Actin Nucleation Research Articles

Membrane nanodomains modulate formin condensation for actin remodeling in Arabidopsis innate immune responses.

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.

Actin nucleator formins regulate the tension-buffering function of caveolin-1

Both the mechanosensitive actin cytoskeleton and caveolae contribute to active processes such as cell migration, morphogenesis, and vesicular trafficking. Although distinct actin components are well studied, how they contribute to cytoplasmic caveolae, especially in the context of mechano-stress, has remained elusive. Here, we identify two actin-associated mobility stereotypes of caveolin-1 (CAV-1)-marked intracellular vesicles, which are characterized as ‘dwelling’ and ‘go and dwelling’. In order to exploit the reason for their distinct dynamics, elongated actin-associated formin functions are perturbed. We find drastically decreased density, increased clustering, and compromised motility of cytoplasmic CAV-1 vesicles resulting from lacking actin nucleator formins by both chemical treatment and RNA silencing of formin genes. Furthermore, hypo-osmosis-stimulated diminishing of CAV-1 is dramatically intensified upon blocking formins. The clustering of CAV-1 vesicles when cells are cultured on soft substrate is also aggravated under formin inhibition condition. Together, we reveal that actin-associated formins are essential for maintaining the dynamic organization of cytoplasmic CAV-1 and importantly its sensitivity upon mechanical challenge. We conclude that tension-controlled actin formins act as a safety valve dampening excessive tension on CAV-1 and safeguarding CAV-1 against mechanical damage.

Role of formin INF2 in human diseases.

Formin proteins catalyze actin nucleation and microfilament polymerization. Inverted formin 2 (INF2) is an atypical diaphanous-related formin characterized by polymerization and depolymerization of actin. Accumulating evidence showed that INF2 is associated with kidney disease focal segmental glomerulosclerosis and cancers, such as colorectal and thyroid cancer where it functions as a tumor suppressor, glioblastoma, breast, prostate, and gastric cancer, via its oncogenic function. However, studies on the underlying molecular mechanisms of the different roles of INF2 in diverse cancers are limited. This review comprehensively describes the structure, biochemical features, and primary pathogenic mutations of INF2.

Rac1 promotes kidney collecting duct integrity by limiting actomyosin activity.

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.

Cytoplasmic pressure maintains epithelial integrity and inhibits cell motility

Cytoplasmic pressure, a function of actomyosin contractility and water flow, can regulate cellular morphology and dynamics. In mesenchymal cells, cytoplasmic pressure powers cell protrusion through physiological three-dimensional extracellular matrices. However, the role of intracellular pressure in epithelial cells is relatively unclear. Here we find that high cytoplasmic pressure is necessary to maintain barrier function, one of the hallmarks of epithelial homeostasis. Further, our data show that decreased cytoplasmic pressure facilitates lamellipodia formation during the epithelial to mesenchymal transition (EMT). Critically, activation of the actin nucleating protein Arp2/3 is required for the reduction in cytoplasmic pressure and lamellipodia formation in response to treatment with hepatocyte growth factor (HGF) to induce EMT. Thus, elevated cytoplasmic pressure functions to maintain epithelial tissue integrity, while reduced cytoplasmic pressure triggers lamellipodia formation and motility during HGF-dependent EMT.

Targeting the actin nucleation promoting factor WASp provides a therapeutic approach for hematopoietic malignancies

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.

Profilin and Mical combine to impair F-actin assembly and promote disassembly and remodeling

Cellular events require the spatiotemporal interplay between actin assembly and actin disassembly. Yet, how different factors promote the integration of these two opposing processes is unclear. In particular, cellular monomeric (G)-actin is complexed with profilin, which inhibits spontaneous actin nucleation but fuels actin filament (F-actin) assembly by elongation-promoting factors (formins, Ena/VASP). In contrast, site-specific F-actin oxidation by Mical promotes F-actin disassembly and release of polymerization-impaired Mical-oxidized (Mox)-G-actin. Here we find that these two opposing processes connect with one another to orchestrate actin/cellular remodeling. Specifically, we find that profilin binds Mox-G-actin, yet these complexes do not fuel elongation factors’-mediated F-actin assembly, but instead inhibit polymerization and promote further Mox-F-actin disassembly. Using Drosophila as a model system, we show that similar profilin–Mical connections occur in vivo – where they underlie F-actin/cellular remodeling that accompanies Semaphorin–Plexin cellular/axon repulsion. Thus, profilin and Mical combine to impair F-actin assembly and promote F-actin disassembly, while concomitantly facilitating cellular remodeling and plasticity.

Cortactin Contributes to Activity-Dependent Modulation of Spine Actin Dynamics and Spatial Memory Formation.

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.

Functional interdependence of the actin nucleator Cobl and Cobl-like in dendritic arbor development

Local actin filament formation is indispensable for development of the dendritic arbor of neurons. We show that, surprisingly, the action of single actin filament-promoting factors was insufficient for powering dendritogenesis. Instead, this required the actin nucleator Cobl and its only evolutionary distant ancestor Cobl-like acting interdependently. This coordination between Cobl-like and Cobl was achieved by physical linkage by syndapins. Syndapin I formed nanodomains at convex plasma membrane areas at the base of protrusive structures and interacted with three motifs in Cobl-like, one of which was Ca2+/calmodulin-regulated. Consistently, syndapin I, Cobl-like's newly identified N terminal calmodulin-binding site and the single Ca2+/calmodulin-responsive syndapin-binding motif all were critical for Cobl-like's functions. In dendritic arbor development, local Ca2+/CaM-controlled actin dynamics thus relies on regulated and physically coordinated interactions of different F-actin formation-promoting factors and only together they have the power to bring about the sophisticated neuronal morphologies required for neuronal network formation in mammals.

Identification of Arp2/3 Complex Subunits as Prognostic Biomarkers for Hepatocellular Carcinoma.

The actin-related protein 2/3 complex (Arp2/3) is a major actin nucleator that has been widely reported and plays an important role in promoting the migration and invasion of various cancers. However, the expression patterns and prognostic values of Arp2/3 subunits in hepatocellular carcinoma (HCC) remain unclear. In this study, The Cancer Genome Atlas (TCGA) and UCSC Xena databases were used to obtain mRNA expression and the corresponding clinical information, respectively. The differential expression and Arp2/3 subunits in HCC were analyzed using the “limma” package of R 4.0.4 software. The prognostic value of each subunit was evaluated using Kaplan–Meier survival analysis and Cox proportional hazards regression analyses. The results revealed that mRNA expression of Arp2/3 members (ACTR2, ACTR3, ARPC1A, APRC1B, ARPC2, ARPC3, ARPC4, ARPC5, and ARPC5L) was upregulated in HCC. Higher expression of Arp2/3 members was significantly correlated with worse overall survival (OS) and shorter progression-free survival (PFS) in HCC patients. Cox proportional hazards regression analyses demonstrated that ACTR3, ARPC2, and ARPC5 were independent prognostic biomarkers of survival in patients with HCC. The relation between tumor immunocyte infiltration and the prognostic subunits was determined using the TIMER 2.0 platform and the GEPIA database. Gene set enrichment analysis (GSEA) was performed to explore the potential mechanisms of prognostic subunits in the carcinogenesis of HCC. The results revealed that ACTR3, ARPC2, and ARPC5 were significantly positively correlated with the infiltration of immune cells in HCC. The GSEA results indicated that ACTR3, ARPC2, and ARPC5 are involved in multiple cancer-related pathways that promote the development of HCC. In brief, various analyses indicated that Arp2/3 complex subunits were significantly upregulated and predicted worse survival in HCC, and they found that ACTR3, ARPC2, and ARPC5 could be used as independent predictors of survival and might be applied as promising molecular targets for diagnosis and therapy of HCC in the future.

CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation.

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.

Novel CTRP8‐RXFP1‐JAK3‐STAT3 axis promotes Cdc42‐dependent actin remodeling for enhanced filopodia formation and motility in human glioblastoma cells

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.

Wiskott-Aldrich Syndrome Protein: Roles in Signal Transduction in T Cells.

Signal transduction regulates the proper function of T cells in an immune response. Upon binding to its specific ligand associated with major histocompatibility complex (MHC) molecules on an antigen presenting cell, the T cell receptor (TCR) initiates intracellular signaling that leads to extensive actin polymerization. Wiskott-Aldrich syndrome protein (WASp) is one of the actin nucleation factors that is recruited to TCR microclusters, where it is activated and regulates actin network formation. Here we highlight the research that has focused on WASp-deficient T cells from both human and mice in TCR-mediated signal transduction. We discuss the role of WASp in proximal TCR signaling as well as in the Ras/Rac-MAPK (mitogen-activated protein kinase), PKC (protein kinase C) and Ca2+-mediated signaling pathways.

Identify Function of WASL in Prognosis of Cervical Cancer Based on Omics Data.

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.

The WAVE complex associates with sites of saddle membrane curvature.

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.

Characterization of a L136P mutation in Formin-like 2 (FMNL2) from a patient with chronic inflammatory bowel disease.

Diaphanous related formins are highly conserved proteins regulated by Rho-GTPases that act as actin nucleation and assembly factors. Here we report the functional characterization of a non-inherited heterozygous FMNL2 p.L136P mutation carried by a patient who presented with severe very early onset inflammatory bowel disease (IBD). We found that the FMNL2 L136P protein displayed subcellular mislocalization and deregulated protein autoinhibition indicating gain-of-function mechanism. Expression of FMNL2 L136P impaired cell spreading as well as filopodia formation. THP-1 macrophages expressing FMNL2 L136P revealed dysregulated podosome formation and a defect in matrix degradation. Our data indicate that the L136P mutation affects cellular actin dynamics in fibroblasts and immune cells such as macrophages.

Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates

Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple "cryptic leading edges" within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as "cell polarization barriers," decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.

Phosphorylation of the WH2 domain in yeast Las17/WASP regulates G-actin binding and protein function during endocytosis

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.

SIK2 orchestrates actin-dependent host response upon Salmonella infection

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Formins

Actin is one of the most abundant proteins in eukaryotes. Discovered in muscle and described as far back as 1887, actin was first purified in 1942. It plays myriad roles in essentially every eukaryotic cell. Actin is central to development, muscle contraction, and cell motility, and it also functions in the nucleus, to name a spectrum of examples. The flexibility of actin function stems from two factors: firstly, it is dynamic, transitioning between monomer and filament, and, secondly, there are hundreds of actin-binding proteins that build and organize specific actin-based structures. Of prime importance are actin nucleators - proteins that stimulate de novo formation of actin filaments. There are three known classes of actin nucleators: the Arp2/3 complex, formins, and tandem WASP homology 2 (WH2) nucleators. Each class nucleates by a distinct mechanism that contributes to the organization of the larger structure being built. Evidence shows that the Arp2/3 complex produces branched actin filaments, remaining bound at the branch point, while formins create linear actin filaments, remaining bound at the growing end. Here, we focus on the formin family of actin nucleators.