Actin Cytoskeletal Remodeling Research Articles

Solo regulates the localization and activity of PDZ-RhoGEF for actin cytoskeletal remodeling in response to substrate stiffness.

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study aimed to identify Solo-interacting proteins using the BioID, a proximal-dependent labeling method, and elucidate the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG colocalized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at the Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling. Furthermore, the purified Solo itself had little or negligible GEF activity, even its GEF-inactive mutant directly activated the GEF activity of PRG through interaction. Moreover, overexpression of the Solo and PRG binding domains, respectively, had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

Transient induction of actin cytoskeletal remodeling associated with dedifferentiation, proliferation, and redifferentiation stimulates cardiac regeneration

The formation of new and functional cardiomyocytes requires a 3-step process: dedifferentiation, proliferation, and redifferentiation, but the critical genes required for efficient dedifferentiation, proliferation, and redifferentiation remain unknown. In our study, a circular trajectory using single-nucleus RNA sequencing of the pericentriolar material 1 positive (PCM1+) cardiomyocyte nuclei from hearts 1 and 3 days after surgery-induced myocardial infarction (MI) on postnatal Day 1 was reconstructed and demonstrated that actin remodeling contributed to the dedifferentiation, proliferation, and redifferentiation of cardiomyocytes after injury. We identified four top actin-remodeling regulators, namely Tmsb4x, Tmsb10, Dmd, and Ctnna3, which we collectively referred to as 2D2P. Transiently expressed changes of 2D2P, using a polycistronic non-integrating lentivirus driven by Tnnt2 (cardiac-specific troponin T) promoters (Tnnt2-2D2P-NIL), efficiently induced transiently proliferative activation and actin remodeling in postnatal Day 7 cardiomyocytes and adult hearts. Furthermore, the intramyocardial delivery of Tnnt2-2D2P-NIL resulted in a sustained improvement in cardiac function without ventricular dilatation, thickened septum, or fatal arrhythmia for at least 4 months. In conclusion, this study highlights the importance of actin remodeling in cardiac regeneration and provides a foundation for new gene-cocktail-therapy approaches to improve cardiac repair and treat heart failure using a novel transient and cardiomyocyte-specific viral construct.

Sphingosine 1-phosphate signaling axis mediates neuropeptide S-induced invasive phenotype of endometriotic cells.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1, S1P3 and S1P5, three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3, given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

Drebrin is Required for Myosin-facilitated Actin Cytoskeletal Remodeling during Pulmonary Alveolar Development.

Alveolar septation increases gas-exchange surface area and requires coordinated cytoskeletal rearrangement in lung fibroblasts (LFs) to balance the demands of contraction and cell migration. We hypothesized that DBN (drebrin), a modulator of the actin cytoskeleton in neuronal dendrites, regulates the remodeling of the LF cytoskeleton. Using mice bearing a transgelin-Cre-targeted deletion of Dbn in pulmonary fibroblasts and pericytes, we examined alterations in alveolar septal outgrowth, LF spreading and migration, and actomyosin function. The alveolar surface area and number of alveoli were reduced, whereas alveolar ducts were enlarged, in mice bearing the dbn deletion (DBNΔ) compared with their littermates bearing only one dbn-Flox allele (control). Cultured DBNΔ LFs were deficient in their responses to substrate rigidity and migrated more slowly. Drebrin was abundant in the actin cortex and lamella, and the actin fiber orientation was less uniform in lamella of DBNΔ LFs, which limited the development of traction forces and altered focal adhesion dynamics. Actin fiber orientation is regulated by contractile NM2 (nonmuscle myosin-2) motors, which help arrange actin stress fibers into thick ventral actin stress fibers. Using fluorescence anisotropy, we observed regional intracellular differences in myosin regulatory light chain phosphorylation in control LFs that were altered by dbn deletion. Using perturbations to induce and then release stalling of NM2 on actin in LFs from both genotypes, we made predictions explaining how DBN interacts with actin and NM2. These studies provide new insight for diseases such as emphysema and pulmonary fibrosis, in which fibroblasts inappropriately respond to mechanical cues in their environment.

Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring.

Elevation of the intermediate amino acid metabolite Homocysteine (Hcy) causes Hyperhomocysteinemia (HHcy), a metabolic disorder frequently associated with mutations in the methionine-cysteine metabolic cycle as well as with nutritional deficiency and aging. The previous literature suggests that HHcy is a strong risk factor for cardiovascular diseases. Severe HHcy is well-established to correlate with vascular pathologies primarily via endothelial cell death. Though moderate HHcy is more prevalent and associated with an increased risk of cardiovascular abnormalities in later part of life, its precise role in endothelial physiology is largely unknown. In this study, we report that moderate elevation of Hcy causes endothelial dysfunction through impairment of their migration and proliferation. We established that unlike severe elevation of Hcy, moderate HHcy is not associated with suppression of endothelial VEGF/VEGFR transcripts and ROS induction. We further showed that moderate HHcy induces a sub-lethal ER stress that causes defective endothelial migration through abnormal actin cytoskeletal remodeling. We also found that sub-lethal increase in Hcy causes endothelial proliferation defect by suppressing mitochondrial respiration and concomitantly increases glycolysis to compensate the consequential ATP loss and maintain overall energy homeostasis. Finally, analyzing a previously published microarray dataset, we confirmed that these hallmarks of moderate HHcy are conserved in adult endothelial cells as well. Thus, we identified adaptive UPR and metabolic rewiring as two key mechanistic signatures in moderate HHcy-associated endothelial dysfunction. As HHcy is clinically associated with enhanced vascular inflammation and hypercoagulability, identifying these mechanistic pathways may serve as future targets to regulate endothelial function and health.

WAVE2 Is a Vital Regulator in Myogenic Differentiation of Progenitor Cells through the Mechanosensitive MRTFA-SRF Axis.

Skeletal myogenesis is an intricate process involving the differentiation of progenitor cells into myofibers, which is regulated by actin cytoskeletal dynamics and myogenic transcription factors. Although recent studies have demonstrated the pivotal roles of actin-binding proteins (ABPs) as mechanosensors and signal transducers, the biological significance of WAVE2 (Wiskott-Aldrich syndrome protein family member 2), an ABP essential for actin polymerization, in myogenic differentiation of progenitor cells has not been investigated. Our study provides important insights into the regulatory roles played by WAVE2 in the myocardin-related transcription factor A (MRTFA)-serum response factor (SRF) signaling axis and differentiation of myoblasts. We demonstrate that WAVE2 expression is induced during myogenic differentiation and plays a pivotal role in actin cytoskeletal remodeling in C2C12 myoblasts. Knockdown of WAVE2 in C2C12 cells reduced filamentous actin levels, increased globular actin accumulation, and impaired the nuclear translocation of MRTFA. Furthermore, WAVE2 depletion in myoblasts inhibited the expression and transcriptional activity of SRF and suppressed cell proliferation in myoblasts. Consequently, WAVE2 knockdown suppressed myogenic regulatory factors (i.e., MyoD, MyoG, and SMYD1) expressions, thereby hindering the differentiation of myoblasts. Thus, this study suggests that WAVE2 is essential for myogenic differentiation of progenitor cells by modulating the mechanosensitive MRTFA-SRF axis.

Autoregulation of the LIM kinases by their PDZ domain

LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the ‘x-Φ-G-Φ’ motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.

NET4 and RabG3 link actin to the tonoplast and facilitate cytoskeletal remodelling during stomatal immunity

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function being downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure involves a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune-triggered osmotic changes and actin cytoskeletal remodelling likely driving compact vacuolar morphologies.

Super-resolution imaging reveals the relationship between CaMKIIβ and drebrin within dendritic spines

Dendritic spines are unique postsynaptic structures that emerge from the dendrites of neurons. They undergo activity-dependent morphological changes known as structural plasticity. The changes involve actin cytoskeletal remodeling, which is regulated by actin-binding proteins. CaMKII is a crucial molecule in synaptic plasticity. Notably, CaMKIIβ subtype is known to bind to filamentous-actin and is closely involved in structural plasticity. We have shown that CaMKIIβ binds to drebrin, and is localized in spines as both drebrin-dependent and drebrin-independent pools. However, the nanoscale relationship between drebrin and CaMKIIβ within dendritic spines has not been clarified. In this study, we used stochastic optical reconstruction microscopy (STORM) to examine the detailed localization of these proteins. STORM imaging showed that CaMKIIβ co-localized with drebrin in the core region of spines, and localized in the submembrane region of spines without drebrin. Interestingly, the dissociation of CaMKIIβ and drebrin in the core region was induced by NMDA receptor activation. In drebrin knockdown neurons, CaMKIIβ was decreased in the core region but not in the submembrane region. Together it indicates that the clustering of CaMKIIβ in the spine core region is dependent on drebrin. These findings suggest that drebrin-dependent CaMKIIβ is in a standby state before its activation.

Sigma-1 Receptor Activation Is Protective against TGFβ2-Induced Extracellular Matrix Changes in Human Trabecular Meshwork Cells.

The trabecular meshwork (TM) route is the principal outflow egress of the aqueous humor. Actin cytoskeletal remodeling in the TM and extracellular matrix (ECM) deposition increase TM stiffness, outflow resistance, and elevate intraocular pressure (IOP). These alterations are strongly linked to transforming growth factor-β2 (TGFβ2), a known profibrotic cytokine that is markedly elevated in the aqueous humor of glaucomatous eyes. Sigma-1 receptor (S1R) has been shown to have neuroprotective effects in the retina, but data are lacking about its role in the TM. In this study, we identified the presence of S1R in mouse TM tissue and investigated the effect of an S1R agonist fluvoxamine (FLU) on TGFβ2-induced human TM cells regarding cell proliferation; ECM-related functions, including F-actin reorganization; and the accumulation of ECM elements. TGFβ2 increased the proliferation, cytoskeletal remodeling, and protein levels of fibronectin, collagen type IV, and connective tissue growth factor, and decreased the level of matrix metalloproteinase-2. Most importantly, FLU reversed all these effects of TGFβ2, suggesting that S1R agonists could be potential candidates for preserving TM function and thus maintaining normal IOP.

Tiam1 coordinates synaptic structural and functional plasticity underpinning the pathophysiology of neuropathic pain

Neuropathic pain is a common, debilitating chronic pain condition caused by damage or a disease affecting the somatosensory nervous system. Understanding the pathophysiological mechanisms underlying neuropathic pain is critical for developing new therapeutic strategies to treat chronic pain effectively. Tiam1 is a Rac1 guanine nucleotide exchange factor (GEF) that promotes dendritic and synaptic growth during hippocampal development by inducing actin cytoskeletal remodeling. Here, using multiple neuropathic pain animal models, we show that Tiam1 coordinates synaptic structural and functional plasticity in the spinal dorsal horn via actin cytoskeleton reorganization and synaptic NMDAR stabilization and that these actions are essential for the initiation, transition, and maintenance of neuropathic pain. Furthermore, an antisense oligonucleotides (ASO) targeting spinal Tiam1 persistently alleviate neuropathic pain sensitivity. Our findings suggest that Tiam1-coordinated synaptic functional and structural plasticity underlies the pathophysiology of neuropathic pain and that intervention of Tiam1-mediated maladaptive synaptic plasticity has long-lasting consequences in neuropathic pain management.

EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons.

Dendritic spines are the postsynaptic compartment of a neuronal synapse and are critical for synaptic connectivity and plasticity. A developmental precursor to dendritic spines, dendritic filopodia (DF), facilitate synapse formation by sampling the environment for suitable axon partners during neurodevelopment and learning. Despite the significance of the actin cytoskeleton in driving these dynamic protrusions, the actin elongation factors involved are not well characterized. We identified the Ena/VASP protein EVL as uniquely required for the morphogenesis and dynamics of DF. Using a combination of genetic and optogenetic manipulations, we demonstrated that EVL promotes protrusive motility through membrane-direct actin polymerization at DF tips. EVL forms a complex at nascent protrusions and DF tips with MIM/MTSS1, an I-BAR protein important for the initiation of DF. We proposed a model in which EVL cooperates with MIM to coalesce and elongate branched actin filaments, establishing the dynamic lamellipodia-like architecture of DF.

DIAPH3 condensates formed by liquid-liquid phase separation act as a regulatory hub for stress-induced actin cytoskeleton remodeling

Membraneless condensates, such as stress granules (SGs) and processing bodies (P-bodies), have attracted wide attention due to their unique feature of rapid response to stress without first requiring nuclear feedback. In this study, we identify diaphanous-related formin 3 (DIAPH3), an actin nucleator, as a scaffold protein to initiate liquid-liquid phase separation (LLPS) and form abundant cytosolic phase-separated DIAPH3 granules (D-granules) in mammalian cells such as HeLa, HEK293, and fibroblasts under various stress conditions. Neither mRNAs nor known stress-associated condensate markers, such as G3BP1, G3BP2, and TIA1 for SGs and DCP1A for P-bodies, are detected in D-granules. Using overexpression and knockout of DIAPH3, pharmacological interventions, and optogenetics, we further demonstrate that stress-induced D-granules spatially sequester DIAPH3 within the condensation to inhibit the assembly of actin filaments in filopodia. This study reveals that D-granules formed by LLPS act as a regulatory hub for actin cytoskeletal remodeling in response to stress.

Integrated bioinformatic analysis to understand the association between phthalate exposure and breast cancer progression

Phthalates have been extensively used as plasticizers while manufacturing plastic-based consumer products. Estradiol mimicking properties and association studies suggest phthalates may contribute to breast cancer (BC). We performed an in-silico analysis and functional studies to understand the association between phthalate exposure and BC progression. Search for phthalate-responsive genes using the comparative toxicogenomics database identified 20 genes as commonly altered in response to multiple phthalates exposure. Of the 20 genes, 12 were significantly differentially expressed between normal and BC samples. In BC samples, 9 out of 20 genes showed a negative correlation between promoter methylation and its expression. AHR, BAX, BCL2, CAT, ESR2, IL6, and PTGS2 expression differed significantly between metastatic and non-metastatic BC samples. Gene set enrichment analysis identified metabolism, ATP-binding cassette transporters, insulin signaling, and type II diabetes as highly enriched pathways. The diagnostic assessment based on 20 genes expression suggested a sensitivity and a specificity >0.91. The aberrantly expressed phthalate interactive gene influenced the overall survival of BC patients. Drug-gene interaction analysis identified 14 genes and 523 candidate drugs, including 19 BC treatment-approved drugs. Di(2-ethylhexyl) phthlate (DEHP) exposure increased the growth, proliferation, and migration of MCF-7 and MDA-MB-231 cells in-vitro. DEHP exposure induced morphological changes, actin cytoskeletal remodeling, increased ROS content, reduced basal level lipid peroxidation, and induced epithelial to mesenchymal transition (EMT). The present approach can help to explore the potentially damaging effects of environmental agents on cancer risk and understand the underlined pathways and molecular mechanisms.

Thromboxane-induced contractile response of mesenteric arterioles is diminished in the older rats and the older hypertensive rats.

Nearly all physiological processes are controlled at some level by G-protein-coupled receptor (GPCR) signaling activity. The thromboxane A2 (TXA2) receptor (TP) is a member of the GPCR family. The ultimate effect of TP receptor activation depends on the availability of specific G proteins, which in turn depend on the cell type, tissue, and disease state. However, the roles of the TXA2-TP signaling pathway executed under disease states are poorly defined. In this study, 16-week-spontaneously hypertensive rats (SHR), the 18-month-SHR (OldSHR), and the age-matched Wistar-Kyoto (WKY) rats were used to study the vasoconstriction of mesenteric resistance artery induced by TP-specific agonist, U-46619. Vasoconstriction induced by U-46619 was significantly attenuated in OldWKY and OldSHR rats, and mesenteric arteries with impaired response to U-46619 responded strongly to the adrenergic receptor agonist, phenylephrine. Similar vascular responses to U-46619 were obtained in endothelium-denuded mesenteric arteries. Accordingly, the expression of TP membrane proteins in mesenteric vessels was decreased, and the endogenous TP competitor, 8, 9-EET, in serum was increased, which was partly responsible for the decreased vascular reactivity of U-46619. Decreased TP membrane expression was associated with TP endocytosis, which involved actin cytoskeletal remodeling, including increased ratio of F-actin/G-actin in OldWKY and OldSHR rats. Hence, we studied the effects of TXA2 and its receptors on blood vessels and found that the TXA2-TP prostaglandin signaling pathway was impaired in older adults, which would facilitate the creation of “precision therapeutics” that possess selective efficacy in diseases.

The non-glycosylated protein of Toxocara canis MUC-1 interacts with proteins of murine macrophages.

Toxocariasis is a neglected parasitic disease caused predominantly by larvae of Toxocara canis. While this zoonotic disease is of major importance in humans and canids, it can also affect a range of other mammalian hosts. It is known that mucins secreted by larvae play key roles in immune recognition and evasion, but very little is understood about the molecular interactions between host cells and T. canis. Here, using an integrative approach (affinity pull-down, mass spectrometry, co-immunoprecipitation and bioinformatics), we identified 219 proteins expressed by a murine macrophage cell line (RAW264.7) that interact with prokaryotically-expressed recombinant protein (rTc-MUC-1) representing the mucin Tc-MUC-1 present in the surface coat of infective larvae of T. canis. Protein-protein interactions between rTc-MUC-1 and an actin binding protein CFL1 as well as the fatty acid binding protein FABP5 of RAW264.7 macrophages were also demonstrated in a human embryonic kidney cell line (HEK 293T). By combing predicted structural information on the protein-protein interaction and functional knowledge of the related protein association networks, we inferred roles for Tc-MUC-1 protein in the regulation of actin cytoskeletal remodelling, and the migration and phagosome formation of macrophage cells. These molecular interactions now require verification in vivo. The experimental approach taken here should be readily applicable to comparative studies of other ascaridoid nematodes (e.g. T. cati, Anisakis simplex, Ascaris suum and Baylisascaris procyonis) whose larvae undergo tissue migration in accidental hosts, including humans.

Transcriptomic underpinnings of high and low mirror aggression zebrafish behaviours

BackgroundAggression is an adaptive behaviour that animals use to protect offspring, defend themselves and obtain resources. Zebrafish, like many other animals, are not able to recognize themselves in the mirror and typically respond to their own reflection with aggression. However, mirror aggression is not an all-or-nothing phenomenon, with some individuals displaying high levels of aggression against their mirror image, while others show none at all. In the current work, we have investigated the genetic basis of mirror aggression by using a classic forward genetics approach - selective breeding for high and low mirror aggression zebrafish (HAZ and LAZ).ResultsWe characterized AB wild-type zebrafish for their response to the mirror image. Both aggressive and non-aggressive fish were inbred over several generations. We found that HAZ were on average more aggressive than the corresponding LAZ across generations and that the most aggressive adult HAZ were less anxious than the least aggressive adult LAZ after prolonged selective breeding. RNAseq analysis of these fish revealed that hundreds of protein-encoding genes with important diverse biological functions such as arsenic metabolism (as3mt), cell migration (arl4ab), immune system activity (ptgr1), actin cytoskeletal remodelling (wdr1), corticogenesis (dgcr2), protein dephosphorylation (ublcp1), sialic acid metabolism (st6galnac3) and ketone body metabolism (aacs) were differentially expressed between HAZ and LAZ, suggesting a strong genetic contribution to this phenotype. DAVID pathway analysis showed that a number of diverse pathways are enriched in HAZ over LAZ including pathways related to immune function, oxidation-reduction processes and cell signalling. In addition, weighted gene co-expression network analysis (WGCNA) identified 12 modules of highly correlated genes that were significantly associated with aggression duration and/or experimental group.ConclusionsThe current study shows that selective breeding based of the mirror aggression phenotype induces strong, heritable changes in behaviour and gene expression within the brain of zebrafish suggesting a strong genetic basis for this behaviour. Our transcriptomic analysis of fish selectively bred for high and low levels of mirror aggression revealed specific transcriptomic signatures induced by selective breeding and mirror aggression and thus provides a large and novel resource of candidate genes for future study.

Spatiotemporal Dynamics and Mechanisms of Actin Cytoskeletal Re-modeling in Cells Perforated by Ultrasound-Driven Microbubbles.

To develop new strategies for improving the efficacy and biosafety of sonoporation-based macromolecule delivery, it is essential to understand the mechanisms underlying plasma membrane re-sealing and function recovery of the cells perforated by ultrasound-driven microbubbles. However, we lack a clear understanding of the spatiotemporal dynamics of the disrupted actin cytoskeleton and its role in the re-sealing of sonoporated cells. Here we used a customized experimental setup for single-pulse ultrasound (133.33-µs duration and 0.70-MPa peak negative pressure) exposure to microbubbles and for real-time recording of single-cell (human umbilical vein endothelial cell) responses by laser confocal microscopic imaging. We found that in reversibly sonoporated cells, the locally disrupted actin cytoskeleton, which was spatially correlated with the perforated plasma membrane, underwent three successive phases (expansion; contraction and re-sealing; and recovery) to re-model and that each phase of the disrupted actin cytoskeleton was approximately synchronized with that of the perforated plasma membrane. Moreover, compared with the closing time of the perforated plasma membrane, the same time was used for the re-sealing of the actin cytoskeleton in mildly sonoporated cells and a longer time was required in moderately sonoporated cells. Further, the generation, directional migration, accumulation and re-polymerization of globular actin polymers during the three phases drove the re-modeling of the actin cytoskeleton. However, in irreversibly sonoporated cells, the actin cytoskeleton, which underwent expansion and no contraction, was progressively de-polymerized and could not be re-sealed. Finally, we found that intracellular calcium transients were essential for the recruitment of globular actin and the re-modeling of the actin cytoskeleton. These results provide new insight into the role of actin cytoskeleton dynamics in the re-sealing of sonoporated cells and serve to guide the design of new strategies for sonoporation-based delivery.

Host genetics of innate immune system in infection.

The innate immune system is critical for initial recognition of infectious pathogens. Herein we highlight pathways critical to innate immunity as defined by the latest genetic findings. Several genes previously known to have recessive diseases now have dominant mutations, while other genes have both germline and somatic mutations now recognized, providing expanded phenotypes associated with each gene. Population-based variants in recognition and signaling directly impact infection susceptibility. Mutations in genes converging on STAT3 highlight signaling behind specific STAT3-related phenotypes. Novel roles for autophagy, actin cytoskeletal remodeling and DNA replication are described. Together these findings highlight both how rapidly our knowledge is advancing and how much there is still to learn about innate immunity.

Mechanosensitive channel inhibition attenuates TGFβ2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFβ2) levels within astrocytes have been described in glaucoma, and TGFβ signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFβ2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFβ2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFβ2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining and immunoblotting for GFAP. Levels of fibronectin and collagen IV deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (F4/80) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFβ2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFβ2 treatment and the presence of f-actin stress fibers. TGFβ2 treatment significantly increased GFAP, fibronectin, and collagen IV levels, and GsMTx4 co-treatment ameliorated GFAP immunoreactivity. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.