Disruption Of F-actin Research Articles

Epigallocatechin-3-gallate improves the quality of maternally aged oocytes.

The decline in female fertility as age advances is intricately linked to the diminished developmental potential of oocytes. Despite this challenge, the strategies available to enhance the quality of aged oocytes remain limited. Epigallocatechin-3-gallate (EGCG), characterised by its anti-inflammatory, antioxidant and tissue protective properties, holds promise as a candidate for improving the quality of maternally aged oocytes. In this study, we explored the precise impact and underlying mechanisms of EGCG on aged oocytes. EGCG exhibited the capacity to enhance the quality of aged oocytes both in vitro and in vivo. Specifically, the application of EGCG in vitro resulted in noteworthy improvements, including an increased rate of first polar body extrusion, enhanced mitochondrial function, refined spindle morphology and a reduction in oxidative stress. These beneficial effects were further validated by the improved fertility observed among aged mice. In addition, our findings propose that EGCG might augment the expression of Arf6. This augmentation, in turn, contributes to the assembly of spindle-associated F-actin, which can contribute to mitigate the aneuploidy induced by the disruption of spindle F-actin within aged oocytes. This work thus contributes not only to understanding the role of EGCG in bolstering oocyte health, but also underscores its potential as a therapeutic intervention to address fertility challenges associated with advanced age.

Tankyrase inhibition interferes with junction remodeling, induces leakiness, and disturbs YAP1/TAZ signaling in the endothelium

Tankyrase inhibitors are increasingly considered for therapeutic use in malignancies that are characterized by high intrinsic β-catenin activity. However, how tankyrase inhibition affects the endothelium after systemic application remains poorly understood. In this study, we aimed to investigate how the tankyrase inhibitor XAV939 affects endothelial cell function and the underlying mechanism involved. Endothelial cell function was analyzed using sprouting angiogenesis, endothelial cell migration, junctional dynamics, and permeability using human umbilical vein endothelial cells (HUVEC) and explanted mouse retina. Underlying signaling was studied using western blot, immunofluorescence, and qPCR in HUVEC in addition to luciferase reporter gene assays in human embryonic kidney cells. XAV939 treatment leads to altered junctional dynamics and permeability as well as impaired endothelial migration. Mechanistically, XAV939 increased stability of the angiomotin-like proteins 1 and 2, which impedes the nuclear translocation of YAP1/TAZ and consequently suppresses TEAD-mediated transcription. Intriguingly, XAV939 disrupts adherens junctions by inducing RhoA-Rho dependent kinase (ROCK)-mediated F-actin bundling, whereas disruption of F-actin bundling through the ROCK inhibitor H1152 restores endothelial cell function. Unexpectedly, this was accompanied by an increase in nuclear TAZ and TEAD-mediated transcription, suggesting differential regulation of YAP1 and TAZ by the actin cytoskeleton in endothelial cells. In conclusion, our findings elucidate the complex relationship between the actin cytoskeleton, YAP1/TAZ signaling, and endothelial cell function and how tankyrase inhibition disturbs this well-balanced signaling.

Abstract P1182: The Genetic Dissection Of Rpl3l As A Causal Gene For Neonatal Dilated Cardiomyopathy And Left Ventricular Noncompaction

Background: Cardiomyopathies are responsible for nearly 10% of cardiac deaths in neonates. Recently, biallelic variants in ribosomal protein L3-like ( RPL3L , NM_005061.2) have been reported in neonates with dilated cardiomyopathy (DCM) and heart failure (HF). This study is aimed to study RPL3L induced genetic pathways and mechanisms involved in the development of DCM. Methods: Whole exome and RNA sequencing, histology, immunohistochemistry, and Western blotting were performed in a human explanted heart. Genetic correlation, expression quantitative trait loci (eQTL) mapping and functional analysis were performed using cardiac gene expression data from the patient as well as human and murine genetic reference populations (GRPs). Results: Two-month-old patient with DCM and HF carrying compound heterozygous RPL3L variants, p.A51T and p.V231F, has been managed on an INTERMACS profile 2 with continuous-flow ventricular assist device and successfully bridged to heart transplantation at the age of 4 months. Morphology of the explanted heart displayed apical deep intertrabecular recesses consistent with left ventricular noncompaction (LVNC). Histology revealed interstitial fibrosis and myocardial hypertrophy. No signs of myocarditis and glycogen or mucopolysaccharides deposits were detected. Immunohistochemistry revealed abnormal clusters of RPL3L protein in the cytosol of cardiomyocytes and disruption of F-actin, Z-disk proteins, MYL4 and dystrophin was evident. In a mouse heart, the Rpl3l level was of 33.45 RPKM, while levels of RPL3L appeared to be 32 RPKM in a human heart compared to its mean expression in other adult tissues of 2.54 (mouse) and 2.36 (human). Systems genetics analysis identified high expression values ranged from 11.31 to 12.16 across murine GRPs of BXD mice with the ~1.8-fold difference. Pathways such as “thermogenesis”, “diabetic cardiomyopathy” and “DCM” significantly associated with RPL3L. e QTL mapping suggested Myl4 (Chr 11) and Sdha (Chr 13) as the upstream regulators of Rpl3l . Conclusions: Compound RPL3L heterozygosity is causal for neonatal DCM and LVNC. The RPL3L is highly expressed in the heart tissue of humans and mice. Genes, Myl4 and Sdha, are strong candidates that regulate expression of Rpl3l in murine heart.

Blocking Store-Operated Ca2+ Entry to Protect HL-1 Cardiomyocytes from Epirubicin-Induced Cardiotoxicity.

Epirubicin (EPI) is one of the most widely used anthracycline chemotherapy drugs, yet its cardiotoxicity severely limits its clinical application. Altered intracellular Ca2+ homeostasis has been shown to contribute to EPI-induced cell death and hypertrophy in the heart. While store-operated Ca2+ entry (SOCE) has recently been linked with cardiac hypertrophy and heart failure, its role in EPI-induced cardiotoxicity remains unknown. Using a publicly available RNA-seq dataset of human iPSC-derived cardiomyocytes, gene analysis showed that cells treated with 2 µM EPI for 48 h had significantly reduced expression of SOCE machinery genes, e.g., Orai1, Orai3, TRPC3, TRPC4, Stim1, and Stim2. Using HL-1, a cardiomyocyte cell line derived from adult mouse atria, and Fura-2, a ratiometric Ca2+ fluorescent dye, this study confirmed that SOCE was indeed significantly reduced in HL-1 cells treated with EPI for 6 h or longer. However, HL-1 cells presented increased SOCE as well as increased reactive oxygen species (ROS) production at 30 min after EPI treatment. EPI-induced apoptosis was evidenced by disruption of F-actin and increased cleavage of caspase-3 protein. The HL-1 cells that survived to 24 h after EPI treatment demonstrated enlarged cell sizes, up-regulated expression of brain natriuretic peptide (a hypertrophy marker), and increased NFAT4 nuclear translocation. Treatment by BTP2, a known SOCE blocker, decreased the initial EPI-enhanced SOCE, rescued HL-1 cells from EPI-induced apoptosis, and reduced NFAT4 nuclear translocation and hypertrophy. This study suggests that EPI may affect SOCE in two phases: the initial enhancement phase and the following cell compensatory reduction phase. Administration of a SOCE blocker at the initial enhancement phase may protect cardiomyocytes from EPI-induced toxicity and hypertrophy.

Actin limits egg aneuploidies associated with female reproductive aging.

Aging-related centromeric cohesion loss underlies premature separation of sister chromatids and egg aneuploidy in reproductively older females. Here, we show that F-actin maintains chromatid association after cohesion deterioration in aged eggs. F-actin disruption in aged mouse eggs exacerbated untimely dissociation of sister chromatids, while its removal in young eggs induced extensive chromatid separation events generally only seen in advanced reproductive ages. In young eggs containing experimentally reduced cohesion, F-actin removal accelerated premature splitting and scattering of sister chromatids in a microtubule dynamics-dependent manner, suggesting that actin counteracts chromatid-pulling spindle forces. Consistently, F-actin stabilization restricted scattering of unpaired chromatids generated by complete degradation of centromeric cohesion proteins. We conclude that actin mitigates egg aneuploidies arising from age-related cohesion depletion by limiting microtubule-driven separation and dispersion of sister chromatids. This is supported by our finding that spindle-associated F-actin structures are disrupted in eggs of reproductively older females.

A Glu-Glu-Tyr Sequence in the Cytoplasmic Tail of the M2 Protein Renders Influenza A Virus Susceptible to Restriction of the Hemagglutinin-M2 Association in Primary Human Macrophages.

Influenza A virus (IAV) assembly at the plasma membrane is orchestrated by at least five viral components, including hemagglutinin (HA), neuraminidase (NA), matrix (M1), the ion channel M2, and viral ribonucleoprotein (vRNP) complexes, although particle formation is observed with expression of only HA and/or NA. While these five viral components are expressed efficiently in primary human monocyte-derived macrophages (MDMs) upon IAV infection, this cell type does not support efficient HA-M2 association and IAV particle assembly at the plasma membrane. Both defects are specific to MDMs and can be reversed upon disruption of F-actin. However, the relationship between the two defects is unclear. Here, we examined whether M2 contributes to particle assembly in MDMs and if so, which region of M2 determines the susceptibility to the MDM-specific and actin-dependent suppression. An analysis using correlative fluorescence and scanning electron microscopy showed that an M2-deficient virus failed to form budding structures at the cell surface even after F-actin was disrupted, indicating that M2 is essential for virus particle formation at the MDM surface. Notably, proximity ligation analysis revealed that a single amino acid substitution in a Glu-Glu-Tyr sequence (residues 74 to 76) in the M2 cytoplasmic tail allowed the HA-M2 association to occur efficiently even in MDMs with intact actin cytoskeleton. This phenotype did not correlate with known phenotypes of the M2 substitution mutants regarding M1 interaction or vRNP packaging in epithelial cells. Overall, our study identified M2 as a target of MDM-specific restriction of IAV assembly, which requires the Glu-Glu-Tyr sequence in the cytoplasmic tail. IMPORTANCE Human MDMs represent a cell type that is nonpermissive to particle formation of influenza A virus (IAV). We previously showed that close proximity association between viral HA and M2 proteins is blocked in MDMs. However, whether MDMs express a restriction factor against IAV assembly or whether they lack a dependency factor promoting assembly remained unknown. In the current study, we determined that the M2 protein is necessary for particle formation in MDMs but is also a molecular target of the MDM-specific suppression of assembly. Substitutions in the M2 cytoplasmic tail alleviated the block in both the HA-M2 association and particle production in MDMs. These findings suggest that MDMs express dependency factors necessary for assembly but also express a factor(s) that inhibits HA-M2 association and particle formation. High conservation of the M2 sequence rendering the susceptibility to the assembly block highlights the potential for M2 as a target of antiviral strategies.

Centrosome-dependent microtubule modifications set the conditions for axon formation.

Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.

Uncovering the F-Actin-Based Nuclear Egress Mechanism of Newly Synthesized Influenza A Virus Ribonucleoprotein Complexes by Single-Particle Tracking.

Nuclear trafficking of viral genome is an essential cellular process in the life cycles of viruses. Despite substantial progress in uncovering a wide variety of complicated mechanisms of virus entry, intracellular transport of viral components, virus assembly, and egress, the temporal and spatial dynamics of viral genes trafficking within the nucleus remains poorly understood. Herein, using single-particle tracking, we explored the real-time dynamic nuclear trafficking of influenza A virus (IAV) genes packaged as the viral ribonucleoprotein complexes (vRNPs) by combining a four-plasmid DNA transfection system for the reconstruction of green fluorescent protein (GFP)-labeled vRNPs and a spinning disk super-resolution fluorescence microscope. We found that IAV infection significantly induced the formation of actin microfilaments (F-actin) in the nucleus. In combination with the fluorescent protein-tagged nuclear F-actin probe, we visualized the directed movement of GFP-labeled vRNPs foci along the nuclear F-actin with a speed of 0.18 μm/s, which is similar to the microfilaments-dependent slow directed motion of IAVs in the cytoplasm. The disruption of nuclear F-actin after treatment with microfilament inhibitors caused a considerable decrease in vRNPs motility and suppressed the nuclear export of newly produced vRNPs, indicating that the slow, directed movement plays a crucial role in facilitating the nuclear egress of vRNPs. Our findings identified a nuclear F-actin-dependent pathway for IAV vRNPs transporting from the nucleus into the cytoplasm, which may in turn uncover a novel target for antiviral treatment.

The prophase oocyte nucleus is a homeostatic G-actin buffer.

ABSTRACTFormation of healthy mammalian eggs from oocytes requires specialised F-actin structures. F-actin disruption produces aneuploid eggs, which are a leading cause of human embryo deaths, genetic disorders and infertility. We found that oocytes contain prominent nuclear F-actin structures that are correlated with meiotic developmental capacity. We demonstrate that nuclear F-actin is a conserved feature of healthy mammalian oocytes and declines significantly with female reproductive ageing. Actin monomers used for nuclear F-actin assembly are sourced from an excess pool in the oocyte cytoplasm. Increasing monomeric G-actin transfer from the cytoplasm to the nucleus or directly enriching the nucleus with monomers led to assembly of stable nuclear F-actin bundles that significantly restrict chromatin mobility. By contrast, reducing G-actin monomer transfer by blocking nuclear import triggered assembly of a dense cytoplasmic F-actin network that is incompatible with healthy oocyte development. Overall, our data suggest that the large oocyte nucleus helps to maintain cytoplasmic F-actin organisation and that defects in this function are linked with reproductive age-related female infertility.This article has an associated First Person interview with Federica Giannini, joint first author of the paper.

Influencing the Actin Dynamics in Plant Cells by Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latrunculins.

This chapter presents an overview of the most common F-actin influencing substances, used to study actin dynamics in living plant cells for studies on morphogenesis, motility, organelle movement, apoptosis, or abiotic stress. These substances can be divided into two major subclasses-F-actin-stabilizing and F-actin-polymerizing substances like jasplakinolide and chondramides and F-actin-severing compounds like cytochalasins and latrunculins. Jasplakinolide, which may have anti-cancer activities, was originally isolated from a marine sponge and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane-permeable F-actin-stabilizing and F-actin-polymerizing agent. Recently an acyclic derivate of jasplakinolide was isolated. Cytochalasins, derived from fungi, show an F-actin-severing function, and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from Red Sea sponges; however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide, isolated from red algae or the cyanobacterium Nostoc, a stable complex with actin dimers is formed, resulting in severing F-actin.For influencing F-actin dynamics in plant cells, only membrane permeable drugs are useful in a broad range. We, however, introduce also the phallotoxins and synthetic derivatives thereof, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes but has also been described in siphonal giant algae. The focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; moreover fluorescence methods for phalloidin- and antibody staining as well as techniques for immunoelectron microscopy are explained.

Abstract MP24: Disruption Of Actin Remodeling Intensifies Aneurysm-promoting Signals In Smooth Muscle Cells

Mutations within proteins coding for extracellular matrix assembly or those in the transforming growth factor beta (TGFβ) signaling pathway lead to aneurysm formation in patients and mice. As the SMC cytoskeletal network is intricately linked to TGFβ and ECM signaling, we sought to determine if disruption of the cytoskeletal filaments would alter how SMCs respond to angiotensin II, an important mediator of aneurysm formation. C57BL/6 mice lacking Tgbfr1 in SMCs (SMC- Tgbr1 iko ) generated by Jiang et al. have been shown to develop rapid and severe aortic aneurysm degeneration with 100% penetrance. Analysis was conducted on aneurysm and control mouse and human aortic tissue sections. We observed that SMC- Tgbr1 iko mice had reduced expression of filamentous (f-) and α-actin protein in the aortic media than control mice. Primary Tgbr1 iko SMCs also had higher levels of pRelA expression, less α-actin mRNA, and less f-actin than control SMCs. Tissue sections from aneurysm patients had reduced f-actin staining relative to controls. Disruption of f-actin with the destabilizer latrunculin A in vitro led to increased RelA and ERK activation in the presence of AngII, which was further augmented in the presence of a neutralizing antibody for TGFβ. In contrast, jasplakinolide—an f-actin stabilizer—prevented TGFβ blockade induced pRelA expression, suggesting that altered/inhibited TGFβ signaling modulates NFkB through actin depolymerization. While stabilization and disruption of microtubules led to RelA activation, no effect was observed on TGFβ blockade-induced pRelA expression. The phosphoantibody array revealed that f-actin disruption in the presence of AngII activated the CREB, AKT1/2/3, ERK1/2, and STAT3 pathways in mouse aortic SMCs. SMC- Tgbr1 iko mice treated with cytochalasin B suffered significantly greater mortality and had increased aortic growth over a 30-day follow-up period. Aortic aneurysms are coincident with reduced f-actin expression in murine and human aortic SMCs. Chemical deconstruction of actin filaments promotes an inflammatory phenotype in mouse SMCs in vitro , and aggravates aneurysm formation in vivo . Aberrant or inhibited TGFβ signaling potentiates NFkB activation in SMCs, which may be dependent on f-actin disassembly.

Insulin-like growth factor-1 regulates the mechanosensitivity of chondrocytes by modulating TRPV4

Both mechanical and biochemical stimulation are required for maintaining the integrity of articular cartilage. However, chondrocytes respond differently to mechanical stimuli in osteoarthritic cartilage when biochemical signaling pathways, such as Insulin-like Growth Factor-1 (IGF-1), are altered. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel is central to chondrocyte mechanotransduction and regulation of cartilage homeostasis. Here, we propose that changes in IGF-1 can modulate TRPV4 channel activity. We demonstrate that physiologic levels of IGF-1 suppress hypotonic-induced TRPV4 currents and intracellular calcium flux by increasing apparent cell stiffness that correlates with actin stress fiber formation. Disruption of F-actin following IGF-1 treatment results in the return of the intracellular calcium response to hypotonic swelling. Using point mutations of the TRPV4 channel at the microtubule-associated protein 7 (MAP-7) site shows that regulation of TRPV4 by actin is mediated via the interaction of actin with the MAP-7 domain of TRPV4. We further highlight that ATP release, a down-stream response to mechanical stimulation in chondrocytes, is mediated by TRPV4 during hypotonic challenge. This response is significantly abrogated with IGF-1 treatment. As chondrocyte mechanosensitivity is greatly altered during osteoarthritis progression, IGF-1 presents as a promising candidate for prevention and treatment of articular cartilage damage.

FHL2 anchors mitochondria to actin and adapts mitochondrial dynamics to glucose supply.

Mitochondrial movement and distribution are fundamental to their function. Here we report a mechanism that regulates mitochondrial movement by anchoring mitochondria to the F-actin cytoskeleton. This mechanism is activated by an increase in glucose influx and the consequent O-GlcNAcylation of TRAK (Milton), a component of the mitochondrial motor-adaptor complex. The protein four and a half LIM domains protein 2 (FHL2) serves as the anchor. FHL2 associates with O-GlcNAcylated TRAK and is both necessary and sufficient to drive the accumulation of F-actin around mitochondria and to arrest mitochondrial movement by anchoring to F-actin. Disruption of F-actin restores mitochondrial movement that had been arrested by either TRAK O-GlcNAcylation or forced direction of FHL2 to mitochondria. This pathway for mitochondrial immobilization is present in both neurons and non-neuronal cells and can thereby adapt mitochondrial dynamics to changes in glucose availability.

Selective mediation of ovarian cancer SKOV3 cells death by pristine carbon quantum dots/Cu2O composite through targeting matrix metalloproteinases, angiogenic cytokines and cytoskeleton

It was shown that some nanomaterials may have anticancer properties, but lack of selectivity is one of challenges, let alone selective suppression of cancer growth by regulating the cellular microenvironment. Herein, we demonstrated for the first time that carbon quantum dots/Cu2O composite (CQDs/Cu2O) selectively inhibited ovarian cancer SKOV3 cells by targeting cellular microenvironment, such as matrix metalloproteinases, angiogenic cytokines and cytoskeleton. The result was showed CQDs/Cu2O possessed anticancer properties against SKOV3 cells with IC50 = 0.85 μg mL−1, which was approximately threefold lower than other tested cancer cells and approximately 12-fold lower than normal cells. Compared with popular anticancer drugs, the IC50 of CQDs/Cu2O was approximately 114-fold and 75-fold lower than the IC50 of commercial artesunate (ART) and oxaliplatin (OXA). Furthermore, CQDs/Cu2O possessed the ability to decrease the expression of MMP-2/9 and induced alterations in the cytoskeleton of SKOV3 cells by disruption of F-actin. It also exhibited stronger antiangiogenic effects than commercial antiangiogenic inhibitor (SU5416) through down-regulating the expression of VEGFR2. In addition, CQDs/Cu2O has a vital function on transcriptional regulation of multiple genes in SKOV3 cells, where 495 genes were up-regulated and 756 genes were down-regulated. It is worth noting that CQDs/Cu2O also regulated angiogenesis-related genes in SKOV3 cells, such as Maspin and TSP1 gene, to suppress angiogenesis. Therefore, CQDs/Cu2O selectively mediated of ovarian cancer SKOV3 cells death mainly through decreasing the expression of MMP-2, MMP-9, F-actin, and VEGFR2, meanwhile CQDs/Cu2O caused apoptosis of SKOV3 via S phase cell cycle arrest. These findings reveal a new application for the use of CQDs/Cu2O composite as potential therapeutic interventions in ovarian cancer SKOV3 cells.

Efficacy of bioactive nanoparticles on tissue-endotoxin induced suppression of stem cell viability, migration and differentiation.

To characterize a lipopolysaccharide (LPS)-treated dentine tissue model (LPS dentine) to analyse the efficacy of polycationic chitosan nanoparticles (CSnp) and/or dexamethasone conjugate chitosan nanoparticles (Dex-CSnp) on the viability/differentiation potential of stem cells from apical papilla (SCAP) when exposed to LPS dentine. A further aim was to understand the effect of macrophage-dependent inflammation on SCAP migration in the presence of LPS dentine. A total of 88 dentine slabs were used. TOF-SIMS analysis was performed amongst the LPS-treated and untreated dentine groups (n=2/group). The study was conducted using four dentine groups: no treatment (control); LPS treatment only; LPS treatment followed by CSnp conditioning; and LPS treatment followed by Dex-CSnp conditioning groups. SCAP adherence, viability, differentiation and biomineralization potential on dentine from different groups were studied using fluorescent and scanning electron microscopy. Inflammation by macrophages in response to LPS dentine was quantified, and effect on SCAP migration was analysed. Statistical analysis was performed using Student's t-test with a significance level of P<0.05. TOF-SIMS analysis confirmed LPS contamination. LPS dentine affected SCAP viability but not adherence to dentine (P<0.001). Conditioning of LPS dentine with either nanoparticles improved SCAP viability (P<0.01) and rescued other LPS related adverse effects on SCAPs, such as F-actin disruption, decrease in differentiation/biomineralization potential. IL-6 produced by macrophages in response to LPS-treated dentine impeded SCAP migration (P<0.001), diminished on CSnp and Dex-CSnp conditioning groups (P<0.01). This study developed an LPS-dentine model and highlighted the ability of CSnp and Dex-CSnp to promote stem cell viability, migration, differentiation potential and reduce inflammation, providing an environment conducive for tissue regeneration/repair.

ER stress induced by ER calcium depletion and UVB irradiation regulates tight junction barrier integrity in human keratinocytes

BackgroundEndoplasmic reticulum (ER) calcium depletion-induced ER stress is a crucial signal for keratinocyte differentiation and barrier homeostasis, but its effects on the epidermal tight junction (TJ) have not been characterized. Ultraviolet B (UVB) causes ER calcium release in keratinocytes and disrupts epidermal TJ, however, the involvement of ER stress in the UVB-induced TJ alterations remains unknown. ObjectivesTo investigate the effect of ER stress by pharmacological ER calcium depletion or UVB on the TJ integrity in normal human epidermal keratinocytes (NHEK). MethodsNHEK were exposed to ER calcium pump inhibitor thapsigargin (Tg) or UVB. ER stress markers and TJ molecules expression, TJ and F-actin structures, and TJ barrier function were analyzed. ResultsTg or UVB exposure dose-dependently triggered unfolded protein response (UPR) in NHEK. Low dose Tg induced the IRE1α-XBP1 pathway and strengthened TJ barrier. Contrary, high dose Tg activated PERK phosphorylation and disrupted TJ by F-actin disorganization. UVB disrupted TJ and F-actin structures dose dependently. IRE1α RNase inhibition induced or exacerbated TJ and F-actin disruption in the presence of low dose Tg or UVB. High dose Tg increased RhoA activity. 4-PBA or Rho kinase (ROCK) inhibitor partially prevented the disruption of TJ and F-actin following high dose Tg or UVB. ConclusionsER stress has bimodal effects on the epidermal TJ depending on its intensity. The IRE1α pathway is critical for the maintenance of TJ integrity during mild ER stress. Severe ER stress-induced UPR or ROCK signalling mediates the disruption of TJ through cytoskeletal disorganization during severe ER stress.

Secreted autotransporter toxin (Sat) induces cell damage during enteroaggregative Escherichia coli infection.

Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77, O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.

Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cγ1.

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas' disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cγ1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

The vimentin network displays remarkable plasticity to support basic cellular functions and reorganizes during cell division. Here, we show that in several cell types vimentin filaments redistribute to the cell cortex during mitosis, forming a robust framework interwoven with cortical actin and affecting its organization. Importantly, the intrinsically disordered tail domain of vimentin is essential for this redistribution, which allows normal mitotic progression. A tailless vimentin mutant forms curly bundles, which remain entangled with dividing chromosomes leading to mitotic catastrophes or asymmetric partitions. Serial deletions of vimentin tail domain gradually impair cortical association and mitosis progression. Disruption of f-actin, but not of microtubules, causes vimentin bundling near the chromosomes. Pathophysiological stimuli, including HIV-protease and lipoxidation, induce similar alterations. Interestingly, full filament formation is dispensable for cortical association, which also occurs in vimentin particles. These results unveil implications of vimentin dynamics in cell division through its interplay with the actin cortex.

MON-002 YI QI QING RE GAO, A TRADITIONAL CHINESE HERBAL FORMULA, ORCHESTRATES ACTIN CYTOSKELETON REORGANIZATION INDUCED BY PUROMYCIN AMINONUCLEOSIDE IN PODOCYTES VIA RHOA/ROCK SIGNALING PATHWAY

Podocyte injury is closely associated with the onset of proteinuria in many kidney diseases. A major mechanism of podocyte injury is the reorganization of actin cytoskeleton, which leads to increased podocyte motility and foot process effacement. Yi Qi Qing Re Gao (YQQRG), a traditional Chinese herbal formula, has been used clinically to treat chronic nephritis for decades with dramatic anti-proteinuria effects. In this study, we investigated the effects of YQQRG on actin cytoskeleton reorganization induced by puromycin aminonucleoside (PAN) and explored the possible mechanisms. Wistar rats were randomly divided into sham group, PAN group, PAN + YQQRG group, and PAN + Cyclosprin A (CsA) group. The PAN model was established by a single intravenous injection of PAN (40mg/kg body weight). Serum biochemical parameters and 24h urinary protein were assessed on days 5, 9, and 14, respectively. Renal tissues were harvested for observations of pathologic alterations and ultrastructural changes. Quantitative expressions of regulatory proteins of cytoskeleton were measured using Western-blot and real-time polymerase chain reaction. In vitro, the conditionally immortalized mouse podocytes (MPC5) were used to investigate the effects of Yi Qi Qing Re Gao-containing serum (YQ-S) on actin cytoskeleton reorganization. Podocytes injury were evaluated by released lactic dehydrogenase (LDH) activity assay. Podocytes apoptosis were determined by Annexin V assay. Wound healing assay and transwell migration assay were used to assess podocyte motility. Immunofluorescence was used to demonstrate the formation and distribution of stress fiber. Western Blot was performed to detect the expression of key proteins of RhoA/ROCK signaling pathway. YQQRG significantly decreased urinary protein level, lowered serum lipid level, elevated serum albumin, and ameliorated renal function in Wistar rats, similar to the effects of CsA. Renal pathological lesions, especially podocytes effacement were obviously attenuated. The experiments in vivo and in vitro both displayed that YQQRG could downregulate the mRNA and protein expressions of α-actinin-4, synaptopodin, vimentin, desmin, and nestin in renal tissues and podocytes. Besides, YQ-S alleviated PAN-induced podocytes injury and apoptosis, and reduced podocyte motility and migration. Disruption of F-actin and disorganization of cytoskeleton were largely recovered by YQ-S. Furthermore, YQ-S significantly antagonized PAN-induced downregulation of the protein expression of RhoA and ROCK, decreased the phosphorylation of MLC, LIMK and cofilin, and increased F-actin/G-actin levels. Addition of Y-27632 (a Rho-kinase inhibitor) could weaken the effects of YQ-S on F-actin reorganization and expressions of ROCK, p-MLC, and p-LIMK. YQQRG alleviates actin cytoskeleton reorganization and attenuates podocytes injury in PAN model in vivo and in vitro, and its mechanism may involve the upregulation of RhoA/ROCK signaling pathway. These findings suggest that YQQRG might be a promising natural drug for the treatment of kidney diseases.