Polymerization Of Cytoskeleton Research Articles

Antagonistic actions of PAK1 and NF2/Merlin drive myelin membrane expansion in oligodendrocytes.

In the central nervous system, the formation of myelin by oligodendrocytes (OLs) relies on the switch from the polymerization of the actin cytoskeleton to its depolymerization. The molecular mechanisms that trigger this switch have yet to be elucidated. Here, we identified P21-activated kinase 1 (PAK1) as a major regulator of actin depolymerization in OLs. Our results demonstrate that PAK1 accumulates in OLs in a kinase-inhibited form, triggering actin disassembly and, consequently, myelin membrane expansion. Remarkably, proteomic analysis of PAK1 binding partners enabled the identification of NF2/Merlin as its endogenous inhibitor. Our findings indicate that Nf2 knockdown in OLs results in PAK1 activation, actin polymerization, and a reduction in OL myelin membrane expansion. This effect is rescued by treatment with a PAK1 inhibitor. We also provide evidence that the specific Pak1 loss-of-function in oligodendroglia stimulates the thickening of myelin sheaths in vivo. Overall, our data indicate that the antagonistic actions of PAK1 and NF2/Merlin on the actin cytoskeleton of the OLs are critical for proper myelin formation. These findings have broad mechanistic and therapeutic implications in demyelinating diseases and neurodevelopmental disorders.

The mammalian actin elongation factor ENAH/MENA contributes to autophagosome formation via its actin regulatory function

ABSTRACT Macroautophagy/autophagy is a catabolic process crucial for degrading cytosolic components and damaged organelles to maintain cellular homeostasis, enabling cells to survive in extreme extracellular environments. ENAH/MENA, a member of the Ena/VASP protein family, functions as a highly efficient actin elongation factor. In this study, our objective was to explore the role of ENAH in the autophagy process. Initially, we demonstrated that depleting ENAH in cancer cells inhibits autophagosome formation. Subsequently, we observed ENAH’s colocalization with MAP1LC3/LC3 during tumor cell starvation, dependent on actin cytoskeleton polymerization and the interaction between ENAH and BECN1 (beclin 1). Additionally, mammalian ATG9A formed a ring-like structure around ENAH-LC3 puncta during starvation, relying on actin cytoskeleton polymerization. Furthermore, ENAH’s EVH1 and EVH2 domains were found to be indispensable for its colocalization with LC3 and BECN1, while the PRD domain played a crucial role in the formation of the ATG9A ring. Finally, our study revealed ENAH-led actin comet tails in autophagosome trafficking. In conclusion, our findings provide initial insights into the regulatory role of the mammalian actin elongation factor ENAH in autophagy. Abbreviations: 3-MA 3-methyladenine; ABPs actin-binding proteins; ATG autophagy related; ATG9A autophagy related 9A; Baf A1 bafilomycin A1; CM complete medium; CytERM endoplasmic reticulum signal-anchor membrane protein; Cyto D cytochalasin D; EBSS Earl’s balanced salt solution; ENAH/MENA ENAH actin regulator; EVH1 Ena/VASP homology 1 domain; EVH2 Ena/VASP homology 2 domain; GAPDH glyceraldehyde-3-phosphate dehydrogenase; Lat B latrunculin B; LC3-I unlipidated form of LC3; LC3-II phosphatidylethanolamine-conjugated form of LC3; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; mEGFP monomeric enhanced green fluorescent protein; mTagBFP2 monomeric Tag blue fluorescent protein 2; OSER organized smooth endoplasmic reticulum; PRD proline-rich domain; PtdIns3K class III phosphatidylinositol 3-kinase; WM wortmannin.

Breaking the symmetry of cell contractility drives tubulogenesis via CXCL1 polarization

The intricate interplay between biomechanical and biochemical pathways in modulating morphogenesis is an interesting research topic. How biomechanical force regulates epithelial cell tubulogenesis remains poorly understood. Here, we established a model of tubulogenesis by culturing renal proximal tubular epithelial cells on a collagen gel while manipulating contractile force. Epithelial cells were dynamically self-organized into tubule-like structures by augmentation of cell protrusions and cell-cell association. Reduction and asymmetric distribution of phosphorylated myosin light chain 2, the actomyosin contractility, in cells grown on soft matrix preceded tube connection. Notably, reducing matrix stiffness via sonication of collagen fibrils and inhibiting actomyosin contractility with blebbistatin promoted tubulogenesis, whereas inhibition of cytoskeleton polymerization suppressed it. CXC chemokine ligand 1 (CXCL1) expression was transcriptionally upregulated in cells undergoing tubulogenesis. Additionally, inhibiting actomyosin contractility facilitated CXCL1 polarization and cell protrusions preceding tube formation. Conversely, inhibiting the CXCL1-CXC receptor 1 pathway hindered cell protrusions and tubulogenesis. Mechanical property asymmetry with cell-collagen fibril interaction patterns at cell protrusions and along the tube structure supported the association of anisotropic contraction with tube formation. Furthermore, suppressing the mechanosensing machinery of integrin subunit beta 1 reduced CXCL1 expression, collagen remodeling, and impaired tubulogenesis. In summary, symmetry breaking of cell contractility on a soft collagen gel promotes CXCL1 polarization at cell protrusions which in turn facilitates cell-cell association and thus tubule connection.

Actin cytoskeleton polymerization and focal adhesion as important factors in the pathomechanism and potential targets of mucopolysaccharidosis treatment

Actin cytoskeleton polymerization and focal adhesion as important factors in the pathomechanism and potential targets of mucopolysaccharidosis treatment

HDAC1/2 control mesothelium/ovarian cancer adhesive interactions impacting on Talin-1-α5β1-integrin-mediated actin cytoskeleton and extracellular matrix protein remodeling

BackgroundPeritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion.MethodsStatic adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum—sensitive/resistant EOC cell lines with MS-275—a Histone deacetylase (HDAC)1–3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software.The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies.Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on β1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination.ResultsTreatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing β1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts.ConclusionOur study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis.

Plectin Promotes the Migration of Hepatocellular Carcinoma Cells Through Inducing F-actin Polymerization

To explore the relationship between the expression of plectin and the migration of hepatocellular carcinoma (HCC) cells and to elucidate the molecular mechanisms by which plectin expression affects the migration of HCC cells. First of all, Western blot was performed to determine the expression of plectin in normal hepatocytes and HCC cells. Secondly, a plectin-downregulated HCC cell strain was established and the control group (shNC group) and shPLEC group were set up. Each group was divided into a vehicle control group (shNC+DMSO group or shPLEC+DMSO group) and a F-actin cytoskeleton polymerization inducer Jasplakinolide group (shNC+Jasp group or shPLEC+Jasp group). Western blot was performed to determine the expression of plectin and epithelial-mesenchymal transition (EMT)-related proteins, including N-cadherin, vimentin, and E-cadherin. HCC cell migration was evaluated by Transwell assay. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to analyze the signaling pathways related to plectin gene. The polymerization of F-actin was analyzed by immunofluorescence assay. Compared with the normal hepatocytes, HCC cells showed high expression of plectin. Compared with those in the shNC group, the expression of plectin in the shPLEC group was decreased (P<0.05), the migration ability of HCC cells was weakened (P<0.05), and the EMT process was inhibited (with the expression of N-cadherin and vimentin being decreased and the expression of E-cadherin being increased) (P<0.05). KEGG analysis showed that the regulation of cytoskeletal F-actin was most closely associated with plectin and cytoskeletal F-actin depolymerized in the shPLEC group. After treatment with Jasplakinolide, an inducer of F-actin cytoskeleton polymerization, the migration ability of HCC cells in the shPLEC+Jasp group was enhanced compared with that of shPLEC+DMSO group (P<0.05) and the EMT process was restored (with the expression of N-cadherin and vimentin being increased and the expression of E-cadherin being decreased) (P<0.05). In addition, the polymerization of cytoskeletal F-actin in HCC cells was also restored. Plectin is highly expressed in HCC cells. Plectin promotes the migration and the EMT of HCC cells through inducing F-actin polymerization.

Phytol and (−)-α-bisabolol Synergistically trigger intrinsic apoptosis through redox and Ca2+ imbalance in non-small cell lung cancer

This study aimed to identify a combination of natural compounds that could enhance lung cancer treatment by targeting multiple pathways and reducing toxicity. The Chow-Talay method's CI value of 0.75 demonstrated that the synergistic combination of phytol and (−)-α-bisabolol (Phy + Bis) exhibited a significant anti-proliferative effect on A549 cells. After 24 h of treatment, Phy + Bis significantly altered the morphology of A549 cells and activated apoptosis. Increased levels of reactive oxygen species (ROS) are evidence that the combination treatments induced oxidative stress. Consequently, there was an increase in intracellular calcium concentration ([Ca2+]i), leading to perturbations in the membrane potential (ΔΨm). Intrinsic apoptosis was triggered by these effects, which resulted in depolarization of the mitochondrial membrane, activation of Cas 9 and Cas 3, upregulation of Bax and Cyt-C, and downregulation of Bcl-2. Phy + Bis also caused G0/G1 phase arrest and cell senescence, with senescent cells expressing more p53. The combined therapy prevented the migration of A549 cells, possibly by interfering with the polymerization of the actin cytoskeleton. Importantly, Phy + Bis did not have any harmful effects on human lung L-132 cells, red blood cells, peripheral blood mononuclear cells, and an in vivo artemia model. Overall, phytol and (−)-α-bisabolol appear to be a promising combination as a safe and effective therapy choice for non-small cell lung cancer.

RhoA suppresses pseudorabies virus replication in vitro

The porcine pseudorabies virus (PRV) is one of the most devastating pathogens and brings great economic losses to the swine industry worldwide. Viruses are intracellular parasites that have evolved numerous strategies to subvert and utilize different host processes for their life cycle. Among the different systems of the host cell, the cytoskeleton is one of the most important which not only facilitate viral invasion and spread into neighboring cells, but also help viruses to evade the host immune system. RhoA is a key regulator of cytoskeleton system that may participate in virus infection. In this study, we characterized the function of RhoA in the PRV replication by chemical drugs treatment, gene knockdown and gene over-expression strategy. Inhibition of RhoA by specific inhibitor and gene knockdown promoted PRV proliferation. On the contrary, overexpression of RhoA or activation of RhoA by chemical drug inhibited PRV infection. Besides, our data demonstrated that PRV infection induced the disruption of actin stress fiber, which was consistent with previous report. In turn, the actin specific inhibitor cytochalasin D markedly disrupted the normal fibrous structure of intracellular actin cytoskeleton and decreased the PRV replication, suggesting that actin cytoskeleton polymerization contributed to PRV replication in vitro. In summary, our data displayed that RhoA was a host restriction factor that inhibited PRV replication, which may deepen our understanding the pathogenesis of PRV and provide further insight into the prevention of PRV infection and the development of anti-viral drugs.

Interplay of Cholesterol and Actin in Neurotransmitter GPCR Signaling: Insights from Chronic Cholesterol Depletion Using Statin.

Serotonin1A receptors are important neurotransmitter receptors in the G protein-coupled receptor (GPCR) family and modulate a variety of neurological, behavioral, and cognitive functions. We recently showed that chronic cholesterol depletion by statins, potent inhibitors of HMG-CoA reductase (the rate-limiting enzyme in cholesterol biosynthesis), leads to polymerization of the actin cytoskeleton that alters lateral diffusion of serotonin1A receptors. However, cellular signaling by the serotonin1A receptor under chronic cholesterol depletion remains unexplored. In this work, we explored signaling by the serotonin1A receptor under statin-treated condition. We show that cAMP signaling by the receptor is reduced upon lovastatin treatment due to reduction in cholesterol as well as polymerization of the actin cytoskeleton. To the best of our knowledge, these results constitute the first report describing the effect of chronic cholesterol depletion on the signaling of a G protein-coupled neuronal receptor. An important message arising from these results is that it is prudent to include the contribution of actin polymerization while analyzing changes in membrane protein function due to chronic cholesterol depletion by statins. Notably, our results show that whereas actin polymerization acts as a negative regulator of cAMP signaling, cholesterol could act as a positive modulator. These results assume significance in view of reports highlighting symptoms of anxiety and depression in humans upon statin administration and the role of serotonin1A receptors in anxiety and depression. Overall, these results reveal a novel role of actin polymerization induced by chronic cholesterol depletion in modulating GPCR signaling, which could act as a potential therapeutic target.

SAT515 Searching For Markers To Identify Angioinvasion In Papillary Thyroid Cancer

Abstract Disclosure: A. Buczyńska: None. M. Kościuszko: None. I. Sidorkiewicz: None. A. Wiatr: None. K. Siewko: None. A. Adamska: None. A. Krętowski: None. A. Popławska-Kita: None. The incidence of Papillary Thyroid Cancer (PTC) has increased significantly over last decades. Despite the great progress in PTC clinical management, the association of angioinvasion with tumor aggressiveness in PTC remains as significant clinical problem, suggesting worse patients prognosis. Therefore cancer metastasis occurs through cell detachment and angioinvasion, epithelial–mesenchymal transition (EMT) and cell migration, changes in cell morphology are initiated through Rho-GTPase-dependent actin cytoskeleton polymerization. 8-hydroxydeoxyguanosine (8-OHdG) is generated accordingly to increased oxidative stress, however has been characterized by paradoxical anti-oxidative, anti-inflammatory and anti-mutagenic effects following Rho-GTPase inhibition, which were noted in various cancer models. Since oxidative stress has been recognized as one of the main risk factors for thyroid cancer development and progression, the total oxidative and antioxidant capacities may also be potentially involved in the process of angioinvasion. Regardless, the identification of angioinvasion in PTC remains controversial mainly due to loose criteria in judging vascular invasion. Thus, searching for laboratory biomarkers of angioinvasion is of paramount importance.In this study, the potential screening utility of oxidative stress-related markers, such as total oxidative capacity (TOC), total antioxidant capacity (TAC) and 8-OHdG was assessed to identify angioinvasion in PTC patients.For the purpose od this study, 50 patients diagnosed with angioinvasive PTC (study group) and 50 patients with very low risk PTC (control group) were enrolled. The TOC, TAC and 8-OHdG concentrations were evaluated using enzyme-linked immunosorbent assay (ELISA). Following the received results, the TAC and 8-OHdG concentrations were decresed among study group comparing to control group (p&amp;lt;0.001; p&amp;lt;0.02, respectively). However, there were no differences in TOC among studied groups (p&amp;gt;0.05). The AUC values for TAC=0.76 and AUC for 8-OHdG= 0.70 (all, p&amp;lt;0.05) suggest their screening utility. The results indicate that angioinvasion process is associated with decreased antioxidant defense in PTC patients which may insight new medical targets in the future. Presentation Date: Saturday, June 17, 2023

Actin Cytoskeleton Polymerization and Focal Adhesion as Important Factors in the Pathomechanism and Potential Targets of Mucopolysaccharidosis Treatment.

The main approach used in the current therapy of mucopolysaccharidosis (MPS) is to reduce the levels of glycosaminoglycans (GAGs) in cells, the deposits considered to be the main cause of the disease. Previous studies have revealed significant differences in the expression of genes encoding proteins involved in many processes, like those related to actin filaments, in MPS cells. Since the regulation of actin filaments is essential for the intracellular transport of specific molecules, the process which may affect the course of MPSs, the aim of this study was to evaluate the changes that occur in the actin cytoskeleton and focal adhesion in cells derived from patients with this disease, as well as in the MPS I mouse model, and to assess whether they could be potential therapeutic targets for different MPS types. Western-blotting, flow cytometry and transcriptomic analyses were employed to address these issues. The levels of the key proteins involved in the studied processes, before and after specific treatment, were assessed. We have also analyzed transcripts whose levels were significantly altered in MPS cells. We identified genes whose expressions were changed in the majority of MPS types and those with particularly highly altered expression. For the first time, significant changes in the expression of genes involved in the actin cytoskeleton structure/functions were revealed which may be considered as an additional element in the pathogenesis of MPSs. Our results suggest the possibility of using the actin cytoskeleton as a potential target in therapeutic approaches for this disease.

Tensile Overload Injures Human Alveolar Epithelial Cells through YAP/F-Actin/MAPK Signaling.

Explosion shockwaves can generate overloaded mechanical forces and induce lung injuries. However, the mechanism of lung injuries caused by tensile overload is still unclear. Flow cytometry was used to detect the apoptosis of human alveolar epithelial cells (BEAS-2B) induced by tensile overload, and cell proliferation was detected using 5-ethynyl-2'-deoxyuridine (EdU). Immunofluorescence and Western blot analysis were used to identify the tensile overload on the actin cytoskeleton, proteins related to the mitogen-activated protein kinase (MAPK) signal pathway, and the Yes-associated protein (YAP). Tensile overload reduced BEAS-2B cell proliferation and increased apoptosis. In terms of the mechanism, we found that tensile overload led to the depolymerization of the actin cytoskeleton, the activation of c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase 1/2 (ERK1/2), and the upregulation of YAP expression. Jasplakinolide (Jasp) treatment promoted the polymerization of the actin cytoskeleton and reduced the phosphorylation of tension-overload-activated JNK and ERK1/2 and the apoptosis of BEAS-2B cells. Moreover, the inhibition of the JNK and ERK1/2 signaling pathways, as well as the expression of YAP, also reduced apoptosis caused by tensile overload. Our study establishes the role of the YAP/F-actin/MAPK axis in tensile-induced BEAS-2B cell injury and proposes new strategies for the treatment and repair of future lung injuries.

Synthesis of New Chromene Derivatives Targeting Triple-Negative Breast Cancer Cells.

Breast cancer continues to be the leading cause of cancer-related deaths among women worldwide. The most aggressive type of breast cancer is triple-negative breast cancer (TNBC). Indeed, not only does TNBC not respond well to several chemotherapeutic agents, but it also frequently develops resistance to various anti-cancer drugs, including taxane mitotic inhibitors. This necessitates the search for newer, more efficacious drugs. In this study, we synthesized two novel chromene derivatives (C1 and C2) and tested their efficacy against a battery of luminal type A and TNBC cell lines. Our results show that C1 and C2 significantly and specifically inhibited TNBC cell viability but had no effect on the luminal A cell type. In addition, these novel compounds induced mitotic arrest, cell multinucleation leading to senescence, and apoptotic cell death through the activation of the extrinsic pathway. We also showed that the underlying mechanisms for these actions of C1 and C2 involved inhibition of microtubule polymerization and disruption of the F-actin cytoskeleton. Furthermore, both compounds significantly attenuated migration of TNBC cells and inhibited angiogenesis in vitro. Finally, we performed an in silico analysis, which revealed that these novel variants bind to the colchicine binding site in β-tubulin. Taken together, our data highlight the potential chemotherapeutic properties of two novel chromene compounds against TNBC.

Stiff Extracellular Matrix Promotes Invasive Behaviors of Trophoblast Cells.

The effect of extracellular matrix (ECM) stiffness on embryonic trophoblast cells invasion during mammalian embryo implantation remains largely unknown. In this study, we investigated the effects of ECM stiffness on various aspects of human trophoblast cell behaviors during cell-ECM interactions. The mechanical microenvironment of the uterus was simulated by fabricating polyacrylamide (PA) hydrogels with different levels of stiffness. The human choriocarcinoma (JAR) cell lineage was used as the trophoblast model. We found that the spreading area of JAR cells, the formation of focal adhesions, and the polymerization of the F-actin cytoskeleton were all facilitated with increased ECM stiffness. Significantly, JAR cells also exhibited durotactic behavior on ECM with a gradient stiffness. Meanwhile, stiffness of the ECM affects the invasion of multicellular JAR spheroids. These results demonstrated that human trophoblast cells are mechanically sensitive, while the mechanical properties of the uterine microenvironment could play an important role in the implantation process.

Dragon’s Blood Protect Rat Blood-Brain Barrier Dysfunction Induced by Simulated Microgravity Effect

Dragon’s blood (DB) has shown a protective effect on neurological diseases. Microgravity (MG) or simulated MG (SMG) can induce blood–brain barrier (BBB) dysfunction, which is a characteristic feature of neurological disorders. This study’s purpose was to evaluate the effect of DB on SMG-induced BBB dysfunction and explore its signaling pathway. Both DB and vitamin C (Vc) were administered orally for tail-suspended rats within 3 weeks. DB and Vc solutions were added to human brain microvascular endothelial cells (HCMEC/D3) cells, which were then exposed to SMG for 24 h. The protective effect of DB was assessed by hematoxylin and eosin and Nissl staining, ultrastructure observation, and permeability in rats. Cell apoptosis and the distribution of tight junction (TJ) and adherens junction (AJ) proteins and filamentous actin (F-actin) were examined in HCMEC/D3. The oxidative stress and inflammation, and TJ and AJ protein expressions were determined in rat brain and HCMEC/D3. The focal adhesion kinase (FAK) signaling pathway proteins were determined. DB protected SMG-induced rat BBB disruption by improving neuronal apoptosis, repairing widened intercellular space, and decreasing BBB permeability. DB effectively relieved SMG-induced HCMEC/D3 damage by inhibiting cell apoptosis and restoring F-actin spindle distribution. High doses of DB upregulated TJ and AJ protein expressions and decreased oxidative stress and proinflammatory cytokine levels in rat brain and HCMEC/D3. DB enhanced the expressions of FAK signal transduction proteins and F-actin/globular actin (G-actin) ratio in rat brain and HCMEC/D3, suggesting that DB promotes actin cytoskeleton polymerization, benefits the endothelial cell–cell and cell–extracellular matrix adhesion, and, in consequence, contributes to BBB integrity.

Mapping of Tilapia Lake Virus entry pathways with inhibitors reveals dependence on dynamin activity and cholesterol but not endosomal acidification.

Tilapia Lake Virus (TiLV) is an emerging virus lethal to tilapia, which threatens the global tilapia aquaculture with severe implications for food security. TiLV possesses similar features to orthomyxoviruses but is classified in the sole and the monotypic genus Tilapinevirus of the family Amnoonviridae. TiLV enveloped virions encapsidate a genome comprising ten segments of single-stranded, negative RNA. Remarkably, nine of TiLV's ten major proteins lack sequence homology to any known viral or cellular proteins. The mode of TiLV entry into tilapia cells is not known. Following the measurement of the entry window of TiLV (∼3h), we applied a panel of inhibitors of known regulators of endocytic functions to map the molecular requirements for TiLV entry. We identified productive entry by quantification of TiLV nucleoprotein expression and the generation of infectious particles. Inhibition of dynamin activity with dynasore or dynole, or depletion of cholesterol with methyl-β-cyclodextrin, strongly inhibited TiLV protein synthesis and infectious virion production. Moreover, inhibition of actin cytoskeleton polymerization with latrunculin A or microtubule polymerization with nocodazole within the entry window resulted in partial inhibition of TiLV infection. In contrast, inhibitors of endosomal acidification (NH4Cl, bafilomycin A1, or chloroquine), an inhibitor of clathrin-coated pit assembly (pitstop 2), and erlotinib-an inhibitor of the endocytic Cyclin G-associated kinase (GAK), did not affect TiLV entry. Altogether, these results suggest that TiLV enters via dynamin-mediated endocytosis in a cholesterol-, cytoskeleton-dependent manner, and clathrin-, pH-independent manner. Thus, despite being an orthomyxo-like virus, when compared to the prototypical orthomyxovirus (influenza A virus), TiLV shows a distinct set of requirements for entry into cells.

Actin restricts cell proliferation and promotes differentiation during planarian regeneration

Actin is an integral component of the cytoskeleton, which plays an important role in various fundamental cellular processes, such as affecting the polarity of embryonic cells during embryonic development in various model organisms. Meanwhile, previous studies have demonstrated that the polymerization of the actin cytoskeleton can affect cell migration, proliferation, and differentiation. Actin polymerization state regulated osteogenic differentiation and affected cell proliferation. However, the function of actin in regenerative biology has not been thoroughly elucidated. The planarian flatworm, which contains a large number of adult somatic stem cells (neoblasts), is an ideal model organism to study regenerative biology. Here, we identified a homolog of actin in planarian Dugesia japonica and found that RNAi targeting actin during planarian regeneration results in the formation of protrusions on the dorsal side, where the division of phospho-H3 mitotic cells is increased. In addition, a decrease in differentiation is observed in regenerating tissues after Djactin RNAi. These results indicate that Djactin functions in proliferation and differentiation control in planarian regeneration.

Uninterrupted dynamic stiffening microenvironment enhances the paracrine function of mesenchymal stem cells for vascularization through chromatin remodeling

The stiffness of extracellular matrix is dynamically stiffened during bone formation. Paracrine of angiogenic factors in mesenchymal stem cells (MSCs) plays an essential role during tissue regeneration. Whether the paracrine of angiogenic factors in MSCs is influenced by the mechanical microenvironment of materials, especially dynamic matrix mechanics of materials largely remains unknown. A matrix dynamic stiffening material previous designed was used here to explore whether matrix dynamic stiffening could influence paracrine of angiogenic factors in MSCs. The cytoskeleton polymerization, chromatin remodeling and histone acetylation in MSCs were analyzed using phalloidin staining, chromatin condensation parameter analysis and immunofluorescent staining. The results found that the paracrine of MSCs cultured on dynamic stiffening material was higher than that on static stiffness materials. The cytoskeletal polymerization and nuclear deformation degrees of MSCs seeded on dynamic stiffening material were significantly higher than those seeded on materials with static stiffness. The dynamic stiffening substrate could promote the cytoskeletal polymerization to increase the nuclear deformation of MSCs and further induce the paracrine of MSCs. These findings will help to provide clinical guidelines for designing vascularized bone repair materials with appropriate mechanical properties.

Regulation of fenestra formation via actin-dynamin2 interaction in rat pituitary endothelial cells.

Endothelial fenestrae are transcellular pores divided by a diaphragm consisting of plasmalemma vesicle-associated protein (PLVAP). They function as a channel for peptide hormones and other substances. Invagination of the plasma membrane is necessary for the fenestra formation. The actin cytoskeleton is essential for scission of endocytic vesicles from the invaginated plasma membrane. Therefore, we examined the involvement of the actin cytoskeleton in fenestra formation in cultured endothelial cells isolated from the anterior lobe (AL) of the rat pituitary, using immunofluorescence and scanning electron microscopy. Inhibition of polymerization and depolymerization of the actin cytoskeleton by latrunculin A and jasplakinolide, respectively, remarkably increased the PLVAP-positive sieve plate area and number of fenestrae. Jasplakinolide significantly affected the arrangement of the fenestra on the cell surface, resulting in parallel serpentine furrows of the fenestra. These results suggest that the actin cytoskeleton not only induces fenestra formation but also regulates cell arrangement. Dynamin is a scission protein of the invaginated plasma membrane and interacts with the actin cytoskeleton. We found that dynamin2 is mainly expressed in the endothelial cells of the rat AL. We then investigated the function of dynamin2 by the treatment with dyngo-4a, a potent inhibitor of dynamin1 and dynamin2, on the fenestra formation. As a result, the PLVAP-positive area is significantly increased by the treatment. These results show that the actin-dynamin2 interaction is essential for the control of the fenestra formation in endothelial cells of rat AL. In conclusion, the actin cytoskeleton and dynamin2 function as regulators of endothelial fenestra formation.

Statin-induced increase in actin polymerization modulates GPCR dynamics and compartmentalization

The function of the actin cytoskeleton in cellular motility and trafficking has been widely studied. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol and its consequences on membrane dynamics are addressed only rarely. In a recent work, we reported that chronic cholesterol depletion using statins leads to significant polymerization of the actin cytoskeleton. In this work, we explore the effect of reorganization of the actin cytoskeleton on membrane dynamics under cholesterol-depleted condition. Specifically, we explore the role of actin cytoskeleton in regulating the dynamics of the serotonin1A receptor, a crucial neurotransmitter G protein-coupled receptor (GPCR) that plays a major role in the generation and modulation of cognitive and behavioral functions. For this, we analyzed the lateral dynamics of the serotonin1A receptor in cholesterol-depleted cells (using statins) by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) measurements. Our results indicate that lateral diffusion parameters of serotonin1A receptors in normal cells are consistent with models describing the diffusion of molecules in a homogeneous membrane. Interestingly, these parameters are altered in cholesterol-depleted cells and the receptor exhibits dynamic confinement. Notably, our results show that statin-induced dynamic confinement could be reversed by destabilization of the actin cytoskeleton. On a broader perspective, these results assume significance in understanding the modulatory role of the membrane environment on the organization and dynamics of GPCRs in diseases caused by altered cholesterol biosynthesis.