Polymerization Of Actin Cytoskeleton Research Articles

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.

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.

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.

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<0.001; p<0.02, respectively). However, there were no differences in TOC among studied groups (p>0.05). The AUC values for TAC=0.76 and AUC for 8-OHdG= 0.70 (all, p<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.

Exploitation of the host exocyst complex by bacterial pathogens.

Intracellular bacterial pathogens remodel the plasma membrane of eukaryotic cells in order to establish infection. A common and well-studied mechanism of plasma membrane remodelling involves bacterial stimulation of polymerization of the host actin cytoskeleton. Here, we discuss recent results showing that several bacterial pathogens also exploit the host vesicular trafficking pathway of 'polarized exocytosis' to expand and reshape specific regions in the plasma membrane during infection. Polarized exocytosis is mediated by an evolutionarily conserved octameric protein complex termed the exocyst. We describe examples in which the bacteria Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Shigella flexneri co-opt the exocyst to promote internalization into human cells or intercellular spread within host tissues. We also discuss results showing that Legionella pneumophila or S. flexneri manipulate exocyst components to modify membrane vacuoles to favour intracellular replication or motility of bacteria. Finally, we propose potential ways that pathogens manipulate exocyst function, discuss how polarized exocytosis might promote infection and highlight the importance of future studies to determine how actin polymerization and polarized exocytosis are coordinated to achieve optimal bacterial infection.

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.

Trabecular Meshwork Cholesterol Levels Regulate Actin Polymerization and Tunneling Nanotubes

Trabecular meshwork (TM) tissue is made of highly contractile mechanosensitive cells regulating aqueous humor (AH) outflow facility and intraocular pressure (IOP). Increased actin‐mediated contraction of TM elevates IOP causing glaucoma and irreversible blindness. This study aims at understanding the role of cholesterol in regulating actin dynamics in human TM (HTM) cells. To test this, HTM cells were treated with SiR‐Actin in serum‐free media overnight and F‐actin changes were recorded using live‐cell time‐lapse (5 min interval) confocal imaging before and during exposure to ‐ A) 10mM methyl‐β‐cyclodextrin (MβCD) for 1h for acute removal of cellular cholesterol, B) 100μM cholesterol‐saturated MβCD (MβCD‐CHOL) for 1h to enrich cholesterol, and C) 10mM MβCD for 1h followed by cholesterol supplementation using MβCD‐CHOL for 1h. F‐actin fluorescence intensity changes were calculated and unpaired t‐test was used for statistical analyses and results were significant if p<0.05 with a sample size of n= 4‐57. HTM cells plated on coverslips were treated as A – C as mentioned above, cholesterol and lipid synthesis inhibitors for 24h ‐ D) 100μM atorvastatin and E) 20μM fatostatin, respectively, F) actin polymerization disruptor ‐ 10μM cytochalasin D (CYTD) for 1h, G) 10μM CYTD for 1h followed by 100μM MβCD‐CHOL for 1h, H) microtubule disruptor ‐ 1μM demecolcine (DEM) for 1h, I) 1μM DEM for 1h followed by 100μM MβCD‐CHOL for 1h. Cells without treatment were used as control. After all these treatments, cells were fixed and immunofluorescence (IF) was performed to check for cholesterol using filipin, F‐actin by phalloidin, focal adhesion (FA) localization based on ‐ vinculin and paxillin distribution. Further F‐actin/G‐actin in vitro assay was performed to quantify the changes in actin polymerization under the conditions A – I compared to the control. The imaging analysis showed cholesterol removal significantly decreased F‐actin fluorescence intensity from 20min sustaining up to 1h and decreased the FA distribution. Cholesterol supplementation led to partial rescue of actin and FA indicating the re‐establishment of actin cytoskeleton and increased tunneling nanotube structures enhancing the cell‐cell communication (Figure 1). Moreover, IF results show that atorvastatin and fatostatin decreased F‐actin and FA distribution. CYTD disrupted F‐actin network leaving remnant filament ends and decreased FA but cholesterol enrichment increased F‐actin branches formed at ~70‐degree angle indicating Arp2/3 activation. DEM treatment increased F‐actin and FA, and cholesterol enrichment induced thick F‐actin filaments. These results were confirmed by quantitative F/G‐actin ratio decrease and increase upon cholesterol removal and enrichment, respectively. Additionally, supplementing cholesterol after removal increased the ratio compared to solely removing cholesterol. Interestingly, F/G‐actin ratio increased under CYTD treatment indicating that CYTD disrupted longer actin filaments but helped polymerization of newly formed actin filaments. Finally, DEM treatment increased F/G‐actin ratio, which was further increased by cholesterol enrichment. This systematic evaluation of cholesterol modulation on TM actin cytoskeleton polymerization identifies the significance of maintaining membrane and cellular cholesterol levels and cellular communication for achieving IOP homeostasis.

OxLDL induces membrane structure rearrangement leading to biomechanics alteration and migration deficiency in macrophage

Cholesterol sequestration from plasma membrane has been shown to induce lipid packing disruption, causing actin cytoskeleton reorganization and polymerization, increasing cell stiffness and inducing lysosomal exocytosis in non-professional phagocytes. Similarly, oxidized form of low-density lipoprotein (oxLDL) has also been shown to disrupt lipid organization and packing in endothelial cells, leading to biomechanics alterations that interfere with membrane injury and repair. For macrophages, much is known about oxLDL effects in cell activation, cytokine production and foam cell formation. However, little is known about its impact in the organization of macrophage membrane structured domains and cellular mechanics, the focus of the present study. Treatment of bone marrow-derived macrophages (BMDM) with oxLDL not only altered membrane structure, and potentially the distribution of raft domains, but also induced actin rearrangement, diffuse integrin distribution and cell shrinkage, similarly to observed upon treatment of these cells with MβCD. Those alterations led to decreased migration efficiency. For both treatments, higher co-localization of actin cytoskeleton and GM1 was observed, indicating a similar mechanism of action involving raft-like domain dynamics. Lastly, like MβCD treatment, oxLDL also induced lysosomal spreading in BMDM. We propose that OxLDL induced re-organization of membrane/cytoskeleton complex in macrophages can be attributed to the insertion of oxysterols into the membrane, which lead to changes in lipid organization and disruption of membrane structure, similar to the effect of cholesterol depletion by MβCD treatment. These results indicate that oxLDL can induce physical alterations in the complex membrane/cytoskeleton of macrophages, leading to significant biomechanical changes that compromise cell behavior.

Scaffold Pore Curvature Influences ΜSC Fate through Differential Cellular Organization and YAP/TAZ Activity.

Tissue engineering aims to repair, restore, and/or replace tissues in the human body as an alternative to grafts and prostheses. Biomaterial scaffolds can be utilized to provide a three-dimensional microenvironment to facilitate tissue regeneration. Previously, we reported that scaffold pore size influences vascularization and extracellular matrix composition both in vivo and in vitro, to ultimately influence tissue phenotype for regenerating cranial suture and bone tissues, which have markedly different tissue properties despite similar multipotent stem cell populations. To rationally design biomaterials for specific cell and tissue fate specification, it is critical to understand the molecular processes governed by cell-biomaterial interactions, which guide cell fate specification. Building on our previous work, in this report we investigated the hypothesis that scaffold pore curvature, the direct consequence of pore size, modulates the differentiation trajectory of mesenchymal stem cells (MSCs) through alterations in the cytoskeleton. First, we demonstrated that sufficiently small pores facilitate cell clustering in subcutaneous explants cultured in vivo, which we previously reported to demonstrate stem tissue phenotype both in vivo and in vitro. Based on this observation, we cultured cell-scaffold constructs in vitro to assess early time point interactions between cells and the matrix as a function of pore size. We demonstrate that principle curvature directly influences nuclear aspect and cell aggregation in vitro. Scaffold pores with a sufficiently low degree of principle curvature enables cell differentiation; pharmacologic inhibition of actin cytoskeleton polymerization in these scaffolds decreased differentiation, indicating a critical role of the cytoskeleton in transducing cues from the scaffold pore microenvironment to the cell nucleus. We fabricated a macropore model, which allows for three-dimensional confocal imaging and demonstrates that a higher principle curvature facilitates cell aggregation and the formation of a potentially protective niche within scaffold macropores which prevents MSC differentiation and retains their stemness. Sufficiently high principle curvature upregulates yes-associated protein (YAP) phosphorylation while decreased principle curvature downregulates YAP phosphorylation and increases YAP nuclear translocation with subsequent transcriptional activation towards an osteogenic differentiation fate. Finally, we demonstrate that the inhibition of the YAP/TAZ pathway causes a defect in differentiation, while YAP/TAZ activation causes premature differentiation in a curvature-dependent way when modulated by verteporfin (VP) and 1-oleyl-lysophosphatidic acid (LPA), respectively, confirming the critical role of biomaterials-mediated YAP/TAZ signaling in cell differentiation and fate specification. Our data support that the principle curvature of scaffold macropores is a critical design criterion which guides the differentiation trajectory of mesenchymal stem cells’ scaffolds. Biomaterial-mediated regulation of YAP/TAZ may significantly contribute to influencing the regenerative outcomes of biomaterials-based tissue engineering strategies through their specific pore design.

The Role of WAVE2 Signaling in Cancer.

The Wiskott–Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE)—WAVE1, WAVE2 and WAVE3 regulate rapid reorganization of cortical actin filaments and have been shown to form a key link between small GTPases and the actin cytoskeleton. Upon receiving upstream signals from Rho-family GTPases, the WASP and WAVE family proteins play a significant role in polymerization of actin cytoskeleton through activation of actin-related protein 2/3 complex (Arp2/3). The Arp2/3 complex, once activated, forms actin-based membrane protrusions essential for cell migration and cancer cell invasion. Thus, by activation of Arp2/3 complex, the WAVE and WASP family proteins, as part of the WAVE regulatory complex (WRC), have been shown to play a critical role in cancer cell invasion and metastasis, drawing significant research interest over recent years. Several studies have highlighted the potential for targeting the genes encoding either part of or a complete protein from the WASP/WAVE family as therapeutic strategies for preventing the invasion and metastasis of cancer cells. WAVE2 is well documented to be associated with the pathogenesis of several human cancers, including lung, liver, pancreatic, prostate, colorectal and breast cancer, as well as other hematologic malignancies. This review focuses mainly on the role of WAVE2 in the development, invasion and metastasis of different types of cancer. This review also summarizes the molecular mechanisms that regulate the activity of WAVE2, as well as those oncogenic pathways that are regulated by WAVE2 to promote the cancer phenotype. Finally, we discuss potential therapeutic strategies that target WAVE2 or the WAVE regulatory complex, aimed at preventing or inhibiting cancer invasion and metastasis.

Cooperativity between β-agonists and c-Abl inhibitors in regulating airway smooth muscle relaxation.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of β-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and β-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and β-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.

RhoA- and Actin-Dependent Functions of Macrophages from the Rodent Cardiac Transplantation Model Perspective -Timing Is the Essence.

Simple SummaryThe functions of animal and human cells depend on the actin cytoskeleton and its regulating protein called the RhoA. The actin cytoskeleton and RhoA also regulate the response of the immune cells such as macrophages to the microbial invasion and/or the presence of a non-self, such as a transplanted organ. The immune response against transplant occurs in several steps. The early step occurring within days post-transplantation is called the acute rejection and the late step, occurring months to years post-transplantation, is called the chronic rejection. In clinical transplantation, acute rejection is easily manageable by the anti-rejection drugs. However, there is no cure for chronic rejection, which is caused by the macrophages entering the transplant and promoting blockage of its blood vessels and destruction of tissue. We discuss here how the inhibition of the RhoA and actin cytoskeleton polymerization in the macrophages, either by genetic interference or pharmacologically, prevents macrophage entry into the transplanted organ and prevents chronic rejection, and also how it affects the anti-microbial function of the macrophages. We also focus on the importance of timing of the macrophage functions in chronic rejection and how the circadian rhythm may affect the anti-chronic rejection and anti-microbial therapies.The small GTPase RhoA, and its down-stream effector ROCK kinase, and the interacting Rac1 and mTORC2 pathways, are the principal regulators of the actin cytoskeleton and actin-related functions in all eukaryotic cells, including the immune cells. As such, they also regulate the phenotypes and functions of macrophages in the immune response and beyond. Here, we review the results of our and other’s studies on the role of the actin and RhoA pathway in shaping the macrophage functions in general and macrophage immune response during the development of chronic (long term) rejection of allografts in the rodent cardiac transplantation model. We focus on the importance of timing of the macrophage functions in chronic rejection and how the circadian rhythm may affect the anti-chronic rejection therapies.

RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization.

Background: In this study, we investigated the mechanism of Rho GTPases signaling on Ang II-mediated cell migration and dedifferentiation in human aortic vascular smooth muscle cells (HA-VSMCs) and an Ang II-infusion mouse model. Methods: Cells were pretreated with different inhibitors or Ang II. Cell migration was detected by Wound healing and Transwell assay. Mice were treated with Ad-RhoA-shRNA virus or Irbesartan or fasudil and then infused with Ang II. Results: Ang II treatment induced HA-VSMCs migration in a dose- and time-dependent manner and reduced the expression of VSMC contractile proteins. These effects were significantly suppressed by the inhibition of Ang II type 1 receptor (AT1 receptor), RhoA, and Rho-associated kinase (ROCK). Furthermore, Ang II treatment promoted the activation of RhoA and ROCK, which was reduced by AT1 receptor inhibition. Meanwhile, Ang II treatment induced F-actin polymerization, which was inhibited after ROCK inhibition. In mice, Ang II infusion increased VSMC migration into the neointima and reduced VSMC differentiation proteins levels, and these effects were shown to be dependent on AT1 receptor and RhoA/ROCK pathway. Conclusion: This study reveals a novel mechanism by which Ang II regulates RhoA/ROCK signaling and actin polymerization via AT1 receptor and then affects VSMC dedifferentiation.

The Recombinant Form of Trypanosoma cruzi P21 Controls Infection by Modulating Host Immune Response.

Trypanosoma cruzi P21 protein (P21) is a putative secreted and immunomodulatory molecule with potent bioactive properties such as induction of phagocytosis and actin cytoskeleton polymerization. Despite the bioactive properties described so far, the action of P21 on parasite replication in muscle cell lineage or T. cruzi parasitism during acute experimental infection is unclear. We observed that recombinant P21 (rP21) decreased the multiplication of T. cruzi in C2C12 myoblasts, phenomenon associated with greater actin polymerization and IFN-γ and IL-4 higher expression. During experimental infection, lower cardiac nests, inflammatory infiltrate and fibrosis were observed in mice infected and treated with rP21. These results were correlated with large expression of IFN-γ counterbalanced by high levels of IL-10, which was consistent with the lower cardiac tissue injury found in these mice. We have also observed that upon stress, such as that induced by the presence of the IFN-γ cytokine, T. cruzi produced more P21. The effect of P21 in controlling the replication of T. cruzi, may indicate an evolutionary mechanism of survival developed by the parasite. Thus, when subjected to different stress conditions, the protozoan produces more P21, which induces T. cruzi latency in the host organism, enabling the protozoan to evade the host's immune system.

Activation of the RhoA-YAP-β-catenin signaling axis promotes the expansion of inner ear progenitor cells in 3D culture.

Cellular mechanotransduction plays an essential role in the development and differentiation of many cell types, but if and how mechanical cues from the extracellular matrix (ECM) influence the fate determination of inner ear progenitor cells (IEPCs) remains largely unknown. In the current study, we compared the biological behavior of IEPCs in Matrigel‐based suspension and encapsulated culture systems, and we found that the mechanical cues from the ECM promote the survival and expansion of IEPCs. Furthermore, we found that the mechanical cues from the ECM induced the accumulation of Ras homolog family member A (RhoA) and caused the polymerization of actin cytoskeleton in IEPCs. These changes in turn resulted in increased Yes‐associated protein (YAP) nuclear localization and enhanced expansion of IEPCs, at least partially through upregulating the canonical Wnt signaling pathway. We therefore provide the first demonstration that the RhoA‐YAP‐β‐catenin signaling axis senses and transduces mechanical cues from the ECM and plays crucial roles in promoting the expansion of IEPCs.

CD20 is dispensable for B-cell receptor signaling but is required for proper actin polymerization, adhesion and migration of malignant B cells.

Surface protein CD20 serves as the critical target of immunotherapy in various B-cell malignancies for decades, however its biological function and regulation remain largely elusive. Better understanding of CD20 function may help to design improved rational therapies to prevent development of resistance. Using CRISPR/Cas9 technique, we have abrogated CD20 expression in five different malignant B-cell lines. We show that CD20 deletion has no effect upon B-cell receptor signaling or calcium flux. Also B-cell survival and proliferation is unaffected in the absence of CD20. On the contrary, we found a strong defect in actin cytoskeleton polymerization and, consequently, defective cell adhesion and migration in response to homeostatic chemokines SDF1α, CCL19 and CCL21. Mechanistically, we could identify a reduction in chemokine-triggered PYK2 activation, a calcium-activated signaling protein involved in activation of MAP kinases and cytoskeleton regulation. These cellular defects in consequence result in a severely disturbed homing of B cells in vivo.

Hippocampal overexpression of SGK1 ameliorates spatial memory, rescues Aβ pathology and actin cytoskeleton polymerization in middle-aged APP/PS1 mice

The increasing occurrence and ineffective treatment of Alzheimer’s disease (AD) has become one of the major challenges of the world. Limited studies have shown that serum- and glucocorticoid-inducible kinase 1 (SGK1) is involved in spatial memory formation and consolidation, but its role in AD-like spatial memory impairment and the related mechanisms are not clear. In this study, we first examined the age-related changes of SGK1 in the hippocampus of female APP/PS1 (AD) mice. Based on the finding and our previous finding that significant spatial memory impairment was detected in 8-month old AD mice, SGK1-overexpressing AAV (oSGK1) was constructed and injected into the hippocampus of 9-month old AD mice. One month later, the behavior alterations, Aβ production and deposit as well as changes of CA1 spine density and selected actin polymerization remodeling proteins were examined. The results showed that significant decrease of SGK1 was detected in 10-month old AD mice. The spatial memory impairment, the production and deposit of Aβ were reversed by oSGK1. Levels of hippocampal ADAM10 (α-secretase) and IDE (Aβ degradase), actin remodeling related proteins Rictor, Rac1, Cdc42 and Profilin-1 were significantly increased after oSGK1 treatment while hippocampal BACE1 (γ-secretase) and Cofilin remained unchanged. Taken together, our findings demonstrated a pivotal role of SGK1 in the treatment of AD-related memory impairment through upregulation of non- amyloidogenic processing of APP and degradation of Aβ, increase in spine plasticity related proteins, indicating increase in hippocampal SGK1 may be a potent therapeutic target against AD.