals the specific requirement of F-actin polymerisation by Fmn2 in neuromuscular development. It also underscores the role of Fmn2 in motor neuropathies, especially as a proportion of individuals harbouring mutations in Fmn2 present with hypotonia.

SCAR/WAVE proteins and Arp2/3 complex assemble branched actin networks at the leading edge. Two isoforms of SCAR/WAVE, WAVE1 and WAVE2, reside at the leading edge, yet it has remained unclear whether they perform similar or distinct roles. Further, there have been conflicting reports about the Arp2/3-independent biochemical activities of WAVE1 on actin filament elongation. To investigate this in vivo, we knocked out WAVE1 and WAVE2 genes, individually and together, in B16-F1 melanoma cells. We demonstrate that WAVE1 and WAVE2 are redundant for lamellipodia formation and motility. However, there is a significant decrease in the rate of leading edge actin extension in WAVE2 KO cells, and an increase in WAVE1 KO cells. The faster rates of actin extension in WAVE1 KO cells are offset by faster retrograde flow, and therefore do not translate into faster lamellipodium protrusion. Thus, WAVE1 restricts the rate of actin extension at the leading edge, and appears to couple actin networks to the membrane to drive protrusion. Overall, these results suggest that WAVE1 and WAVE2 have redundant roles in promoting Arp2/3-dependent actin nucleation and lamellipodia formation, but distinct roles in controlling actin network extension and harnessing network growth to cell protrusion.

Migration of meiosis-I (MI) spindle from the cell center to a sub-cortical location is a critical step for mouse oocytes to undergo asymmetric meiotic cell division. In this study, we investigate the mechanism by which formin-2 (FMN2) orchestrates the initial movement of MI spindle. By defining protein domains responsible for targeting FMN2, we show that spindle-periphery localized FMN2 is required for spindle migration. The spindle-peripheral FMN2 nucleates short actin bundles from vesicles derived likely from the endoplasmic reticulum (ER) and concentrated in a layer outside the spindle. This layer is in turn surrounded by mitochondria. A model based on polymerizing actin filaments pushing against mitochondria, thus generating a counter force on the spindle, demonstrated an inherent ability of this system to break symmetry and evolve directional spindle motion. The model is further supported through experiments involving spatially biasing actin nucleation via optogenetics and disruption of mitochondrial distribution and dynamics., Mammalian oocytes divide asymmetrically during meiotic maturation. Here, the authors show that spindle movement away from oocyte center depends on actin filaments nucleated from the spindle periphery pushing against surrounding mitochondria, which polarizes spontaneously to produce directional spindle motion.

Profilins are abundant cytosolic proteins that are universally expressed in eukaryotes and that regulate actin filament elongation by binding to both monomeric actin (G-actin) and formin proteins. The atypical profilin Arabidopsis AtPRF3 has been reported to cooperate with canonical profilin isoforms in suppressing formin-mediated actin polymerization during plant innate immunity responses. AtPRF3 has a 37-amino acid-long N-terminal extension (NTE), and its suppressive effect on actin assembly is derived from enhanced interaction with the polyproline (Poly-P) of the formin AtFH1. However, the molecular mechanism remains unclear. Here, we solved the crystal structures of AtPRF3Δ22 and AtPRF3Δ37, as well as AtPRF2 apo form and in complex with AtFH1 Poly-P at 1.5-3.6 A resolutions. By combining these structures with molecular modeling, we found that AtPRF3Δ22 NTE has high plasticity, with a primary "closed" conformation that can adopt an open conformation that enables Poly-P binding. Furthermore, using molecular dynamics simulation and free-energy calculations of protein-protein binding, along with experimental validation, we show that the AtPRF3Δ22 binds to Poly-P in an adaptive manner, thereby enabling different binding modes that maintain the interaction through disordered sequences. Together, our structural and simulation results suggest that the dynamic conformational changes of the AtPRF3 NTE upon Poly-P binding modulate their interactions to fine-tune formin-mediated actin assembly.

Protein recycling through the endolysosomal system relies on molecular assemblies that interact with cargo proteins, membranes, and effector molecules. Among them, the COMMD/CCDC22/CCDC93 (CCC) complex plays a critical role in recycling events. While CCC is closely associated with retriever, a cargo recognition complex, its mechanism of action remains unexplained. Herein we show that CCC and retriever are closely linked through sharing a common subunit (VPS35L), yet the integrity of CCC, but not retriever, is required to maintain normal endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P). CCC complex depletion leads to elevated PI(3)P levels, enhanced recruitment and activation of WASH (an actin nucleation promoting factor), excess endosomal F-actin and trapping of internalized receptors. Mechanistically, we find that CCC regulates the phosphorylation and endosomal recruitment of the PI(3)P phosphatase MTMR2. Taken together, we show that the regulation of PI(3)P levels by the CCC complex is critical to protein recycling in the endosomal compartment., Recycling of proteins that have entered the endosome is essential to homeostasis. The COMMD/CCDC22/CCDC93 (CCC) complex is regulator of recycling but the molecular mechanisms are unclear. Here, the authors report that the CCC complex regulates endosomal recycling by maintaining PI3P levels on endosomal membranes.

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)P2. 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.

Schrank and Gautier discuss the generation and function of nuclear domains during DNA repair with a special focus on nuclear actin polymerization., Eukaryotic nuclei are organized into nuclear domains that unite loci sharing a common function. These domains are essential for diverse processes including (1) the formation of topologically associated domains (TADs) that coordinate replication and transcription, (2) the formation of specialized transcription and splicing factories, and (3) the clustering of DNA double-strand breaks (DSBs), which concentrates damaged DNA for repair. The generation of nuclear domains requires forces that are beginning to be identified. In the case of DNA DSBs, DNA movement and clustering are driven by actin filament nucleators. Furthermore, RNAs and low-complexity protein domains such as RNA-binding proteins also accumulate around sites of transcription and repair. The link between liquid–liquid phase separation and actin nucleation in the formation of nuclear domains is still unknown. This review discusses DSB repair domain formation as a model for functional nuclear domains in other genomic contexts.

Formins are evolutionarily conserved eukaryotic proteins engaged in actin nucleation and other aspects of cytoskeletal organization. Angiosperms have two formin clades with multiple paralogs; typical plant Class I formins are integral membrane proteins that can anchor cytoskeletal structures to membranes. For the main Arabidopsis housekeeping Class I formin, FH1 (At3g25500), plasmalemma localization was documented in heterologous expression and overexpression studies. We previously showed that loss of FH1 function increases cotyledon epidermal pavement cell shape complexity via modification of actin and microtubule organization and dynamics. Here we employ transgenic Arabidopsis expressing green fluorescent protein-tagged FH1 (FH1-GFP) from its native promoter to investigate in vivo behaviour of this formin using advanced microscopy techniques. The fusion protein is functional, since its expression complements the fh1 loss-of-function mutant phenotype. Accidental overexpression of FH1-GFP results in a decrease in trichome branch number, while fh1 mutation has the opposite effect, indicating a general role of this formin in controlling cell shape complexity. Consistent with previous reports, FH1-GFP associates with membranes. However, the protein exhibits surprising actin- and secretory pathway-dependent dynamic localization and relocates between cellular endomembranes and the plasmalemma during cell division and differentiation in root tissues, with transient tonoplast localization at the transition/elongation zones border. FH1-GFP also accumulates in actin-rich regions of cortical cytoplasm and associates with plasmodesmata in both the cotyledon epidermis and root tissues. Together with previous reports from metazoan systems, this suggests that formins might have an ancestral role at cell-cell junctions.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is one of the leading global causes of virus-induced encephalitis. The infectious life-cycle of viruses is heavily dependent on the host membrane trafficking network. Here, we have performed a RNA-interference-based screen using a siRNA panel targeting 136 membrane trafficking proteins to identify the key regulators of JEV infection in HeLa cells. We identified 35 proteins whose siRNA depletion restricts JEV replication by over twofold. We observe that JEV infection in HeLa cells is largely dependent on components of the clathrin-mediated endocytic (CME) pathway. Proteins involved in actin-filament-based processes, specifically CDC42 and members of the ARP2/3 complex are crucial for establishment of infection. Pharmacological pertubations of actin polymerization, a small molecule inhibitor of actin nucleation by the ARP2/3 complex - CK-548 - and the inhibitor of neural Wiskott-Aldrich syndrome proteins- Wiskostatin- inhibited JEV replication, highlighting the important role of the dynamic actin network. Other proteins involved in cargo-recognition for CME and endomembrane system organization were also validated as essential host factors for virus replication.

Dynamic molecular clusters are assembled through weak multivalent interactions and are platforms for cellular functions, especially receptor-mediated signaling. Clustering is also a prerequisite for liquid-liquid phase separation. But it is not well understood how molecular structure and cellular organization control clustering. Using coarse-grain kinetic Langevin dynamics, we performed computational experiments on a prototypical ternary system modeled after membrane-bound nephrin, the adaptor Nck1 and the actin nucleation promoting factor NWASP. Steady state cluster size distributions favored stoichiometries that optimized binding (stoichiometry matching), but still were quite broad. At high concentrations, the system can be driven beyond the saturation boundary such that cluster size is limited only by the number of available molecules. This behavior would be predictive of phase separation. Domains close to binding sites sterically inhibited clustering much less than terminal domains because the latter effectively restrict access to the cluster interior. Increased flexibility of interacting molecules diminished clustering by shielding binding sites within compact conformations. Membrane association of nephrin increased the cluster size distribution in a density-dependent manner. These properties provide insights into how molecular ensembles function to localize and amplify cell signaling.

Postsynaptic structures on excitatory neurons, dendritic spines, are actin-rich. It is well known that actin-binding proteins regulate actin dynamics and by this means orchestrate structural plasticity during the development of the brain, as well as synaptic plasticity mediating learning and memory processes. The actin-binding protein cortactin is localized to pre- and postsynaptic structures and translocates in a stimulus-dependent manner between spines and the dendritic compartment, thereby indicating a crucial role for synaptic plasticity and neuronal function. While it is known that cortactin directly binds F-actin, the Arp2/3 complex important for actin nucleation and branching as well as other factors involved in synaptic plasticity processes, its precise role in modulating actin remodeling in neurons needs to be deciphered. In this study, we characterized the general neuronal function of cortactin in knockout mice. Interestingly, we found that the loss of cortactin leads to deficits in hippocampus-dependent spatial memory formation. This impairment is correlated with a prominent dysregulation of functional and structural plasticity. Additional evidence shows impaired long-term potentiation in cortactin knockout mice together with a complete absence of structural spine plasticity. These phenotypes might at least in part be explained by alterations in the activity-dependent modulation of synaptic actin in cortactin-deficient neurons.

SUMMARYThe assembly and maintenance of actin-based mechanosensitive stereocilia in the cochlea is critical for lifelong hearing. Myosin-15 (MYO15) is hypothesized to modulate stereocilia height by trafficking actin regulatory proteins to their tip compartments, where actin polymerization must be precisely controlled during development. We identified a mutation (p.D1647G) in the MYO15 motor-domain that initially maintained trafficking, but caused progressive hearing loss by stunting stereocilia growth, revealing an additional function for MYO15. Consistent with its maintenance of tip trafficking in vivo, purified p.D1647G MYO15 modestly reduced actin-stimulated ATPase activity in vitro. Using ensemble and single-filament fluorescence in vitro assays, we demonstrated that wild-type MYO15 directly accelerated actin filament polymerization by driving nucleation, whilst p.D1647G MYO15 blocked this activity. Collectively, our studies suggest direct actin nucleation by MYO15 at the stereocilia tip is necessary for elongation in vivo, and that this is a primary mechanism disrupted in DFNB3 hereditary human hearing loss.

The Scar/WAVE complex drives actin nucleation during cell migration. Interestingly, the same complex is important in forming membrane ruffles during macropinocytosis, a process mediating nutrient uptake and membrane receptor trafficking. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A, has not been characterized. Here, we implicate CYRI-A as a key regulator of macropinosome formation and integrin internalization. We find that CYRI-A is transiently recruited to nascent macropinosomes, dependent on PI3K and RAC1 activity. CYRI-A recruitment precedes RAB5A recruitment but follows sharply after RAC1 and actin signaling, consistent with it being a local inhibitor of actin polymerization. Depletion of both CYRI-A and -B results in enhanced surface expression of the α5β1 integrin via reduced internalization. CYRI depletion enhanced migration, invasion, and anchorage-independent growth in 3D. Thus, CYRI-A is a dynamic regulator of macropinocytosis, functioning together with CYRI-B to regulate integrin trafficking. ispartof: JOURNAL OF CELL BIOLOGY vol:220 issue:9 ispartof: location:United States status: published

The local rules that control cell shape and movement are not understood. Here, Pipathsouk et al. discover a novel nanoscale organization of a key actin regulator that could explain how cellular protrusions expand and self-straighten and how transcellular holes are repaired., How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.

ObjectiveTo clarify the clinical relevance of WASP like actin nucleation promoting factor (WASL) in patients with cervical cancer and associated mechanisms.Methods and MaterialsWe obtained high prediction accuracy and determined the correlation between the expression of WASL and the clinical characteristics of cervical cancer patients. Differentially expressed genes (DEGs) were identified using microarray. Gene ontology (GO) enrichment analysis and gene set enrichment analysis (GSEA) were performed to determine potentially relevant mechanisms related to the prognostication ability of WASL expression.ResultsChi-square test and multivariable logistic regression analysis suggested that lower expression of WASL was associated with lower pathological stage (chi-square test: p = 0.022, chi-square = 9.613; logistic regression: OR = 0.869, 95% CI: 0.756–0.991, p = 0.041). Patients in the WASL high expression group have worse overall survival (OS) [hazard ratio (HR): 0.555, 95% CI: 0.348–0.884, log-rank p = 0.012] and recurrence-free survival (RFS) (HR = 0.449, 95% CI: 0.215–0.934, log-rank p = 0.028) compared with those in the WASL low expression group. Univariate and multivariable Cox proportional hazards regression model suggested that WASL expression was an independent prognostic factor for predicting OS and RFS in cervical cancer. DEGs were mostly enriched GO terms related to DNA replication or the proliferation of tumor cells. The results of GSEA suggested samples in the WASL knockdown group were enriched in glycolysis, TNF-α signaling via NFkB, mTORC1 signaling, and Wnt/β-catenin signaling.ConclusionsWASL expression was associated with the pathological stage, and it might be an independent prognostication factor in patients with cervical cancer. Knockdown of WASL might be correlated with biological processes such as glycolysis, TNFα signaling, mTOR signaling, and Wnt/β-catenin signaling.