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At least 73 records · Page 4

The structural OFF and ON states of myosin can be decoupled from the biochemical super- and disordered-relaxed states

Abstract There is a growing awareness that both thick-filament and classical thin-filament regulations play central roles in modulating muscle contraction. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside either in a high-energy-consuming disordered-relaxed (DRX) state available for binding actin to generate force or in an energy-sparing super-relaxed (SRX) state unavailable for actin binding. X-ray diffraction studies have shown that the majority of myosin heads are in a quasi-helically ordered OFF state in a resting muscle and that this helical ordering is lost when myosin heads are turned ON for contraction. It has been assumed that myosin heads in SRX and DRX states are equivalent to the OFF and ON states, respectively, and the terms have been used interchangeably. In this study, we use X-ray diffraction and ATP turnover assays to track the structural and biochemical transitions of myosin heads, respectively, induced with either omecamtiv mecarbil (OM) or piperine in relaxed porcine myocardium. We find that while OM and piperine induce dramatic shifts of myosin heads from the OFF to the ON state, there are no appreciable changes in the population of myosin heads in the SRX and DRX states in both unloaded and loaded preparations. Our results show that biochemically defined SRX and DRX can be decoupled from structurally defined OFF and ON states. In summary, while SRX/DRX and OFF/ON transitions can be correlated in some cases, these two phenomena are measured using different approaches, reflect different properties of the thick filament, and should be investigated and interpreted separately.

Jani, Vivek P. (ORCID:0000000238114973)↗

Zone plate-based extreme ultraviolet mask microscope with through-pellicle imaging capability

Mirror-based and zone plate-based imaging systems are being used in actinic extreme ultraviolet (EUV) reticle review tools. With regard to zone plates, a short working distance is advantageous in terms of the required spectral bandwidth, manufacturability, and potential throughput and imaging performance. Zone plates therefore typically have a short working distance. The industry has adopted the use of an EUV pellicle to protect the photomask. Imaging photomask through-pellicle requires a working distance larger than 2.5 mm. A zone-plate-based EUV mask microscope with a 3-mm working distance has been commissioned at beamline 11.3.2 of the Advanced Light Source. Through-pellicle imaging at an exposure time of 2 s is demonstrated. The instrument achieves an image contrast of 95% on large features on a photomask with a tantalum-based absorber. Imaging down to 45-nm half pitch (mask scale) is demonstrated. A NILS of 2.55 is achieved on 60-nm half-pitch (mask scale) lines and spaces. These results demonstrate that zone-plate-based imaging systems can meet the requirements of an actinic EUV mask review tool in terms of imaging performance and throughput in an instrument compatible with EUV pellicles.

Extreme ultraviolet↗

Identification and selection of optimal reference genes for qPCR-based gene expression analysis in Fucus distichus under various abiotic stresses

Quantitative gene expression analysis is an important tool in the scientist’s belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown alga Fucus distichus . These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter’s unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage.

59 BASIC BIOLOGICAL SCIENCES↗

Electrochemically modulated single-molecule localization microscopy for in vitro imaging cytoskeletal protein structures

A new concept of electrochemically modulated single-molecule localization super-resolution imaging is developed. Applications of single-molecule localization super-resolution microscopy have been limited due to insufficient availability of qualified fluorophores with favorable low duty cycles. The key for the new concept is that the “On” state of a redox-active fluorophore with unfavorable high duty cycle could be driven to “Off” state by electrochemical potential modulation and thus become available for single-molecule localization imaging. The new concept was carried out using redox-active cresyl violet with unfavorable high duty cycle as a model fluorophore by synchronizing electrochemical potential scanning with a single-molecule localization microscope. The two cytoskeletal protein structures, the microtubules from porcine brain and the actins from rabbit muscle, were selected as the model target structures for the conceptual imaging in vitro. The super-resolution images of microtubules and actins were obtained from precise single-molecule localizations determined by modulating the On/Off states of single fluorophore molecules on the cytoskeletal proteins via electrochemical potential scanning. Importantly, this method could allow more fluorophores even with unfavorable photophysical properties to become available for a wider and more extensive application of single-molecule localization microscopy.

electrochemical modulation↗

Macrolides: From Toxins to Therapeutics

Macrolides are a diverse class of hydrophobic compounds characterized by a macrocyclic lactone ring and distinguished by variable side chains/groups. Some of the most well characterized macrolides are toxins produced by marine bacteria, sea sponges, and other species. Many marine macrolide toxins act as biomimetic molecules to natural actin-binding proteins, affecting actin polymerization, while other toxins act on different cytoskeletal components. The disruption of natural cytoskeletal processes affects cell motility and cytokinesis, and can result in cellular death. While many macrolides are toxic in nature, others have been shown to display therapeutic properties. Indeed, some of the most well known antibiotic compounds, including erythromycin, are macrolides. In addition to antibiotic properties, macrolides have been shown to display antiviral, antiparasitic, antifungal, and immunosuppressive actions. Here, we review each functional class of macrolides for their common structures, mechanisms of action, pharmacology, and human cellular targets.

59 BASIC BIOLOGICAL SCIENCES↗

FHOD formin and SRF promote post-embryonic striated muscle growth through separate pathways in C. elegans

Highlights: • Reduced SRF or formin FHOD-1 activity similarly inhibit muscle growth in worms. • Formin does not promote worm SRF-dependent muscle gene transcription. • Proteasome-driven myosin proteolysis is increased in worm muscle lacking formin. Previous work with cultured cells has shown transcription of muscle genes by serum response factor (SRF) can be stimulated by actin polymerization driven by proteins of the formin family. However, it is not clear if endogenous formins similarly promote SRF-dependent transcription during muscle development in vivo. We tested whether formin activity promotes SRF-dependent transcription in striated muscle in the simple animal model, Caenorhabditis elegans. Our lab has shown FHOD-1 is the only formin that directly promotes sarcomere formation in the worm's striated muscle. We show here FHOD-1 and SRF homolog UNC-120 both support muscle growth and also muscle myosin II heavy chain A expression. However, while a hypomorphic unc-120 allele blunts expression of a set of striated muscle genes, these genes are largely upregulated or unchanged by absence of FHOD-1. Instead, pharmacological inhibition of the proteasome restores myosin protein levels in worms lacking FHOD-1, suggesting elevated proteolysis accounts for their myosin deficit. Interestingly, proteasome inhibition does not restore normal muscle growth to fhod-1(Δ) mutants, suggesting formin contributes to muscle growth by some alternative mechanism. Overall, we find SRF does not depend on formin to promote muscle gene transcription in a simple in vivo system.

60 APPLIED LIFE SCIENCES↗

Surface distribution of heterogenous clathrin assemblies in resorbing osteoclasts

Highlights: • Platinum replica microscopy has revealed new structural data on the clathrins on the ventralmembrane of osteoclasts. • Various clathrins were visualized, suggesting that they followed variations in the surface topography of the apatite. • Most of the flat clathrins may participate in the adhesive and mechnosensory functions in co-operation with podosomes. • The present findings in osteoclast biology reveal potential novel drug targets or treatment regimens. Osteoclasts seeded on either glass coverslips or apatite pellets have at least two morphologically distinct substrate adhesion sites: actin-based adhesion structures including podosome belts and sealing zones, and adjacent clathrin sheets. Clathrin-coated structures are exclusively localized at the podosome belts and sealing zone, in both of which the plasma membrane forms a tight attachment to the substrate surface. When cultured on apatite osteoclasts can degrade the apatite leading to the formation of resorption lacunae. The sealing zone divides the ventral membrane into different domains, outside and inside of the sealing zones. The former facing the smooth-surfaced intact apatite contains relatively solitary or networks of larger flat clathrin structures; and the latter, facing the rough-surfaced degraded apatite in the resorption lacunae contain clathrin in various shapes and sizes. Clathrin assemblies on the membrane domain facing not only a resorption lacuna, or trails but also intact apatite indeed were observed to be heterogeneous in size and intensity, suggesting that they appeared to follow variations in the surface topography of the apatite surface. These results provide a detailed insight into the flat clathrin sheets that have been suggested to be the sites of adhesion and mechanosensing in co-operation with podosomes.

60 APPLIED LIFE SCIENCES↗

NF-κB activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization in mice

Highlights: • IMD0354 treatment reduced the microglia infiltration, fluorescein leakage and size of the laser CNV spots. • IKK2 inhibition suppressed phospho-IκBα, VEGF-A, and COX-2 in the Laser CNV mouse eyes. • IMD-0354 treated eyes demonstrated reduced microglial migration to the laser injury site. • In vitro, IKK2 inhibition reduced NF-κB activation, COX-2, Actin-F presence, and the microglia cells' migration capacity. To evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV).

60 APPLIED LIFE SCIENCES↗

Downregulation of fascin in the first trimester placental villi is associated with early recurrent miscarriage

Highlights: • Fascin is downregulated in the placentae of pregnancies complicated by early recurrent miscarriage. • Knockdown of fascin decreased HTR-8/SVneo cells' proliferation, migration and invasion. • Fascin knockdown decreased phosphorylated ERK1/2 and phosphorylated STAT3 protein level in HTR-8/SVneo cells. • Hypoxia reoxygenation decreased fascin, N-Cadherin and Vimentin protein levels to affect HTR-8/SVneo cells' invasion. Inadequate trophoblast proliferation, shallow invasion and exaggerated rate of trophoblast apoptosis are implicated in early recurrent miscarriage (ERM). However, the mechanistic bases of this association have not been fully established. We aimed at investigating the involvement of fascin, an actin-bundling protein, in trophoblast activities and ERM. We found that fascin was downregulated in the cytotrophoblasts (CTBs) and distal cytotrophoblasts (DCTs) of ERM placentae. Knockdown of fascin altered cellular and nucleolar morphology, and inhibited the proliferation but increased apoptosis of trophoblastic HTR8/SVneo cells. Furthermore, fascin knockdown decreased the expression of transcription factors such as Snail1/2, Twist and Zeb1/2, mesenchymal molecules such as Vimentin and N-cadherin, and the protein expression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylates signal transducer and activator of transcript 3 (STAT3). Exposure of HTR-8/SVneo cells to hypoxia reoxygenation (H/R) decreased fascin expression to affect the cells’ invasion. Our results indicate for the first time that the downregulation of fascin is involved in the pathogenesis of early recurrent miscarriage; and hence a potential therapeutic target against the disease.

60 APPLIED LIFE SCIENCES↗

Functional characterization of adult human trabecular meshwork stem cells

We earlier identified native human trabecular meshwork stem cells (TMSCs) based on two-parameters- high ABCG2 expression and high nucleus to cytoplasmic ratio. The TMSCs also expressed p75 and AnkyrinG. Based on the high expression of ABCG2 and p75, the TMSCs were identified to be located in the Schwalbe's line region of the TM. In continuation, the current study aimed at elucidating the functional characteristics of human TMSCs. Upon culturing, only a small proportion of TM cells (0.96 ± 0.21% in <30 years) expressing stem cell markers ABCG2 and p75 adhered to the culture dish. This proportion significantly reduced with ageing (0.32 ± 0.23% in 30–60 years and 0.35 ± 0.04% in >60 years). Characterization of the primary TM cultures identified 7.00 ± 1.80% of stem cells with label retaining property. Further, cultured cells had the ability to form TM spheres (0.82 ± 0.23%) which consisted of high ABCG2 and p75 positive cells. Upon dexamethasone induction, 86.00 ± 14.87% and 64.60 ± 7.24% of the cells derived from the TM spheres expressed myocilin and exhibited cross linked actin networks respectively, indicating differentiation of the TMSCs in the sphere to TM cells. In addition, the sphere derived TM cells also possessed phagocytic potential (13.28 ± 3.30%) equivalent to primary TM cells (16.33 ± 4.04%) which was evident upon internalization of zymosan particles. In conclusion, this study has established that a proportion of cultured TM cells had the label retaining property as well as sphere forming ability of adult stem cells. Thus, these results confirm the presence of adult stem cells in the human TM that might be responsible for the maintenance of tissue homeostasis.

60 APPLIED LIFE SCIENCES↗

PPARα agonist exerts protective effects in podocyte injury via inhibition of the ANGPTL3 pathway

Peroxisome proliferator-activated receptor α (PPARα) activation has been reported to exert protective effects on podocytes, whereas angiopoietin-like 3 (ANGPTL3) has been shown to exert significant pathogenic effects on these cells. This study aimed to investigate the link between the protective effects of PPARα activation and the pathogenic effects of ANGPTL3 in podocytes. Both PPARα and ANGPTL3 were expressed in cultured podocytes. PPARα mRNA and protein levels decreased whereas ANGPTL3 mRNA and protein levels increased in a time-dependent manner in podocytes treated with puromycin aminonucleoside (PAN). Gemfibrozil, a pharmacological agonist of PPARα, increased PPARα levels and activity in podocytes. The drug also decreased ANGPTL3 levels by potentially weakening ANGPTL3 promoter activity in both normal and PAN-treated podocytes. Furthermore, gemfibrozil significantly decreased PAN-induced apoptosis and F-actin rearrangement. Primary podocytes from Angptl3-knockout mice were cultured. There was no significant difference between Angptl3{sup −/−} podocytes treated with or without gemfibrozil in the lamellipodia numbers after PAN treatment. The results suggested that the protective effects of gemfibrozil on podocytes were not exerted following knockout of the Angptl3 gene. This study identified a novel mechanism of the PPARα agonist gemfibrozil that exerts its protective effects by inhibiting PAN-induced apoptosis and cytoskeleton rearrangements through inhibition of ANGPTL3 expression.

60 APPLIED LIFE SCIENCES↗

Serum amyloid A1: Innocent bystander or active participant in cell migration in triple-negative breast cancer?

Highlights: • SAA1 knockdown reduced cell viability in triple-negative breast cancer cells. • SAA1 knockdown induced a S to G{sub 2}/M cell cycle shift in MDA-MB-231 cells. • SAA1 silencing inhibited cell migration in MDA-MB-231 and HCC70 cells. • SAA1 silencing altered epithelial-to-mesenchymal transition markers in MDA-MB-231 cells. The Serum Amyloid A (SAA) family of proteins is associated with various pathological conditions, including cancer. However, their role in cancer is incompletely understood. Here, we investigated the role of SAA1 in cell cycle regulation, apoptosis, survival signaling, metabolism, and metastasis in models of triple-negative breast cancer (TNBC), using RNAi. Our data show that in untransformed epithelial cells (MCF12A), the knockdown of SAA1 induces the expression of cell cycle regulators (MCM2, p53), the activation of DNA repair (PARP synthesis), and survival signaling (NFκB). In contrast, knockdown of SAA1 in the TNBC cell line (MDA-MB-231) induced the expression p16 and shifted cells in the cell cycle from the S to G{sub 2}/M phase, without the activation of DNA repair. Moreover, in SAA1-deficient MDA-MB-231 and HCC70 cells, metabolism (NADH oxidation) continually increased while cell migration (% wound closure and the rate of wound closure) decreased. However, silencing of SAA1 altered epithelial and mesenchymal markers in MCF12A (E-cadherin, Laminin 1β, Vimentin) and MDA-MB-231 (α-Smooth muscle actin) cells, associated with the metastatic program of epithelial-mesenchymal transition. Nonetheless, our data provide evidence that SAA1 could potentially serve as a therapeutic target in TNBC.

60 APPLIED LIFE SCIENCES↗

AGTR1 blocker attenuates activation of Tenon's capsule fibroblasts after glaucoma filtration surgery via the NF-κB signaling pathway

Activation of Tenon's capsule fibroblasts limits the success rate of glaucoma filtration surgery (GFS), the most efficacious therapy for patients with glaucoma. Angiotensin type 1 receptor (AGTR1) is involved in tissues remodeling and fibrogenesis. However, whether AGTR1 is involved in the progress of fibrogenesis after GFS is not fully elucidated. The aim of this study was to investigate the role of an AGTR1 in scar formation after GFS and the potential anti-fibrosis effect of AGTR1 blocker. AGTR1 expression level was increased in subconjunctival tissues in a rat model of GFS and transforming growth factor-beta 2 (TGF-β2)-induced human Tenon's capsule fibroblasts (HTFs). AGTR1 blocker treatment suppressed TGF-β2-induced HTF migration and α-smooth muscle actin (α-SMA) and fibronectin (FN) expression. AGTR1 blocker treatment also attenuated collagen deposition and α-SMA and FN expression in subconjunctival tissues of the rat model after GFS. Moreover, AGTR1 blocker decreased TGF-β2-induced P65 phosphorylation, P65 nuclear translocation, and nuclear factor kappa B (NF-κB) luciferase activity. Additionally, BAY 11–7082 (an NF-κB inhibitor) significantly suppressed HTF fibrosis. In conclusion, our results indicate that AGTR1 is involved in scar formation after GFS. The AGTR1 blocker attenuates subconjunctival fibrosis after GFS by inhibiting the NF-κB signaling pathway. These findings indicate that targeting AGTR1 is a potential approach to attenuate fibrosis after GFS.

60 APPLIED LIFE SCIENCES↗

Effect of docetaxel on mechanical properties of ovarian cancer cells

Docetaxel could inhibit the proliferation of tumor cells by targeting microtubules. The extension of cellular microtubules plays an important role in the invasion and metastasis of tumor cells. This paper aims to study the distribution and mechanical properties of cytoskeletal proteins with low concentration of docetaxel. MTT assay was used to detect the minimum drug activity concentration of docetaxel on SKOV-3 cells, fluorescence staining was used to analyze the distribution of cytoskeleton proteins, scanning electron microscopy(SEM) was used to observe the morphology of single cells, and atomic force microscopy(AFM) was used to determine the microstructure and mechanical properties of cells. The results showed that the IC{sub 10} of docetaxel was 1 ng/ml. Docetaxel can effectively inhibit the formation of cell pseudopodia, hinder the indirectness between cells, reduce the cell extension area, and make the cells malformed. In addition, when AFM analyzes the effects of drugs on cell microstructure and mechanical properties, the average cell surface roughness and cell height are positively correlated with the concentration of docetaxel. Especially when the concentration was 100 ng/ml, the adhesion decreased by 37.04% and Young's modulus increased by 1.57 times compared with the control group. This may be because docetaxel leads to microtubule remodeling and membrane protein aggregation, which affects cell microstructure and increases cell strength, leading to significant changes in the mechanical properties of ovarian cells. This is of great significance to the study of the formation mechanism of tumor cell invasion and migration activities mediated by actin.

60 APPLIED LIFE SCIENCES↗

Insights into posttranslational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP

Here, mice with skeletal muscle-specific and inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and, thus, whether they could underlie the rapid loss of muscle contractile function. Just 4–5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was ~90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a ~65% reduction in in vivo ankle dorsiflexion torque, as compared with wild type (WT; i.e., Cre negative) littermates. Despite this profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial-specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume, or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca 2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca 2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice.

59 BASIC BIOLOGICAL SCIENCES↗

A positive feedback loop involving the Spa2 SHD domain contributes to focal polarization

Focal polarization is necessary for finely arranged cell-cell interactions. The yeast mating projection, with its punctate polarisome, is a good model system for this process. We explored the critical role of the polarisome scaffold protein Spa2 during yeast mating with a hypothesis motivated by mathematical modeling and tested by in vivo experiments. Our simulations predicted that two positive feedback loops generate focal polarization, including a novel feedback pathway involving the N-terminal domain of Spa2. We characterized the latter using loss-of-function and gain-of-function mutants. The N-terminal region contains a Spa2 Homology Domain (SHD) which is conserved from yeast to humans, and when mutated largely reproduced the spa2Δ phenotype. Our work together with published data show that the SHD domain recruits Msb3/4 that stimulates Sec4-mediated transport of Bud6 to the polarisome. There, Bud6 activates Bni1-catalyzed actin cable formation, recruiting more Spa2 and completing the positive feedback loop. We demonstrate that disrupting this loop at any point results in morphological defects. Gain-of-function perturbations partially restored focal polarization in a spa2 loss-of-function mutant without restoring localization of upstream components, thus supporting the pathway order. Thus, we have collected data consistent with a novel positive feedback loop that contributes to focal polarization during pheromone-induced polarization in yeast.

59 BASIC BIOLOGICAL SCIENCES↗

Regulation of Cell Wall Assembly: Myosin and Exocyst Involvement in Cellulose Synthase Delivery to the Plasma Membrane

Cellulose is the most abundant biopolymer on the planet and is produced in the primary and secondary cell wall of terrestrial plants by a plasma membrane (PM)-localized, multimeric protein complex. The catalytic enzyme, or cellulose synthase, belongs to a multigene family known as CESA. It is generally accepted that the behavior and trajectories of cellulose synthase complexes are oriented by the position of cortical microtubules, but how exactly these complexes are delivered and recycled from the PM remains poorly understood. Limited evidence suggests that microtubules determine the site for delivery of new complexes; however, abolishing microtubules with the inhibitor oryzalin has absolutely no effect on rates of delivery. Thus, there is a pressing need to explore the contribution of another component of the cortical cytoskeleton, actin filaments and the associated motor protein myosin, to the delivery and dynamics of CESA at the PM.

59 BASIC BIOLOGICAL SCIENCES↗