Quantitation of F-actin in cytoskeletal reorganization: Context, methodology and implications.

The actin cytoskeleton is involved in a large number of cellular signaling events in addition to providing structural integrity to the cell. Actin polymerization is a key event during cellular signaling. Although the role of actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, the reorganization of the actin cytoskeleton upon signaling has been rarely explored due to lack of suitable assays. Keeping in mind this lacuna, we developed a confocal microscopy based approach that relies on high magnification imaging of cellular F-actin, followed by image reconstruction using commercially available software. In this review, we discuss the context and relevance of actin quantitation, followed by a detailed hands-on approach of the methodology involved with specific points on troubleshooting and useful precautions. In the latter part of the review, we elucidate the method by discussing applications of actin quantitation from our work in several important problems in contemporary membrane biology ranging from pathogen entry into host cells, to GPCR signaling and membrane-cytoskeleton interaction. We envision that future discovery of cell-permeable novel fluorescent probes, in combination with genetically encoded actin-binding reporters, would allow real-time visualization of actin cytoskeleton dynamics to gain deeper insights into active cellular processes in health and disease.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells.

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of -35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

The role of the Arp2/3 complex in shaping the dynamics and structures of branched actomyosin networks.

Actomyosin networks give cells the ability to move and divide. These networks contract and expand while being driven by active energy-consuming processes such as motor protein walking and actin polymerization. Actin dynamics is also regulated by actin-binding proteins, such as the actin-related protein 2/3 (Arp2/3) complex. This complex generates branched filaments, thereby changing the overall organization of the network. In this work, the spatiotemporal patterns of dynamical actin assembly accompanying the branching-induced reorganization caused by Arp2/3 were studied using a computational model (mechanochemical dynamics of active networks [MEDYAN]); this model simulates actomyosin network dynamics as a result of chemical reactions whose rates are modulated by rapid mechanical equilibration. We show that branched actomyosin networks relax significantly more slowly than do unbranched networks. Also, branched networks undergo rare convulsive movements, "avalanches," that release strain in the network. These avalanches are associated with the more heterogeneous distribution of mechanically linked filaments displayed by branched networks. These far-from-equilibrium events arising from the marginal stability of growing actomyosin networks provide a possible mechanism of the "cytoquakes" recently seen in experiments.

The PYR-PP2C-CKL2 module regulates ABA-mediated actin reorganization during stomatal closure.

Limiting water loss by reducing transpiration helps plants survive when water is limited. Under drought stress, abscisic acid (ABA)-mediated gene expression and anion channel activation regulate stomatal closure and stress responses. ABA-induced actin reorganization also affects stomatal closure, but the underlying molecular mechanism remains unclear. In this study, we discovered that under nonstress conditions, the clade A PP2C phosphatases, such as ABI1 and ABI2, interact with CKL2 and inhibit its kinase activity in Arabidopsis. Under drought stress, CKL2 kinase activity was released through the formation of a complex containing ABA, PP2C and a PYR1/PYL/RCAR family (PYL) receptor. The activated CKL2 regulating actin reorganization is another important process to maintain stomatal closure besides ABA-activated SnRK2 signaling. Moreover, CKL2 phosphorylated PYR1-LIKE 1, ABI1 and ABI2 at amino acid residues conserved among PYLs and PP2Cs, and stabilized ABI1 protein. Our results reveal that ABA signaling regulates actin reorganization to maintain stomatal closure during drought stress, and the feedback regulation of PYL1, ABI1 and ABI2 by the CKL2 kinase might fine-tune ABA signaling and affect plant ABA responses.

The effects of 808-nm near-infrared laser light irradiation on actin cytoskeleton reorganization in bone marrow mesenchymal stem cells.

Tailoring the cell organelles and thus changing cell homeostatic behavior has permitted the discovery of fascinating metabolic features enabling enhanced viability, differentiation, or quenching inflammation. Recently, photobiomodulation (PBM) has been accredited as an effective cell manipulation technique with promising therapeutic potential. In this prospective, in vitro results revealed that 808-nm laser light emitted by a hand-piece with a flat-top profile at an irradiation set up of 60 J/cm2 (1 W, 1 W/cm2; 60 s, continuous wave) regulates bone marrow stromal cell (BMSC) differentiation toward osteogenesis. Considering the importance of actin cytoskeleton reorganization, which controls a range of cell metabolic activities, comprising shape change, proliferation and differentiation, the aim of the current work is to assess whether PBM therapy, using a flat-top hand-piece at higher-fluence irradiation on BMSCs, is able to switch photon signals into the stimulation of biochemical/differentiating pathways involving key activators that regulate de novo actin polymerization. Namely, for the first time, we unearthed the role of the flat-top hand-piece at higher-fluence irradiation on cytoskeletal characteristics of BMSCs. These novel findings meet the needs of novel therapeutically protocols provided by laser treatment and the manipulation of BMSCs as anti-inflammatory, osteo-inductive platforms.

Norcantharidin Suppresses YD-15 Cell Invasion Through Inhibition of FAK/Paxillin and F-Actin Reorganization.

Norcantharidin (NCTD), a demethylated derivative of cantharidin, has been reported to exhibit activity against various types of cancers. However, the anti-invasive effects of NCTD and its molecular mechanism in human mucoepidermoid carcinoma (MEC) remain incompletely elucidated. Clonogenic, wound healing, invasion, zymography, western blotting and immunocytochemistry assays were performed in YD-15 cells to investigate the anti-invasive effect of NCTD and its molecular mechanism of action. The inhibitory effects of NCTD on invasiveness were compared with those of a novel focal adhesion kinase (FAK) kinase inhibitor, PF-562271. NCTD markedly suppressed the colony formation, migration, and invasion of YD-15 cells as well as the activities of MMP-2 and MMP-9. It disrupted F-actin reorganization through suppressing the FAK/Paxillin axis. Moreover, NCTD exhibited a powerful anti-invasive effect compared with that of PF-562271 in YD-15 cells. Collectively, these results suggest that NCTD has a potential anti-invasive activity against YD-15 cells. This study may clarify the impact of NCTD on migration and invasion of human MEC cells.

CED-10-WASP-Arp2/3 signaling axis regulates apoptotic cell corpse engulfment in C. elegans.

Efficient clearance of apoptotic cells is essential for tissue homeostasis in metazoans. Genetic studies in Caenorhabditis elegans have identified signaling cascades that activate CED-10/Rac1 GTPase and promote actin cytoskeletal rearrangement during apoptotic cell engulfment. However, the molecular connection between CED-10 activation and actin reorganization remains elusive. Here, we provide evidence that CED-10 binds to the Arp2/3 nucleation promoting factor WASP; CED-10 recruits WASP and Arp2/3 to apoptotic cell corpses in the phagocytes. The loss of WASP and Arp2/3 impaired cell corpse engulfment. Furthermore, we uncover that a WASP-activating factor SEM-5/GRB2 functions in the phagocytes to promote cell corpse clearance. Together, our results suggest CED-10 reorganizes the actin cytoskeleton by recruiting the WASP-Arp2/3 actin nucleation factors during apoptotic cell engulfment.

Bitter tastant quinine modulates glucagon-like peptide-1 exocytosis from clonal GLUTag enteroendocrine L cells via actin reorganization.

Enteroendocrine L cells in the gastrointestinal tract secrete glucagon-like peptide-1 (GLP-1), which plays an important role in glucose homeostasis. Here we investigated the effect of bitter tastant quinine on GLP-1 secretion using clonal GLUTag mouse enteroendocrine L cells. We found that GLUTag cells expressed putative quinine receptors at mRNA levels. Although application of quinine resulted in an increase of intracellular Ca2+ levels, which was mediated by Ca2+ release from the endoplasmic reticulum and Ca2+ influx through voltage-sensitive Ca2+ channels, quinine had little effect on GLP-1 secretion. Total internal reflection fluorescence microscopy and immunocytochemistry revealed that GLP-1-containing vesicles remained unfused with the plasma membrane and facilitated actin polymerization beneath the plasma membrane after application of quinine, respectively. Interestingly, application of forskolin together with quinine induced GLP-1 exocytosis from the cells. These results suggest that quinine does not induce GLP-1 secretion because it facilitates Ca2+ increase and actin reorganization but not cAMP increase, and both Ca2+ and cAMP are essential for GLP-1 secretion.

Bacterial metabolite S-equol modulates glucagon-like peptide-1 secretion from enteroendocrine L cell line GLUTag cells via actin polymerization.

S-equol is one of gut bacterial metabolites produced from soybean isoflavone daizein. While S-equol is known to promote glucose-induced insulin secretion from pancreatic ß cells, whether S-equol affects glucagon-like peptide-1 (GLP-1) secretion from enteroendoceine L cells remains unclear. Here we assessed the effect of S-equol on GLP-1 secretion from mouse enteroendocrine L cell line GLUTag cells. GLUTag cells expressed GPR30 and estrogen receptors, which are putative S-equol receptors. Application of S-equol induced an increase in intracellular Ca2+ levels via GPR30. However, S-equol did not enhance GLP-1 exocytosis, and long-term treatment of S-equol suppressed GLP-1 secretion. Moreover, immunocytochemistry revealed that S-equol increased the density of cortical actin filaments via G12/13 signaling under GPR30. These data suggest that S-equol prevents GLP-1 secretion as a result of competing regulation between Ca2+ mobilization and actin reorganization.

Calcineurin is an adaptor required for assembly of the TCR signaling complex.

The serine/threonine phosphatase calcineurin is a component of the T cell receptor (TCR) signalosome, where it promotes T cell activation by dephosphorylating LckS59. Using small interfering RNA (siRNA)-mediated knockdown and CRISPR-Cas9-targeted genetic disruption of the calcineurin A chain α and ß isoforms, we find that calcineurin also functions as an adaptor in TCR-signaled human T cells. Unlike inhibition of its phosphatase activity, in the absence of calcineurin A, TCR signaling results in attenuated actin rearrangement, markedly reduced TCR-Lck microcluster formation and recruitment of the adaptor RhoH, and diminished phosphorylation of critical targets downstream of Lck such as TCRζ and ZAP-70. Reconstitution of deficient T cells with either calcineurin Aα or Aß restores TCR microcluster formation and signaling, as does reconstitution with a phosphatase-inactive Aα chain. These results assign a non-enzymatic adaptor function to calcineurin in the formation and stabilization of a functional TCR signaling complex.

Changes in the intra- and extra-mechanical environment of the nucleus in Saos-2 osteoblastic cells during bone differentiation process: Nuclear shrinkage and stiffening in cell differentiation.

Osteogenic differentiation has been reportedly regulated by various mechanical stresses, including fluid shear stress and tensile and compressive loading. The promotion of osteoblastic differentiation by these mechanical stresses is accompanied by reorganization of the F-actin cytoskeleton, which is deeply involved in intracellular forces and the mechanical environment. However, there is limited information about the effect on the mechanical environment of the intracellular nucleus, such as the mechanical properties of the nucleus and intracellular forces exerted on the nucleus, which have recently been found to be directly involved in various cellular functions. Here, we investigated the changes in the intracellular force applied to the nucleus and the effect on nuclear morphology and mechanical properties during osteogenic differentiation in human osteoblast-like cells (Saos-2). We carried out cell morphological analyses with confocal fluorescence microscopy, nuclear indentation test with atomic force microscopy (AFM), and fluorescence recovery after photobleaching (FRAP) for intranuclear DNA. The results revealed that a significant reorganization of the F-actin cytoskeleton from the nuclear surfaces to the cell periphery occurred in the osteogenic differentiation processes, simultaneously with the reduction of compressive forces to the nucleus. Such changes also facilitated nuclear shrinkage and stiffening, and further intranuclear chromatin compaction. The results indicate that the reduction of the intracellular compressive force due to reorganization of the F-actin cytoskeleton affects the intra- and extra-mechanical environment of the nucleus, and this change may affect gene expression and DNA replication in the osteogenic differentiation process.

"Non-cytotoxic" doses of metal-organic framework nanoparticles increase endothelial permeability by inducing actin reorganization.

Cytotoxicity of nanoparticles is routinely characterized by biochemical assays such as cell viability and membrane integrity assays. However, these approaches overlook cellular biophysical properties including changes in the actin cytoskeleton, cell stiffness, and cell morphology, particularly when cells are exposed to "non-cytotoxic" doses of nanoparticles. Zeolitic imidazolate framework-8 nanoparticles (ZIF-8 NPs), a member of metal-organic framework family, has received increasing interest in various fields such as environmental and biomedical sciences. ZIF-8 NPs may enter the blood circulation system after unintended oral and inhalational exposure or intended intravenous injection for diagnostic and therapeutic applications, yet the effect of ZIF-8 NPs on vascular endothelial cells is not well understood. Here, the biophysical impact of "non-cytotoxic" dose ZIF-8 NPs on human aortic endothelial cells (HAECs) is investigated. We demonstrate that "non-cytotoxic" doses of ZIF-8 NPs, pre-defined by a series of biochemical assays, can increase the endothelial permeability of HAEC monolayers by causing cell junction disruption and intercellular gap formation, which can be attributed to actin reorganization within adjacent HAECs. Nanomechanical atomic force microscopy and super resolution fluorescence microscopy further confirm that "non-cytotoxic" doses of ZIF-8 NPs change the actin structure and cell morphology of HAECs at the single cell level. Finally, the underlying mechanism of actin reorganization induced by the "non-cytotoxic" dose ZIF-8 NPs is elucidated. Together, this study indicates that the "non-cytotoxic" doses of ZIF-8 NPs, intentionally or unintentionally introduced into blood circulation, may still pose a threat to human health, considering increased endothelial permeability is essential to the progression of a variety of diseases. From a broad view of cytotoxicity evaluation, it is important to consider the biophysical properties of cells, since they can serve as novel and more sensitive markers to assess nanomaterial's cytotoxicity.

Hierarchical morphogenesis of swallowtail butterfly wing scale nanostructures.

The study of color patterns in the animal integument is a fundamental question in biology, with many lepidopteran species being exemplary models in this endeavor due to their relative simplicity and elegance. While significant advances have been made in unraveling the cellular and molecular basis of lepidopteran pigmentary coloration, the morphogenesis of wing scale nanostructures involved in structural color production is not well understood. Contemporary research on this topic largely focuses on a few nymphalid model taxa (e.g., Bicyclus, Heliconius), despite an overwhelming diversity in the hierarchical nanostructural organization of lepidopteran wing scales. Here, we present a time-resolved, comparative developmental study of hierarchical scale nanostructures in Parides eurimedes and five other papilionid species. Our results uphold the putative conserved role of F-actin bundles in acting as spacers between developing ridges, as previously documented in several nymphalid species. Interestingly, while ridges are developing in P. eurimedes, plasma membrane manifests irregular mesh-like crossribs characteristic of Papilionidae, which delineate the accretion of cuticle into rows of planar disks in between ridges. Once the ridges have grown, disintegrating F-actin bundles appear to reorganize into a network that supports the invagination of plasma membrane underlying the disks, subsequently forming an extruded honeycomb lattice. Our results uncover a previously undocumented role for F-actin in the morphogenesis of complex wing scale nanostructures, likely specific to Papilionidae.

External pH Variations Modify Proliferation, Erythrophagocytosis, Cytoskeleton Remodeling, and Cell Morphology of Entamoeba histolytica Trophozoites.

To infect the human host, Entamoeba histolytica carries out processes requiring cytoskeleton remodeling, which involves reorganizing the actin fibers. However, little is known about the external influence factors, e.g., the pH, on the parasite's cytoskeleton remodeling or cell morphology. Such influence becomes relevant given the pH gradient that the amoeba cope with when going through the human colonic mucus during infection. Therefore, we analyzed the proliferation, the reorganization of the actin fibers, and other actin structures and cell shape during adhesion to fibronectin and erythrophagocytosis in trophozoites at different external pH conditions (6.0, 6.5, 6.8, 7.5, 8.0). We found that the best condition of external pH to perform such functions was 6.8. At acid pH, the trophozoites presented better-defined actin fibers that formed a more compact network, while at alkaline pH, the fibers reorganized, forming a looser and less defined network. Similarly, the number of actin dots also changed from acid to alkaline pH. In conclusion, the external pH alters the proliferation of the amoebas and promotes the dynamic restructuration of their cytoskeleton, allowing them to carry out their functions.

Transcriptome sequencing analysis of the role of ß-catenin in F-actin reorganization in embryonic palatal mesenchymal cells.

Background: Palatogenesis is a highly regulated and coordinated developmental process that is coordinated by multiple transcription factors and signaling pathways. Our previous studies identified that defective palatal shelf reorientation due to aberrant mesenchymal ß-catenin signaling is associated with Filamentous actin (F-actin) dysregulation. Herein, the underlying mechanism of mesenchymal ß-catenin in regulating F-actin cytoskeleton reorganization is further investigated. Methods: Firstly, ß-catenin silenced and overexpressed mouse embryonic palatal mesenchymal (MEPM) cells were established by adenovirus-mediated transduction. Subsequently, we compared transcriptomes of negative control (NC) group, ß-catenin knockdown (KD) group, or ß-catenin overexpression group respectively using RNA-sequencing (RNA-seq), and differentially expressed genes (DEGs) screened were further identified by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, in vivo experiments further verified the expression change of critical molecules. Results: We discovered 184 and 522 DEGs in the knockdown and overexpression groups compared to the NC group, respectively (adjusted P<0.05; |fold change| >2.0). Among these, 106 DEGs were altered in both groups. Gene Ontology (GO) enrichment analysis relating to biological functions identified cytokine-cytokine receptor interaction, and positive modulation of cellular migration. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment assessment indicated that these DEGs were chiefly linked by the regulation of signaling receptor activity and chemokine signaling pathways. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the similar expression trend of serum amyloid A3 (Saa3) and CXC-chemokine ligand 5 (Cxcl5) possibly involved in cytoskeletal rearrangement with RNA-seq data. Experiments in vivo displayed that no significant expression change of Saa3 and Cxcl5 was observed in ß-catenin knockout and overexpressed mouse models. Conclusions: Our study provides an expression landscape of DEGs in ß-catenin silenced and overexpressed MEPM cells, which emphasizes the important role of processes such as chemotactic factor and cell migration. Our data gain deeper insight into genes associated with F-actin reorganization that is regulated by ß-catenin either directly or by another route, which will contribute to further investigation of the exact mechanism of mesenchymal ß-catenin/F-actin in palatal shelf reorientation.

Treponema denticola-Induced RASA4 Upregulation Mediates Cytoskeletal Dysfunction and MMP-2 Activity in Periodontal Fibroblasts.

The periodontal complex consists of the periodontal ligament (PDL), alveolar bone, and cementum, which work together to turn mechanical load into biological responses that are responsible for maintaining a homeostatic environment. However oral microbes, under conditions of dysbiosis, may challenge the actin dynamic properties of the PDL in the context of periodontal disease. To study this process, we examined host-microbial interactions in the context of the periodontium via molecular and functional cell assays and showed that human PDL cell interactions with Treponema denticola induce actin depolymerization through a novel actin reorganization signaling mechanism. This actin reorganization mechanism and loss of cell adhesion is a pathological response characterized by an initial upregulation of RASA4 mRNA expression resulting in an increase in matrix metalloproteinase-2 activity. This mechanism is specific to the T. denticola effector protein, dentilisin, thereby uncovering a novel effect for Treponema denticola-mediated RASA4 transcriptional activation and actin depolymerization in primary human PDL cells.

Kainic Acid Activates TRPV1 via a Phospholipase C/PIP2-Dependent Mechanism in Vitro.

Kainic acid (KA) is an excitotoxic glutamate analogue produced by a marine seaweed. It elicits neuronal excitotoxicity leading to epilepsy in rodents. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1), a nonselective cation channel protein, by capsaicin, prevents KA-induced seizures in a mouse model of temporal lobe epilepsy. However, the precise mechanism behind this protective effect of capsaicin remains unclear. In order to analyze the direct effect of KA on TRPV1, we evaluated the ability of KA to activate TRPV1 and analyzed its binding to TRPV1 using a molecular modeling approach. In vitro, KA activates a Ca2+ influx into TRPV1 expressing HEK293 cells but not in contsrol HEK293 cells. Pretreatment with either capsaicin (1 M) or capsazepine (10 M; TRPV1 antagonist) prevents the effect of KA. Pharmacological inhibition of phospholipase C (PLC) by U73122 or overexpression of phosphatidylinositol 5 phosphatase (Synaptojanin 1; Synj-1) counters the effect of KA. Further, KA treatment causes actin reorganization in HEKTRPV1 cells and PLC inhibition by U73122 prevents this. Molecular modeling data revealed that KA binds to TRPV1 and prebinding with capsaicin prevents the binding of KA to TRPV1. Consistently, the lack of effect of KA in activating chicken TRPV1, which is insensitive to capsaicin, suggests that there is a significant overlap between the sites of KA and capsaicin activation of TRPV1. However, PLC inhibition did not suppress TRPV1 activation by capsaicin. Collectively, our data suggest that KA binds to and activates TRPV1 and causes actin reorganization via PLC-dependent mechanism in vitro. We propose that KA mediates Ca2+ induced toxicity possibly by activating TRPV1. Therefore, inhibiting TRPV1 will be a beneficial strategy in abating Ca2+-induced neurotoxicity.

Human Immunodeficiency Virus (HIV) Infection and Use of Illicit Substances Promote Secretion of Semen Exosomes that Enhance Monocyte Adhesion and Induce Actin Reorganization and Chemotactic Migration.

Semen exosomes (SE) from HIV-uninfected (HIV-) individuals potently inhibit HIV infection in vitro. However, morphological changes in target cells in response to SE have not been characterized or have the effect of HIV infection or the use of illicit substances, specifically psychostimulants, on the function of SE been elucidated. The objective of this study was to evaluate the effect of HIV infection, psychostimulant use, and both together on SE-mediated regulation of monocyte function. SE were isolated from semen of HIV- and HIV-infected (HIV+) antiretroviral therapy (ART)-naive participants who reported either using or not using psychostimulants. The SE samples were thus designated as HIV-Drug-, HIV-Drug+, HIV+Drug-, and HIV+Drug+. U937 monocytes were treated with different SEs and analyzed for changes in transcriptome, morphometrics, actin reorganization, adhesion, and chemotaxis. HIV infection and/or use of psychostimulants had minimal effects on the physical characteristics of SE. However, different SEs had diverse effects on the messenger RNA signature of monocytes and rapidly induced monocyte adhesion and spreading. SE from HIV infected or psychostimulants users but not HIV-Drug- SE, stimulated actin reorganization, leading to the formation of filopodia-like structures and membrane ruffles containing F-actin and vinculin that in some cases were colocalized. All SE stimulated monocyte chemotaxis to HIV secretome and activated the secretion of matrix metalloproteinases, a phenotype exacerbated by HIV infection and psychostimulant use. SE-directed regulation of cellular morphometrics and chemotaxis depended on the donor clinical status because HIV infection and psychostimulant use altered SE function. Although our inclusion criteria specified the use of cocaine, humans are poly-drug and alcohol users and our study participants used psychostimulants, marijuana, opiates, and alcohol. Thus, it is possible that the effects observed in this study may be due to one of these other substances or due to an interaction between different substances.

Corticotropin-releasing hormone and urocortin promote phagocytosis of rat macrophages through convergent but distinct pathways.

AIMS: Phagocytosis plays essential roles during inflammation and immune response. This study aims to explore the underlying mechanism of corticotropin-releasing hormone (CRH) and urocortin (UCN)-promoted phagocytosis of rat macrophages. MAIN METHODS: To induce phagocytosis, rat macrophages were incubated with carboxylated fluorescent microspheres. The phagocytosis activity was evaluated by flow cytometric analysis. Actin reorganization was determined by immunostaining with TRITC-labeled phalloidin and transmission electron microscopy (TEM) analysis. Protein expressions of p-RhoA, p-Rac1, p-extracellular signal-related kinase (ERK)1/2 and GAPDH were examined by Western blotting. Protein kinase C (PKC) and protein kinase A (PKA) activities were examined using PreTag non-radio activity assay. KEY FINDINGS: Administration of CRH or UCN alone significantly enhanced phagocytosis of microspheres by rat macrophages, as well as actin reorganization. Ligation of CRH and UCN with CRH receptor increased the phosphorylation of both RhoA and Rac1. Inhibition of RhoA/Rac1 signal pathway suppressed CRH- or UCN-enhanced phagocytosis and actin reorganization. Blockage of PKA signal by MDL-12330A decreased CRH or UCN-promoted p-RhoA and p-Rac1 expressions. Blockage of PKC signal by cholerythine choride decreased CRH or UCN-promoted p-Rac1 expression and UCN-promoted p-RhoA expression, but increased the CRH-induced p-RhoA expression. ERK1/2 was also activated and served as upstream factor of RhoA/Rac1 signal pathway. SIGNIFICANCE: The results reveal that CRH and UCN promote phagocytosis of rat macrophages through convergent but dissociable pathways. PKA/PKC-ERK1/2-RhoA/Rac1 signal pathway plays an essential role in CRH- and UCN-enhanced phagocytosis.