Search Results (53)

Migrastatic strategies are considered as candidate therapeutic approaches to suppress cancer invasion into local surrounding tissues and metastatic spread. The F-actin cytoskeleton is responsible for key properties regulating (cancer) cell migration. The cortical F-actin network controls cell stiffness, which, in turn, determines cell migration strategies and efficiency. Moreover, the dynamic remodeling of F-actin networks mediating filopodia, lamellipodia, and F-actin stress fibers is crucial for cell migration. Here, we have used a conditional knockout approach to delete cofilin, an F-actin-binding protein that controls severing. We find that the deletion of cofilin prevents the migration of cancer cells from tumoroids into the surrounding extracellular matrix without affecting their viability. This identifies cofilin as a candidate target to suppress metastatic spread. Pharmacological inhibitors interfering with F-actin dynamics have been developed but their effects are pleiotropic, including severe toxicity, and their impact on 3D tumor cell migration has not been tested or separated from this toxicity. Using concentration ranges of a panel of inhibitors, we select cytochalasins based on the suppression of 2D migration at non-toxic concentrations. We then show that these attenuate the escape of tumor cells from tumoroids and their migration into the surrounding extracellular matrix without toxicity in 3D cultures. This effect is accompanied by suppression of cell stiffness at such non-toxic concentrations, as measured by acoustic force spectroscopy. These findings identify cytochalasins B and D as candidate migrastatic drugs to suppress metastatic spread. Full article

Both the livestock and biomedical fields require a large supply of high-quality mature oocytes. However, the in vitro maturation (IVM) process often leads to an accumulation of reactive oxygen species (ROS), which can cause defects in oocyte meiosis and embryo development, ultimately compromising oocyte quality. Urolithin A (UA), known for its antioxidant properties, has not been thoroughly investigated for its potential to mitigate the negative effects of oxidative stress during the in vitro culturing of oocytes, and its underlying mechanism is not well understood. In this study, an in vitro oxidative stress model was established using porcine oocytes treated with H₂O₂, followed by exposure to varying concentrations of UA. The results revealed that 30 μM UA significantly improved both the quality of oocyte culture and the developmental potential of the resulting embryos. UA was found to enhance oocyte autophagy, reduce oxidative stress-induced mitochondrial damage, and restore mitochondrial function. Additionally, it lowered ROS and DNA damage levels in the oocytes, maintained proper spindle/chromosome alignment and actin cytoskeleton structure, promoted nuclear maturation, prevented abnormal cortical granule distribution, and supported oocyte cytoplasmic maturation. As a result, UA alleviated oxidative stress-induced defects in oocyte maturation and cumulus cell expansion, thereby improving the developmental potential and quality of parthenogenetic embryos. After supplementation with UA, pig parthenogenetic embryo pluripotency-related genes (Nanog and Sox2) and antiapoptotic genes (Bcl2) were upregulated, while proapoptotic genes (Bax) were downregulated. In conclusion, this study suggests that adding UA during IVM can effectively mitigate the adverse effects of oxidative stress on porcine oocytes, presenting a promising strategy for enhancing their developmental potential in vitro. Full article

The actin cytoskeleton plays an important role in morphological changes of ameloblasts during the formation of enamel, which is indispensable for teeth to withstand wear, fracture and caries progression. This study reveals that the actin nucleator Cobl is expressed in ameloblasts of mandibular molars during amelogenesis. Cobl expression was particularly pronounced during the secretory phase of the enamel-forming cells. Cobl colocalized with actin filaments at the cell cortex. Importantly, our analyses show an influence of Cobl on both ameloblast morphology and cytoskeletal organization as well as on enamel composition. At P0, Cobl knock-out causes an increased height of ameloblasts and an increased F-actin content at the apical membrane. During the maturation phase, the F-actin density at the apical membrane was instead significantly reduced when compared to WT mice. At the same time, Cobl-deficient mice showed an increased carbon content of the enamel and an increased enamel surface of mandibular molars. These findings demonstrate a decisive influence of the actin nucleator Cobl on the actin cytoskeleton and the morphology of ameloblasts during amelogenesis. Our work thus expands the understanding of the regulation of the actin cytoskeleton during amelogenesis and helps to further elucidate the complex processes of enamel formation during tooth development. Full article

RAC3 encodes a small GTPase of the Rho family that plays a critical role in actin cytoskeleton remodeling and intracellular signaling regulation. Pathogenic variants in RAC3, all of which reported thus far affect conserved residues within its functional domains, have been linked to neurodevelopmental disorders characterized by diverse phenotypic features, including structural brain anomalies and facial dysmorphism (NEDBAF). Recently, a novel de novo RAC3 variant (NM_005052.3): c.196C>T, p.R66W was identified in a prenatal case with fetal akinesia deformation sequence (a spectrum of conditions that interfere with the fetus’s ability to move), and complex brain malformations featuring corpus callosum agenesis, diencephalosynapsis, kinked brainstem, and vermian hypoplasia. To investigate the mechanisms underlying the association between RAC3 deficiency and this unique, distinct clinical phenotype, we explored the pathophysiological significance of the p.R66W variant in brain development. Biochemical assays revealed a modest enhancement in intrinsic GDP/GTP exchange activity and an inhibitory effect on GTP hydrolysis. Transient expression studies in COS7 cells demonstrated that RAC3-R66W interacts with the downstream effectors PAK1, MLK2, and N-WASP but fails to activate SRF-, AP1-, and NFkB-mediated transcription. Additionally, overexpression of RAC3-R66W significantly impaired differentiation in primary cultured hippocampal neurons. Acute expression of RAC3-R66W in vivo by in utero electroporation resulted in impairments in cortical neuron migration and axonal elongation during corticogenesis. Collectively, these findings suggest that the p.R66W variant may function as an activated version in specific signaling pathways, leading to a distinctive and severe prenatal phenotype through variant-specific mechanisms. Full article

We have previously reported that the calcineurin inhibitor macrolide immunosuppressant Tacrolimus (TAC, FK506) can promote the migration and invasion of the human-derived extravillous trophoblast cells conducive to preventing implantation failure in immune-complicated gestations manifesting recurrent implantation failure. Although the exact mode of action of TAC in promoting implantation has yet to be elucidated, the integral association of its binding protein FKBP12 with the inositol triphosphate receptor (IP3R) regulated intracellular calcium [Ca²⁺]i channels in the endoplasmic reticulum (ER), suggesting that TAC can mediate its action through ER release of [Ca²⁺]i. Using the immortalized human-derived first-trimester extravillous trophoblast cells HTR8/SVneo, our data indicated that TAC can increase [Ca²⁺]I, as measured by fluorescent live-cell imaging using Fluo-4. Concomitantly, the treatment of HTR8/SVneo with TAC resulted in a major dynamic reorganization in the actin cytoskeleton, favoring a predominant distribution of cortical F-actin networks in these trophoblasts. Notably, the findings that TAC was unable to recover [Ca²⁺]i in the presence of the IP3R inhibitor 2-APB indicate that this receptor may play a crucial role in the mechanism of action of TAC. Taken together, our results suggest that TAC has the potential to influence trophoblast migration through downstream [Ca²⁺]i-mediated intracellular events and mechanisms involved in trophoblast migration, such as F-actin redistribution. Further research into the mono-therapeutic use of TAC in promoting trophoblast growth and differentiation in clinical settings of assisted reproduction is warranted. Full article

In nephrotic syndrome, the podocyte filtration structures are damaged in a process called foot process effacement. This is mediated by the actin cytoskeleton; however, which actins are involved and how they interact with other filtration components, like the basement membrane, remains poorly understood. Here, we used the well-established Drosophila pericardial nephrocyte—the equivalent of podocytes in flies—knockdown models (RNAi) to study the interplay of the actin cytoskeleton (Act5C, Act57B, Act42A, and Act87E), alpha- and beta-integrin (basement membrane), and the slit diaphragm (Sns and Pyd). Knockdown of an actin gene led to variations of formation of actin stress fibers, the internalization of Sns, and a disrupted slit diaphragm cortical pattern. Notably, deficiency of Act5C, which resulted in complete absence of nephrocytes, could be partially mitigated by overexpressing Act42A or Act87E, suggesting at least partial functional redundancy. Integrin localized near the actin cytoskeleton as well as slit diaphragm components, but when the nephrocyte cytoskeleton or slit diaphragm was disrupted, this switched to colocalization, both at the surface and internalized in aggregates. Altogether, the data show that the interdependence of the slit diaphragm, actin cytoskeleton, and integrins is key to the structure and function of the Drosophila nephrocyte. Full article

With the rising demand for medical implants and the dominance of implant-associated failures including infections, extensive research has been prompted into the development of novel biomaterials that can offer desirable characteristics. This study develops and evaluates new titanium-based alloys containing gallium additions with the aim of offering beneficial antibacterial properties while having a reduced stiffness level to minimise the effect of stress shielding when in contact with bone. The focus is on the microstructure, mechanical properties, antimicrobial activity, and cytocompatibility to inform the suitability of the designed alloys as biometals. Novel Ti-33Nb-xGa alloys (x = 3, 5 wt%) were produced via casting followed by homogenisation treatment, where all results were compared to the currently employed alloy Ti-6Al-4V. Optical microscopy, scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) results depicted a single beta (β) phase microstructure in both Ga-containing alloys, where Ti-33Nb-5Ga was also dominated by dendritic alpha (α) phase grains in a β-phase matrix. EDS analysis indicated that the α-phase dendrites in Ti-33Nb-5Ga were enriched with titanium, while the β-phase was richer in niobium and gallium elements. Mechanical properties were measured using nanoindentation and microhardness methods, where the Young’s modulus for Ti-33Nb-3Ga and Ti-33Nb-5Ga was found to be 75.4 ± 2.4 and 67.2 ± 1.6 GPa, respectively, a significant reduction of 37% and 44% with respect to Ti-6Al-4V. This reduction helps address the disproportionate Young’s modulus between titanium implant components and cortical bone. Importantly, both alloys successfully achieved superior antimicrobial properties against Gram-negative P. aeruginosa and Gram-positive S. aureus bacteria. Antibacterial efficacy was noted at up to 90 ± 5% for the 3 wt% alloy and 95 ± 3% for the 5 wt% alloy. These findings signify a substantial enhancement of the antimicrobial performance when compared to Ti-6Al-4V which exhibited very small rates (up to 6.3 ± 1.5%). No cytotoxicity was observed in hGF cell lines over 24 h. Cell morphology and cytoskeleton distribution appeared to depict typical morphology with a prominent nucleus, elongated fibroblastic spindle-shaped morphology, and F-actin filamentous stress fibres in a well-defined structure of parallel bundles along the cellular axis. The developed alloys in this work have shown very promising results and are suggested to be further examined towards the use of orthopaedic implant components. Full article

The cell membrane is frequently subjected to damage, either through physical or chemical means. The swift restoration of the cell membrane’s integrity is crucial to prevent the leakage of intracellular materials and the uncontrolled influx of extracellular ions. Consequently, wound repair plays a vital role in cell survival, akin to the importance of DNA repair. The mechanisms involved in wound repair encompass a series of events, including ion influx, membrane patch formation, endocytosis, exocytosis, recruitment of the actin cytoskeleton, and the elimination of damaged membrane sections. Despite the absence of a universally accepted general model, diverse molecular models have been proposed for wound repair in different organisms. Traditional wound methods not only damage the cell membrane but also impact intracellular structures, including the underlying cortical actin networks, microtubules, and organelles. In contrast, the more recent improved laserporation selectively targets the cell membrane. Studies on Dictyostelium cells utilizing this method have introduced a novel perspective on the wound repair mechanism. This review commences by detailing methods for inducing wounds and subsequently reviews recent developments in the field. Full article

The “Wattle and Daub” model of cyst wall formation in Entamoeba invadens has been used to explain encystment in Entamoeba histolytica, the causal agent of amoebiasis, and this process could be a potential target for new antiamoebic drugs. In this study, we studied the morphological stages of chitin wall formation in E. invadens in more detail using fluorescent chitin-binding dyes and the immunolocalization of cyst wall proteins. It was found that chitin deposition was mainly initiated on the cell surface at a specific point or at different points at the same time. The cystic wall grew outward and gradually covered the entire surface of the cyst over time, following the model of Wattle and Daub. The onset of chitin deposition was guided by the localization of chitin synthase 1 to the plasma membrane, occurring on the basis of the Jacob lectin in the cell membrane. During encystation, F-actin was reorganized into the cortical region within the early stages of encystation and remained intact until the completion of the chitin wall. The disruption of actin polymerization in the cortical region inhibited proper wall formation, producing wall-less cysts or cysts with defective chitin walls, indicating the importance of the cortical actin cytoskeleton for proper cyst wall formation. Full article

Epithelial ovarian cancer is the most lethal gynecological malignant tumor. Although debulking surgery, chemotherapy, and PARP inhibitors have greatly improved survival, the prognosis for patients with advanced EOC without HRD is still poor. LLGL2, as a cell polarity factor, is involved in maintaining cell polarity and asymmetric cell division. In the study of zebrafish development, LLGL2 regulated the proliferation and migration of epidermal cells and the formation of cortical F-actin. However, the role of LLGL2 in ovarian cancer has not been described. Our study found, through bioinformatics analysis, that low expression of LLGL2 was significantly associated with a more advanced stage and a higher grade of EOC and a poorer survival of patients. Functional experiments that involved LLGL2 overexpression and knockdown showed that LLGL2 inhibited the migration and invasion abilities of ovarian cancer cells in vitro, without affecting their proliferation. LLGL2-overexpressing mice had fewer metastatic implant foci than the controls in vivo. Mechanistically, immunoprecipitation combined with mass spectrometry analysis suggested that LLGL2 regulated cytoskeletal remodeling by interacting with ACTN1. LLGL2 altered the intracellular localization and function of ACTN1 without changing its protein and mRNA levels. Collectively, we uncovered that LLGL2 impaired actin filament aggregation into bundles by interacting with ACTN1, which led to cytoskeleton remodeling and inhibition of the invasion and metastasis of ovarian cancer cells. Full article

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca²⁺ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca²⁺ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca²⁺ response at fertilization and the subsequent embryonic development. Full article

Purpose: Inducible Slc4a11 KO leads to corneal edema by disruption of the pump and barrier functions of the corneal endothelium (CE). The loss of Slc4a11 NH₃-activated mitochondrial uncoupling leads to mitochondrial membrane potential hyperpolarization-induced oxidative stress. The goal of this study was to investigate the link between oxidative stress and the failure of pump and barrier functions and to test different approaches to revert the process. Methods: Mice which were homozygous for Slc4a11 Flox and Estrogen receptor –Cre Recombinase fusion protein alleles at 8 weeks of age were fed Tamoxifen (Tm)-enriched chow (0.4 g/Kg) for 2 weeks, and controls were fed normal chow. During the initial 14 days, Slc4a11 expression, corneal thickness (CT), stromal [lactate], Na⁺-K⁺ ATPase activity, mitochondrial superoxide levels, expression of lactate transporters, and activity of key kinases were assessed. In addition, barrier function was assessed by fluorescein permeability, ZO-1 tight junction integrity, and cortical cytoskeleton F-actin morphology. Results: Tm induced a rapid decay in Slc4a11 expression that was 84% complete at 7 days and 96% complete at 14 days of treatment. Superoxide levels increased significantly by day 7; CT and fluorescein permeability by day 14. Tight junction ZO-1 distribution and the cortical cytoskeleton were disrupted at day 14, concomitant with decreased expression of Cldn1, yet with increased tyrosine phosphorylation. Stromal lactate increased by 60%, Na⁺-K⁺ ATPase activity decreased by 40%, and expression of lactate transporters MCT2 and MCT4 significantly decreased, but MCT1 was unchanged at 14 days. Src kinase was activated, but not Rock, PKCα, JNK, or P38Mapk. Mitochondrial antioxidant Visomitin (SkQ1, mitochondrial targeted antioxidant) and Src kinase inhibitor eCF506 significantly slowed the increase in CT, with concomitant decreased stromal lactate retention, improved barrier function, reduced Src activation and Cldn1 phosphorylation, and rescued MCT2 and MCT4 expression. Conclusions: Slc4a11 KO-induced CE oxidative stress triggered increased Src kinase activity that resulted in perturbation of the pump components and barrier function of the CE. Full article

Microglial activation and failure of the antioxidant defense mechanisms are major hallmarks in different brain injuries, particularly traumatic brain injury (TBI). Cofilin is a cytoskeleton-associated protein involved in actin binding and severing. In our previous studies, we identified the putative role of cofilin in mediating microglial activation and apoptosis in ischemic and hemorrhagic conditions. Others have highlighted the involvement of cofilin in ROS production and the resultant neuronal death; however, more studies are needed to delineate the role of cofilin in oxidative stress conditions. The present study aims to investigate the cellular and molecular effects of cofilin in TBI using both in vitro and in vivo models as well as the first-in-class small-molecule cofilin inhibitor (CI). An in vitro H₂O₂-induced oxidative stress model was used in two different types of cells, human neuroblastoma (SH-SY5Y) and microglia (HMC3), along with an in vivo controlled cortical impact model of TBI. Our results show that treatment with H₂O₂ increases the expression of cofilin and slingshot-1 (SSH-1), an upstream regulator of cofilin, in microglial cells, which was significantly reduced in the CI-treated group. Cofilin inhibition significantly attenuated H₂O₂-induced microglial activation by reducing the release of proinflammatory mediators. Furthermore, we demonstrate that CI protects against H₂O₂-induced ROS accumulation and neuronal cytotoxicity, activates the AKT signaling pathway by increasing its phosphorylation, and modulates mitochondrial-related apoptogenic factors. The expression of NF-E2-related factor 2 (Nrf2) and its associated antioxidant enzymes were also increased in CI-treated SY-SY5Y. In the mice model of TBI, CI significantly activated the Nrf2 and reduced the expression of oxidative/nitrosative stress markers at the protein and gene levels. Together, our data suggest that cofilin inhibition provides a neuroprotective effect in in vitro and in vivo TBI mice models by inhibiting oxidative stress and inflammatory responses, the pivotal mechanisms involved in TBI-induced brain damage. Full article

Stimulation of melanocytes and murine melanoma cells with αMSH plus the PI3K inhibitor LY294002 resulted in ROS increase, oxidative DNA damage, and pigment retention. We performed cellular and molecular biology assays (Western blot, FACS, immunofluorescence analysis, scratch assay) on murine and human melanoma cells. Treatment with αMSH plus LY294002 altered cortical actin architecture. Given that cytoskeleton integrity requires energy, we next evaluated ATP levels and we observed a drop in ATP after exposure to αMSH plus LY294002. To evaluate if the αMSH-activated PI3K pathway could modulate energy metabolism, we focused on glucose uptake by analyzing the expression of the Glut-1 glucose translocator. Compared with cells treated with αMSH alone, those exposed to combined treatment showed a reduction of Glut-1 on the plasma membrane. This metabolic alteration was associated with changes in mitochondrial mass. A significant decrease of the cell migratory potential was also observed. We demonstrated that the αMSH-dependent PI3K pathway acts as a regulator of energy metabolism via glucose uptake, influencing the actin cytoskeleton, which is involved in melanosome release and cell motility. Hence, these results could constitute the basis for innovative therapeutical strategies. Full article

Mast cells (MCs) are the main participants in the control of immune reactions associated with inflammation, allergies, defense against pathogens, and tumor growth. Bioactive lipids are lipophilic compounds able to modulate MC activation. Here, we explored some of the effects of the bioactive lipid lysophosphatidylinositol (LPI) on MCs. Utilizing murine bone marrow-derived mast cells (BMMCs), we found that LPI did not cause degranulation, but slightly increased FcεRI-dependent β-hexosaminidase release. However, LPI induced strong chemotaxis together with changes in LIM kinase (LIMK) and cofilin phosphorylation. LPI also promoted modifications to actin cytoskeleton dynamics that were detected by an increase in cell size and interruptions in the continuity of the cortical actin ring. The chemotaxis and cortical actin ring changes were dependent on GPR55 receptor activation, since the specific agonist O1602 mimicked the effects of LPI and the selective antagonist ML193 prevented them. The LPI and O1602-dependent stimulation of BMMC also led to VEGF, TNF, IL-1α, and IL-1β mRNA accumulation, but, in contrast with chemotaxis-related processes, the effects on cytokine transcription were dependent on GPR55 and cannabinoid (CB) 2 receptors, since they were sensitive to ML193 and to the specific CB2 receptor antagonist AM630. Remarkably, GPR55-dependent BMMC chemotaxis was observed towards conditioned media from distinct mouse and human cancer cells. Our data suggest that LPI induces the chemotaxis of MCs and leads to cytokine production in MC in vitro with the differential participation of GPR55 and CB2 receptors. These effects could play a significant role in the recruitment of MCs to tumors and the production of MC-derived pro-angiogenic factors in the tumor microenvironment. Full article