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Three-dimensional matrices often contain highly structured adhesive tracks that require cells to turn corners and bridge non-adhesive areas. Here, we investigate these complex processes using micropatterned cell adhesive frames. Spreading kinetics on these matrices depend strongly on initial adhesive position and are predicted by a cellular Potts model (CPM), which reflects a balance between adhesion and intracellular tension. As cells spread, new stress fibers (SFs) assemble periodically and parallel to the leading edge, with spatial intervals of ∼2.5 μm, temporal intervals of ∼15 min, and characteristic lifetimes of ∼50 min. By incorporating these rules into the CPM, we can successfully predict SF network architecture. Moreover, we observe broadly similar behavior when we culture cells on arrays of discrete collagen fibers. Our findings show that ECM geometry and initial cell position strongly determine cell spreading and that cells encode a memory of their spreading history through SF network organization., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Nephrotoxicity is a critical adverse event that leads to discontinuation of kinase inhibitor (KI) treatment. Here we show, through meta-analyses of FDA Adverse Event Reporting System, that dasatinib is associated with high risk for glomerular toxicity that is uncoupled from hypertension, suggesting a direct link between dasatinib and podocytes. We further investigate the cellular effects of dasatinib and other comparable KIs with varying risks of nephrotoxicity. Dasatinib treated podocytes show significant changes in focal adhesions, actin cytoskeleton, and morphology that are not observed with other KIs. We use phosphoproteomics and kinome profiling to identify the molecular mechanisms of dasatinib-induced injury to the actin cytoskeleton, and atomic force microscopy to quantify impairment to cellular biomechanics. Furthermore, chronic administration of dasatinib in mice causes reversible glomerular dysfunction, loss of stress fibers, and foot process effacement. We conclude that dasatinib induces nephrotoxicity through altered podocyte actin cytoskeleton, leading to injurious cellular biomechanics.

Reactive oxygen species (ROS)-induced oxidative stress leads to neuron damage and is involved in the pathogenesis of chronic inflammation in neurodegenerative diseases (NDs), such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. Researchers, therefore, are looking for antiinflammatory drugs and gene therapy approaches to slow down or even prevent neurological disorders. Combining therapeutics has shown a synergistic effect in the treatment of human diseases. Many nanocarriers could be designed for the simultaneous codelivery of drugs with genes to fight diseases. However, only a few researches have been performed in NDs. In this study, we developed a mesoporous silica nanoparticle (MSN)-based approach for neurodegenerative therapy. This MSN-based platform involved multiple designs in the targeted codelivery of (1) curcumin, a natural antioxidant product, to protect ROS-induced cell damage and (2) plasmid RhoG-DsRed, which is associated with the formation of lamellipodia and filopodia for promoting neurite outgrowth. At the same time, TAT peptide was introduced to the plasmid RhoG-DsRed via electrostatic interaction to elevate the efficiency of nonendocytic pathways and the nuclear plasmid delivery of RhoG-DsRed in cells for enhanced gene expression. Besides, such a plasmid RhoG-DsRed/TAT complex could work as a noncovalent gatekeeper. The release of curcumin inside the channel of the MSN could be triggered when the complex was dissociated from the MSN surface. Taken together, this MSN-based platform combining genetic and pharmacological manipulations of an actin cytoskeleton as well as oxidative stress provides an attractive way for ND therapy.

The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down; depletion of mDia1 and mDia3 individually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent.

Background: During sexual reproduction, pollen grains land on the stigma, rehydrate and produce pollen tubes that grow through the female transmitting-tract tissue allowing the delivery of the two sperm cells to the ovule and the production of healthy seeds. Because pollen tubes are single cells that expand by tip-polarized growth, they represent a good model to study the growth dynamics, cell wall deposition and intracellular machineries. Aiming to understand this complex machinery, we used a low throughput chemical screen approach in order to isolate new tip-growth disruptors. The effect of a chemical inhibitor of monogalactosyldiacylglycerol synthases, galvestine-1, was also investigated. The present work further characterizes their effects on the tip-growth and intracellular dynamics of pollen tubes., Results: Two small compounds among 258 were isolated based on their abilities to perturb pollen tube growth. They were found to disrupt in vitro pollen tube growth of tobacco, tomato and Arabidopsis thaliana. We show that these 3 compounds induced abnormal phenotypes (bulging and/or enlarged pollen tubes) and reduced pollen tube length in a dose dependent manner. Pollen germination was significantly reduced after treatment with the two compounds isolated from the screen. They also affected cell wall material deposition in pollen tubes. The compounds decreased anion superoxide accumulation, disorganized actin filaments and RIC4 dynamics suggesting that they may affect vesicular trafficking at the pollen tube tip., Conclusion: These molecules may alter directly or indirectly ROP1 activity, a key regulator of pollen tube growth and vesicular trafficking and therefore represent good tools to further study cellular dynamics during polarized-cell growth.

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human intestinal epithelium, leading to increased intestinal permeability, impaired restitution, and increased cell death. Although there are numerous animal models of NEC, response to injury and therapeutic interventions may be highly variable between species. Furthermore, it is ethically challenging to study disease pathophysiology or novel therapeutic agents directly in human subjects, especially children. Therefore, it is highly desirable to develop a novel model of NEC using human tissue. Enteroids are 3-dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiologic interactions, cell signaling, and host-pathogen defense. In this manuscript we describe a protocol that cultures human enteroids after isolating intestinal stem cells from patients undergoing bowel resection. The crypt cells are cultured in media containing growth factors that encourage differentiation into the various cell types native of the human intestinal epithelium. These cells are grown in a synthetic, collagenous mix of proteins that serve as a scaffold, mimicking the extra-cellular basement membrane. As a result, enteroids develop apical-basolateral polarity. Co-administration of lipopolysaccharide (LPS) in media causes an inflammatory response in the enteroids, leading to histologic, genetic, and protein expression alterations similar to those seen in human NEC. An experimental model of NEC using human tissue may provide a more accurate platform for drug and treatment testing prior to human trials, as we strive to identify a cure for this disease.

Mercury is a toxic metal that is found ubiquitously in the environment. Humans are exposed to different forms of mercury via ingestion, inhalation, and/or dermal absorption. Following exposure, mercuric ions may gain access to target cells and subsequently lead to cellular intoxication. The mechanisms by which mercury accumulation leads to cellular injury and death are not understood fully. Therefore, purpose of this study was to identify the specific intracellular mechanisms that are altered by exposure to inorganic mercury (Hg 2+ ). Normal rat kidney (NRK) cells were exposed to a physiologically relevant form of Hg 2+ , as a conjugate of cysteine (10 μM or 50 μM). Alterations in oxidative stress were estimated by measuring lipid peroxidation and mitochondrial oxidative stress. Alterations in actin and tubulin were measured using specific fluorescent dyes. Calcium levels were measured using Fluo-3 AM Calcium Indicator while autophagy was identified with Premo ™ Autophagy Sensor LC3B-GFP. The current findings show that exposure to Hg 2+ leads to enhanced oxidative stress, alterations in cytoskeletal structure, increases in intracellular calcium, and enhanced autophagy. We have established a more complete understanding of intoxication and cellular injury induced by a relevant form of Hg 2+ in proximal tubule cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Silicate-substituted hydroxyapatite scaffolds containing multiscale porosity are manufactured. Model parts containing macropores of five cross-sectional geometries (circle, square, rhombus, star and triangle) and two sizes are shaped by microstereolithography. Three open microporosity contents (0.5, 23 or 37 vol%) are introduced in the ceramic. MC3T3-E1 pre-osteoblasts are seeded onto these scaffolds. Analysis of cell colonization inside the macropores after 7 and 14 days of cultivation shows that the cellular filling is proportional to the macropore size and strongly influenced by macropore shape. Straight edges and convex surfaces are detrimental. High aspect ratios, the absence of reentrant angles and the presence of acute angles, by creating concavities and minimizing flat surfaces, facilitate cell colonization. Rhombus and triangle cross-sections are thus particularly favorable, while square and star geometries are the least favored. An increase in the microporosity content strongly impairs cell growth in the macropores. The data are statistically analyzed using a principal components analysis that shows that macro- and microtopographical parameters of scaffolds must be collectively considered with correlated interactions to understand cell behavior. The results indicate the important cell sensing of topography during the initial step of cell adhesion and proliferation and evidence the need for an optimized scaffold design., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cadmium (Cd) is considered a possible etiological factor in neurodegenerative diseases. However, the exact mechanism by which Cd induces neurotoxicity is not well elucidated. In this study, Neuro-2a cells were treated with 0, 10, 20, and 40 μM cadmium chloride for 24 hours to investigate the effects of Cd on the cytoskeleton of nerve cells. MTT assay and ELISA assay were used to examine cell viability and release of lactate dehydrogenase (LDH) from cells, respectively. Results showed that Cd reduced cell viability and increased the release of LDH in a dose-dependent manner (P < 0.05). The morphology of treated cell was damaged as indicated by cell collapse and dimensionality reduction. Moreover, the axonal spines and normal features of Cd-treated neurons disappeared. We checked the ultrastructure of Neuro-2a cells and found that Cd-induced swelling, membrane damage, overflow of cytoplasm contents, and cell fragmentation. Damaged mitochondria, expanded endoplasmic reticulum, and abnormal microfilaments were found in Cd-treated cells rather than in untreated cells. Compared with the control group, the relative release of glutamate in the supernatant after Cd treatment was reduced, indicating that Cd exposure could reduce the release of glutamate by inhibiting the function of nerve-2a cells. Cd decreased the mRNA and protein expression levels of cytoskeletal proteins including DBN, SYP, and TAU, which might promote cytoskeleton alterations in Cd-treated cells. In conclusion, Cd-induced actin cytoskeleton alterations and dysfunction of cultured neurons. The results of the present study provide new insights for the investigation of Cd-induced neurotoxicity., (© 2019 Wiley Periodicals, Inc.)

The small GTPase Rho and its downstream effector, Rho-kinase (ROCK), regulate various cellular functions, including organization of the actin cytoskeleton, cell adhesion and migration. A pro-inflammatory lipid mediator, lysophosphatidic acid (LPA), is a potent activator of the Rho/ROCK signalling pathway and has been shown to induce the expression of chemokines and cell adhesion molecules (CAMs). In the present study, we aimed to elucidate the precise mechanism by which ROCK regulates LPA-induced expressions and functions of chemokines and CAMs. We observed that ROCK blockade reduced LPA-induced phosphorylation of IκBα and inhibited NF-κB RelA/p65 phosphorylation, leading to attenuation of RelA/p65 nuclear translocation. Furthermore, small interfering RNA-mediated ROCK isoform knockdown experiments revealed that LPA induces the expression of monocyte chemoattractant protein-1 (MCP-1) and E-selectin via ROCK2 in human aortic endothelial cells (HAECs). Importantly, we found that ROCK2 but not ROCK1 controls LPA-induced monocytic migration and monocyte adhesion toward endothelial cells. These findings demonstrate that ROCK2 is a key regulator of endothelial inflammation. We conclude that targeting endothelial ROCK2 is potentially effective in attenuation of atherosclerosis.

Vascular smooth muscle cells (VSMCs) have two distinct phenotypes: contractile and synthetic. The major difference between these phenotypes lies in the magnitude of the contractile force produced by the cell. Although traction force microscopy (TFM) is often used to evaluate cellular contractile force, this method requires complex preprocessing and a sufficiently compliant substrate. To evaluate the contractile force and the phenotype of living VSMCs with minimal effort and in a manner independent of the substrate stiffness, we propose a photoelasticity-based method using retardation, which is related to the difference between the first and second principal stresses and their orientation. The results demonstrate that actin filaments co-localize with areas of high retardation in cells, indicating that the retardation of VSMCs is promoted by actin filaments. The retardation of cells treated with calyculin A and Y-27632 tended to be larger and smaller, respectively, than that of control cells. Cell traction force significantly correlates with total cell retardation (r 2  = 0.38). The retardation of contractile VSMCs (passage 2) was significantly higher than that of synthetic VSMCs (passage 12). These results indicate that cell retardation can be used to assess cell contractile force and, thus, determine the phenotype of VSMCs.

Adipose-derived multipotent stem/progenitor cells (ASPCs) were shown to be ideal candidates for cell-based regenerative therapies. Yet, despite their huge potential, successful clinical applications are still rare. It was suggested that the efficacy of ASPCs at the recipient site depends on the vehicle of cell delivery. In this study, for preparation of a murine critical-size nerve defect model, we assessed the commercially available fibrin gel (ARTISS) as a potential cell carrier. In a thorough in vitro analysis, we investigated cell-fibrin interactions and analyzed the distribution and the long-term behavior of ASPCs cultivated in fibrin gel under normoxic and hypoxic conditions. ASPCs attached to the surface of a thin fibrin layer (two-dimensional condition) and spread with the abundant formation of actin stress fibers. Cells cultured within a fibrin matrix (three-dimensional condition) displayed a uniform distribution and formed interconnected networks while exhibiting strong cell-matrix interactions. Using time-lapse analysis, cells were found to migrate out of the gel and subsequently proliferated robustly both under hypoxic and normoxic conditions. During 14 days of culture in fibrin gel, ASPCs showed high viability, metabolic, and remodeling activities. At the end of the culture period, the fibrin matrix was degraded entirely accompanied by an upregulation of matrix metalloproteinases. In conclusion, fibrin gel stands out as a valuable biomaterial for delivering vital and active cells to damaged tissues. As a direct proof, ASPCs carried in a fibrin matrix will be evaluated in a murine critically sized peripheral nerve repair model., (© 2018 John Wiley & Sons, Ltd.)

Senescent cells undergo structural and functional changes that affect essentially every aspect of cell physiology. To date, the impact of senescence on the cytoskeleton is poorly understood. This study evaluated the cytoskeleton in two independent cellular models of kidney epithelium senescence. Our work identified multiple senescence-related alterations that impact microtubules and filamentous actin during interphase. Both filamentous systems reorganized profoundly when cells became senescent. As such, microtubule stability increased during senescence, making these filaments more resistant to disassembly in the cold or by nocodazole. Microtubule stabilization was accompanied by enhanced α-tubulin acetylation on lysine 40 and the depletion of HDAC6, the major deacetylase for α-tubulin lysine 40. Rho-associated kinase Rock1 is an upstream regulator that modulates key properties of the cytoplasmic cytoskeleton. Our research shows that Rock1 concentrations were reduced significantly in senescent cells, and we revealed a mechanistic link between microtubule stabilization and Rock1 depletion. Thus, Rock1 overexpression partially restored the cold sensitivity of microtubules in cells undergoing senescence. Additional components relevant to microtubules were affected by senescence. Specifically, we uncovered the senescence-related loss of the microtubule nucleating protein γ-tubulin and aberrant formation of γ-tubulin foci. Concomitant with the alterations of microtubule and actin filaments, senescent cells displayed functional changes. In particular, cell migration was impaired significantly in senescent cells. Taken together, our study identified new senescence-associated deficiencies of the microtubule and actin cytoskeleton, provided insights into the underlying molecular mechanisms and demonstrated functional consequences that are important to the physiology and function of renal epithelial cells.

In our ongoing search for new bioactive fungal metabolites, two new cytochalasans were isolated from stromata of the hypoxylaceous ascomycete Hypoxylon fragiforme . Their structures were elucidated via high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. Together with 23 additional cytochalasans isolated from ascomata and mycelial cultures of different Ascomycota, they were tested on their ability to disrupt the actin cytoskeleton of mammal cells in a preliminary structure⁻activity relationship study. Out of all structural features, the presence of hydroxyl group at the C7 and C18 residues, as well as their stereochemistry, were determined as important factors affecting the potential to disrupt the actin cytoskeleton. Moreover, reversibility of the actin disrupting effects was tested, revealing no direct correlations between potency and reversibility in the tested compound group. Since the diverse bioactivity of cytochalasans is interesting for various applications in eukaryotes, the exact effect on eukaryotic cells will need to be determined, e.g., by follow-up studies involving medicinal chemistry and by inclusion of additional natural cytochalasans. The results are also discussed in relation to previous studies in the literature, including a recent report on the anti-Biofilm activities of essentially the same panel of compounds against the pathogenic bacterium, Staphylococcus aureus .

Cell fusion as a rare event was observed following the co-culture of human MDA-MB-231 cherry breast cancer cells or benign neoplastic MCF10A cherry breast epithelial cells together with different mesenchymal stroma/stem-like cells (MSC GFP ) cultures, respectively, resulting in the generation of double-fluorescing hybrid cells. Analysis of potential molecular mechanisms for the formation of cancer hybrid cells revealed cytoskeletal components, including F-actin. Thus, a sub-lethal concentration of cytochalasin D, which blocks elongation of actin filaments, was able to significantly reduce cancer hybrid cell formation. Simultaneously, cell cycle progression of the different co-cultures remained unaffected following treatment with cytochalasin D, indicating continued proliferation. Moreover, exposure to 50 nM cytochalasin D revealed little if any effect on the expression of various integrins and cell adhesion molecules in the different co-cultures. However, LC-MS proteome analysis of the different control co-cultures compared to corresponding cytochalasin-treated co-cultures demonstrated predominant differences in the expression of actin-associated cytoskeletal proteins. In addition, the requirement of structured actin to provide an appropriate cytoskeletal network for enabling subsequent fusion processes was also substantiated by the actin filament disrupting latrunculin B, which inhibits the fusion process between the breast cancer populations and mesenchymal stroma/stem-like cells (MSC). Together, these findings suggest an important role of distinct actin structures and associated cytoskeletal components during cell fusion and the formation of breast cancer hybrid cells.

The mechanisms behind the anti-tumoral effects of cannabinoids by impacting the migratory activity of tumor cells are only partially understood. Previous studies demonstrated that cannabinoids altered the organization of the actin cytoskeleton in various cell types. As actin is one of the main contributors to cell motility and is postulated to be linked to tumor invasion, we tested the following hypothesizes: 1) Can cannabinoids alter cell motility in a cannabinoid receptor dependent manner? 2) Are these alterations associated with reorganizations in the actin cytoskeleton? 3) If so, what are the underlying molecular mechanisms? Three different glioblastoma cell lines were treated with specific cannabinoid receptor 1 and 2 agonists and antagonists. Afterwards, we measured changes in cell motility using live cell imaging and alterations of the actin structure in fixed cells. Additionally, the protein amount of phosphorylated p44/42 mitogen-activated protein kinase (MAPK), focal adhesion kinases (FAK) and phosphorylated FAK (pFAK) over time were measured. Cannabinoids induced changes in cell motility, morphology and actin organization in a receptor and cell line dependent manner. No significant changes were observed in the analyzed signaling molecules. Cannabinoids can principally induce changes in the actin cytoskeleton and motility of glioblastoma cell lines. Additionally, single cell motility of glioblastoma is independent of their morphology. Furthermore, the observed effects seem to be independent of p44/42 MAPK and pFAK pathways., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Faramarz Dehhghani is an editor of PLOS ONE.

The pathological significance of Tau (encoded by MAPT ) in mechanisms driving cell migration in glioblastoma is unclear. By using an shRNA approach to deplete microtubule-stabilizing Tau in U87 cells, we determined its impact on cytoskeletal coordination during migration. We demonstrated here that the motility of these Tau-knockdown cells (shTau cells) was significantly (36%) lower than that of control cells. The shTau cells displayed a slightly changed motility in the presence of nocodazole, which inhibits microtubule formation. Such reduced motility of shTau cells was characterized by a 28% lower number of microtubule bundles at the non-adhesive edges of the tails. In accordance with Tau-stabilized microtubules being required for cell movement, measurements of the front, body and rear section displacements of cells showed inefficient tail retraction in shTau cells. The tail retraction was restored by treatment with Y27632, an inhibitor of Rho-ROCK signaling. Moreover, we clearly identified that shTau cells displayed relocation of the active phosphorylated form of p190-RhoGAP (also known as ARHGAP35), which inhibits Rho-ROCK signaling, and focal adhesion kinase (FAK, also known as PTK2) in cell bodies. In conclusion, our findings indicate that Tau governs the remodeling of microtubule and actin networks for the retraction of the tail of cells, which is necessary for effective migration., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Methamphetamine (METH) is a psychostimulant with severe neurotoxicity, which is related to an increase of blood-brain barrier (BBB) permeability. However, the exact mechanisms have not been fully illuminated. In the present study, male Sprague Dawley rats were treated with METH or saline with 8 injections (i.p.) at 12-h intervals and sacrificed 24 h after the last METH injection. To evaluate BBB permeability, 6 rats were administered with Evans blue (EB) by tail vein injection 1 h prior to sacrifice. EB levels significantly increased in both left and right frontal lobes in METH-treated rats, suggesting increase of BBB permeability, which was proved by the rearrangement of F-actin cytoskeleton and decreased expressions of tight junction (TJ) proteins in hippocampus. Over-expressions of RhoA, ROCK, myosin light chain (MLC), cofilin, phosphorylation (p)-MLC, p-cofilin and matrix metalloproteinase (MMP)-9 were observed, indicating activated RhoA/ROCK pathway. Rat brain microvascular endothelial cells (RBMECs) were isolated and treated with inhibitors of RhoA and ROCK followed by METH. Pretreatments of the inhibitors significantly decreased expressions of RhoA, ROCK, MLC, cofilin, p-MLC and p-cofilin, increased expressions of TJ proteins, suppressed F-actin cytoskeleton rearrangement and reduced the permeability of RBMECs. These results suggested that METH increased BBB permeability through activating the RhoA/ROCK pathway, which resulted in F-actin cytoskeleton rearrangement and down-regulation of TJ proteins., (Copyright © 2018 Elsevier Inc. All rights reserved.)

There is a growing interest in the fact that mechanical signals may be as important as biological signals in evaluating cell viability. To investigate the alterations in biomechanics, nanomorphology and biological apoptotic signals during early apoptosis, an apoptosis model was established for cervical cancer HeLa cells induced by cytochalasin B (CB). The cellular mechanical properties, geometry, morphology and expression of key apoptotic proteins were systematically analyzed. The findings indicated a marked decline in cellular elastic modulus and volume and a considerable increase in surface roughness occurring prior to the activation of biological apoptosis signals (such as phosphatidylserine exposure or activation of CD95/Fas). Moreover, the depolymerization of filamentous actin aggravated the intracellular crowding degree, which induced the redistribution of different‑sized protein molecules and protrusions across the cell membrane arising from excluded volume interactions. Statistical analysis revealed that the disassembly of the actin cytoskeleton was negatively correlated with the cellular elastic modulus and volume, but was positively correlated with surface roughness and CD95/Fas activation. The results of the present study suggest that compared with biological signals, mechanical and geometrical reconstruction is more sensitive during apoptosis and the increase in cell surface roughness arises from the redistribution of biophysical molecules. These results contribute to our in‑depth understanding of the apoptosis mechanisms of cancer cells mediated by cytochalasin B.

Hypertension (HTN), i.e. abnormally high blood pressure, is a major risk factor for heart attack, stroke, and kidney failure. The Epithelial Sodium Channel (ENaC), one of the main transporters regulates blood pressure by tightly controlling the sodium reabsorption along the nephron. Recently, we have shown an α-ENaC overexpression in platelets from hypertensive patients compared to platelets from normotensive subjects, suggesting it makes a contribution to the activation state of platelets and the physiopathology of hypertension. However, the involvement of the α-ENaC localized in neutrophils to this disease remains unknown. Neutrophils are the first leukocytes to be recruited to an inflammatory site and are equipped with a strong ability to eliminate intra- or extracellular pathogens using reactive oxygen species or antibacterial proteins contained in their granules. Using the Western blotting (Wb), flow cytometry, and qRT-PCR approaches; we determined α-ENaC neutrophil overexpression at the protein and messenger RNA (mRNA) levels. By confocal and cytometry analysis, we determined the α-ENaC distribution and the heterogeneity of HTN neutrophils population, respectively. Immunoprecipitation and Wb assays demonstrated the presence of both α-ENaC and caveolin-1 phosphorylated forms, compared with neutrophils from healthy individuals. Although neutrophils from hypertensive subjects circulating in an activated state were exhibiting important oxidative stress and modifications registered by confocal, atomic force, and scanning electron microscope, they conserved their defense capabilities. The features described above for neutrophils from hypertensive patients could be attributed to α-ENaC overexpression, as its drug inhibition diminished their activation state modulating the actin cytoskeleton reorganization triggered during the activation process., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13 Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13 Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13 Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13 Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Advanced glycation end products (AGE) play a causative role in the development of aberrant phenotypes of intraglomerular mesangial cells, contributing to acute/chronic glomerulonephritis. The aim of this study was to explore mechanistic effects of the flavonoid chrysin present in bee propolis and herbs on actin dynamics, focal adhesion, and the migration of AGE-exposed mesangial cells. The in vitro study cultured human mesangial cells exposed to 33 mM glucose and 100 μg/mL AGE-bovine serum albumin (AGE-BSA) for up to 5 days in the absence and presence of 1⁻20 μM chrysin. The in vivo study employed db/db mice orally administrated for 10 weeks with 10 mg/kg chrysin. The presence of ≥10 μM chrysin attenuated mesangial F-actin induction and bundle formation enhanced by AGE. Chrysin reduced the mesangial induction of α-smooth muscle actin (α-SMA) by glucose, and diminished the tissue α-SMA level in diabetic kidneys, indicating its blockade of mesangial proliferation. The treatment of chrysin inhibited the activation of vinculin and paxillin and the induction of cortactin, ARP2/3, fascin-1, and Ena/VASP-like protein in AGE-exposed mesangial cells. Oral administration of chrysin diminished tissue levels of cortactin and fascin-1 elevated in diabetic mouse kidneys. Mesangial cell motility was enhanced by AGE, which was markedly attenuated by adding chrysin to cells. On the other hand, chrysin dampened the induction of autophagy-related genes of beclin-1, LC3 I/II, Atg3, and Atg7 in mesangial cells exposed to AGE and in diabetic kidneys. Furthermore, chrysin reduced the mTOR activation in AGE-exposed mesangial cells and diabetic kidneys. The induction of mesangial F-actin, cortactin, and fascin-1 by AGE was deterred by the inhibition of autophagy and mTOR. Thus, chrysin may encumber diabetes-associated formation of actin bundling and focal adhesion and mesangial cell motility through disturbing autophagy and mTOR pathway., Competing Interests: The authors declare no conflict of interest.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca 2+ -activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca 2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms., (© 2018 Breithaupt et al.)

Background: Three-dimensional (3D) printing with a direct metal fabrication (DMF) technology has been innovatively introduced in the field of surface treatment of prostheses. The purpose of this study was to determine whether such modifications on the surface of cobalt-chromium (CoCr) alloy by titanium powder coating using DMF improves the osseointegration ability of CoCr alloy., Methods: We compared the in vitro and in vivo ability of cells to adhere to DMF-coated CoCr alloy with machining. Biological and morphological responses to human osteoblast cell lines were examined by measuring cell proliferation rate and observing expression of actin filament. For in vivo study, we inserted different specimens in each medulla of the distal femurs of rabbit. After 3 months, the distal femurs were harvested, and a push-out test and histomorphometric analyses were performed., Results: The cell proliferation rate and cell adhesion in the DMF group were higher compared with those in the machined group. Human osteoblast cells on the DMF-coated surface were more strongly adhered and well-proliferated compared with those on the other surface. In the in vivo test, there was a significant difference in the ultimate shear strength between the DMF and machined groups (2.49 MPa vs. 0.87 MPa, respectively, p  = 0.001). In the histomorphometric analysis, there was a significant difference in the mean bone-to-implant contact percentages between the DMF and machined groups (72.3 ± 6.2% vs. 47.6 ± 6.9%, respectively, p  < 0.001)., Conclusion: Titanium coating of CoCr alloy with 3D metal printing provides optimal surface characteristics and a good biological surface both in vitro and in vivo ., Competing Interests: The authors declare that they have no conflict of interest.The animal experiment procedures were approved by the institutional animal care and use committee of The Catholic University of Korea (CUMC-2014-0109-03).

Background/aims: Renal injury related to hypertension is characterized by glomerular and tubulointerstitial damage. The overactivation of the renin-angiotensin system mainly by angiotensin II (AII) seems to be a main contributor to progressive renal fibrosis. Epithelial to mesenchymal transition (EMT) is a mechanism that promotes renal fibrosis. Owing to heat shock protein 70 (Hsp70) cytoprotective properties, the chaperone exhibits an important potential as a therapeutic target. We investigate the role of Hsp70 on Angiotensin II induced epithelial mesenchymal transition within the Losartan effect in proximal tubule cells (PTCs) from a genetic model of hypertension in rats (SHR)., Methods: Primary cell culture of PTCs from SHR and Wistar Kyoto (WKY) rats were stimulated with AII, treated with Losartan (L), (L+AII) or untreated (C c ). The functional Hsp70 role in Losartan effect, after silencing its expression by cell transfection, was determined by Immunofluorescence; Western blotting; Gelatin Zymography assays; Scratch wound assays; flow cytometry; and Live Cell Time-lapse microscopy., Results: (L) and (L+AII) treatments induced highly organized actin filaments and increased cortical actin in SHR PTCs. However, SHR PTCs (C c ) and (AII) treated cells showed disorganized actin. After Hsp72 knockdown in SHR PTCs, (L) was unable to stabilize the actin cytoskeleton. We demonstrated that (L) and (L+AII) increased E-cadherin levels and decreased vinculin, α-SMA, vimentin, pERK, p38 and Smad2-3 activation compared to (AII) and (C c ) SHR PTCs. Moreover, (L) inhibited MMP-2 and MMP-9 secretion, reduced migration and cellular displacement, stabilizing intercellular junctions. Notably, (L) treatment in shHsp72 knockdown SHR PTCs showed results similar to SHR PTCs (C c )., Conclusion: Our results demonstrate that Losartan through Hsp70 inhibits the EMT induced by AII in proximal tubule cells derived from SHR., Competing Interests: The authors have no conflicts of interests to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Exendin-4 is a protein of the GLP-1 family currently used to treat diabetes. Recently, a greater number of biological properties have been associated with the GLP-1 family. Our data shows that exendin-4 treatment significantly increases the cytoskeleton rearrangement, which leads to an increasingly differentiated phenotype and reduced cell migration. We also found that exendin-4 could prevent SH-SY5Y and PC12 cells from Nogo-A-Δ20 mediated spreading inhibition and neurite collapse. Western blot analysis indicated that exendin-4 treatment both reduced the expression and activation of RhoA via the PI3K signaling pathway. These data suggest that exendin-4 may protect nerve regeneration by preventing the inhibition of Nogo-A via down-regulating RhoA expression and activation., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The glomerular podocytes control filtration barrier permeability in the kidney, and their disturbance underlies the pathogenesis of idiopathic nephrotic syndrome (INS), a kidney disease that predominantly occurs in children. In this study, we found that the interleukin-7 receptor (IL-7R) was induced in the glomeruli of adriamycin (ADR)-induced mouse nephropathy, a rodent model of nephrotic syndrome. In addition, IL-7R was also induced by ADR in mouse podocytes cultured in vitro. Functionally, we discovered that IL-7R activation through the stimulation of recombinant IL-7 induced apoptosis of podocytes, and moreover, IL-7 stimulation inhibited nephrin activation and caused actin cytoskeleton disorganization, indicating that IL-7 stimulation induces podocyte injury. Furthermore, IL-7 stimulation impaired the filtration barrier function of podocyte monolayer. Together, these results identify IL-7 and its receptor IL-7R as potential regulators of podocyte function, which might offer a novel therapeutic target in the treatment of INS., (Copyright © 2018. Published by Elsevier Inc.)

Background: Three-dimensional (3D) floating culture clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. Previous studies have demonstrated that C-MSCs can be transplanted into bony lesions without an artificial scaffold to induce bone regeneration. Moreover, osteoinductive medium (OIM)-treated C-MSCs (OIM-C-MSCs) have shown rapid and increased new bone formation in vivo. To apply OIM-C-MSCs for novel bone regenerative cell therapy, their cellular properties at the molecular level must be elucidated. The transcriptional co-activators yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) have been recognized as key players in the mechanotransduction cascade, controlling cell lineage commitment in MSCs. It is plausible that 3D C-MSCs/OIM-C-MSCs cultured in floating conditions could provide distinct microenvironments compared to conventional 2D culture systems and thereby induce unique mechanotransduction cascades. Therefore, this study investigated the YAP/TAZ activity in 3D-cultured C-MSCs/OIM-C-MSCs in floating conditions., Methods: Human bone marrow-derived MSCs were cultured in growth medium supplemented with ascorbic acid. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The sheet was rolled to make round clumps of cells. Then, YAP/TAZ activity, filamentous actin (F-actin) integrity, collagen type I (COL1) production, and the differentiation potency in 3D floating culture C-MSCs/OIM-C-MSCs were analyzed., Results: C-MSCs cultured in floating conditions lost their actin cytoskeleton to downregulate YAP/TAZ activity, which directed cells to undergo adipogenesis/chondrogenesis. OIM treatment induced abundant COL1 deposition, which facilitated Intβ1-dependent actin fiber formation and YAP/TAZ activity to elevate the expression levels of osteogenic master transcriptional factor runt-related transcription factor 2 (RUNX2) mRNA in C-MSCs. Importantly, elevation of YAP/TAZ activity via OIM was associated with COL1 deposition and F-actin integrity, suggesting a positive feedback loop in OIM-C-MSCs., Conclusion: These findings suggest that OIM-C-MSCs, which form a unique microenvironment that maintains high YAP/TAZ activity, can serve as better candidates for bone regenerative cell therapy than C-MSCs.

Hydrogen peroxide (H 2 O 2 ) acts as a signaling molecule in cells by oxidising cysteine residues in regulatory proteins such as phosphatases, kinases and transcription factors. It is unclear exactly how many of these proteins are specifically targeted by H 2 O 2 because they appear too unreactive to be directly oxidised. One proposal is that peroxiredoxins (Prxs) initially react with H 2 O 2 and then oxidise adjacent proteins via a thiol relay mechanism. The aim of this study was to identify constitutive interaction partners of Prx2 in Jurkat T-lymphoma cells, in which thiol protein oxidation occurs at low micromolar concentrations of H 2 O 2 . Immunoprecipitation and proximity ligation assays identified a physical interaction between collapsin response mediator protein 2 (CRMP2) and cytoplasmic Prx2. CRMP2 regulates microtubule structure during lymphocyte migration and neuronal development. Exposure of Jurkat cells to low micromolar levels of H 2 O 2 caused rapid and reversible oxidation of CRMP2, in parallel with Prx2 oxidation, despite purified recombinant CRMP2 protein reacting slowly with H 2 O 2 (k~1 M -1 s -1 ). Lowering Prx expression should inhibit oxidation of proteins oxidised by a relay mechanism, however knockout of Prx2 had no effect on CRMP2 oxidation. CRMP2 also interacted with Prx1, suggesting redundancy in single knockout cells. Prx 1 and 2 double knockout Jurkat cells were not viable. An interaction between Prx2 and CRMP2 was also detected in other human and rodent cells, including primary neurons. However, low concentrations of H 2 O 2 did not cause CRMP2 oxidation in these cells. This indicates a cell-type specific mechanism for promoting CRMP2 oxidation in Jurkat cells, with insufficient evidence to attribute oxidation to a Prx-dependent redox relay., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Magnetic nanoparticles are regarded as a promising drug delivery vehicle with the improved efficacy and lowered side effects for antitumor therapy. Herein, the poly lactic-co-glycolic acid (PLGA) modified magnetic nanoplatform was synthesized using superparamagnetic γ-Fe 2 O 3 nanoparticles (MNPs) as a core, and then labelled with polyethylenimine (PEI)-conjugated fluorescein isothiocyanate (FITC), and simultaneously loaded with antitumor drug paclitaxel (PTX) for theranostic analysis of antitumor effects investigated in human brain glioblastoma U251 cells. As a result, the prepared PEI-PLGA-MNPs showed a relatively round sphere with an average size of 80 nm approximately, and the FITC-labeling PEI-PLGA-MNPs were efficiently endocytosed by the U251 cells for cellular imaging. Moreover, the fabricated PEI-PLGA-PTX-MNPs also demonstrated an inhibition of the targeted cell proliferation and migration, and a programmed cell death, via both apoptosis modulating by a burst of reactive oxygen species (ROS) and autophagy with accumulation of autophagosomes and LC3-II signals detected in the treated glioblastoma U251 cells after uptaking. Therefore, the constructed nanoplatform could be effectively applied for simultaneous cellular imaging and drug delivery in human brain glioblastoma treatment in future., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ric-8A is a pleiotropic guanine nucleotide exchange factor involved in the activation of various heterotrimeric G-protein pathways during adulthood and early development. Here, we sought to determine the downstream effectors of Ric-8A during the migration of the vertebrate cranial neural crest (NC) cells. We show that the Gα13 knockdown phenocopies the Ric-8A morphant condition, causing actin cytoskeleton alteration, protrusion instability, and a strong reduction in the number and dynamics of focal adhesions. In addition, the overexpression of Gα13 is sufficient to rescue Ric-8A-depleted cells. Ric-8A and Gα13 physically interact and colocalize in protrusions of the cells leading edge. The focal adhesion kinase FAK colocalizes and interacts with the endogenous Gα13, and a constitutively active form of Src efficiently rescues the Gα13 morphant phenotype in NC cells. We propose that Ric-8A-mediated Gα13 signalling is required for proper cranial NC cell migration by regulating focal adhesion dynamics and protrusion formation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The aberrant regulation of Wnt secretion is implicated in various neurological diseases. However, the mechanisms of Wnt release are still largely unknown. Here we describe the role of a C. elegans tetraspan protein, HIC-1, in maintaining normal Wnt release. We show that HIC-1 is expressed in cholinergic synapses and that mutants in hic-1 show increased levels of the acetylcholine receptor AChR/ACR-16. Our results suggest that HIC-1 maintains normal AChR/ACR-16 levels by regulating normal Wnt release from presynaptic neurons, as hic-1 mutants show an increase in secreted Wnt from cholinergic neurons. We further show that HIC-1 affects Wnt secretion by modulating the actin cytoskeleton through its interaction with the actin-binding protein NAB-1. In summary, we describe a protein, HIC-1, that functions as a neuromodulator by affecting postsynaptic AChR/ACR-16 levels by regulating presynaptic Wnt release from cholinergic motor neurons., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

An intramolecular Diels-Alder (IMDA) reaction efficiently accelerated by Schreiner's thiourea is reported, to build a functionalized cytochalasin scaffold (periconiasin series) for biological purposes. DFT calculation highlighted a unique multidentate cooperative hydrogen bonding in this catalysis. The deprotection end game afforded a collection of diverse structures and showed the peculiar reactivity of the Diels-Alder cycloadducts upon functionalization. Biological studies revealed strong cytotoxicity of a few compounds on breast cancer cell lines while actin polymerization is preserved., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cell⁻substrate interaction plays an important role in intracellular behavior and function. Adherent cell mechanics is directly regulated by the substrate mechanics. However, previous studies on the effect of substrate mechanics only focused on the stiffness relation between the substrate and the cells, and how the substrate stiffness affects the time-scale and length-scale of the cell mechanics has not yet been studied. The absence of this information directly limits the in-depth understanding of the cellular mechanotransduction process. In this study, the effect of substrate mechanics on the nonlinear biomechanical behavior of living cells was investigated using indentation-based atomic force microscopy. The mechanical properties and their nonlinearities of the cells cultured on four substrates with distinct mechanical properties were thoroughly investigated. Furthermore, the actin filament (F-actin) cytoskeleton of the cells was fluorescently stained to investigate the adaptation of F-actin cytoskeleton structure to the substrate mechanics. It was found that living cells sense and adapt to substrate mechanics: the cellular Young's modulus, shear modulus, apparent viscosity, and their nonlinearities (mechanical property vs. measurement depth relation) were adapted to the substrates' nonlinear mechanics. Moreover, the positive correlation between the cellular poroelasticity and the indentation remained the same regardless of the substrate stiffness nonlinearity, but was indeed more pronounced for the cells seeded on the softer substrates. Comparison of the F-actin cytoskeleton morphology confirmed that the substrate affects the cell mechanics by regulating the intracellular structure.

Objective- Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor-related protein 1) maintains vessel wall integrity, and smLRP1 -/- mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results- Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca 2+ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1 -/- mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1 -/- mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1 -/- aortic rings and cultured vascular smooth muscle cells. Conclusions- These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.

Known azole antifungal resistance mechanisms include mitochondrial dysfunction and overexpression of the sterol biosynthetic target enzyme and multidrug efflux pumps. Here, we identify, through a genetic screen, the vacuolar membrane-resident phosphatidylinositol 3-phosphate 5-kinase (CgFab1) to be a novel determinant of azole tolerance. We demonstrate for the first time that fluconazole promotes actin cytoskeleton reorganization in the emerging, inherently less azole-susceptible fungal pathogen Candida glabrata, and genetic or chemical perturbation of actin structures results in intracellular sterol accumulation and azole susceptibility. Further, CgFAB1 disruption impaired vacuole homeostasis and actin organization, and the F-actin-stabilizing compound jasplakinolide rescued azole toxicity in cytoskeleton defective-mutants including the Cgfab1Δ mutant. In vitro assays revealed that the actin depolymerization factor CgCof1 binds to multiple lipids including phosphatidylinositol 3,5-bisphosphate. Consistently, CgCof1 distribution along with the actin filament-capping protein CgCap2 was altered upon both CgFAB1 disruption and fluconazole exposure. Altogether, these data implicate CgFab1 in azole tolerance through actin network remodeling. Finally, we also show that actin polymerization inhibition rendered fluconazole fully and partially fungicidal in azole-susceptible and azole-resistant C. glabrata clinical isolates, respectively, thereby, underscoring the role of fluconazole-effectuated actin remodeling in azole resistance., (© 2018 The Authors Molecular Microbiology Published by John Wiley & Sons Ltd.)

Tunneling nanotubes (TNTs) play an important role in cell-cell communication. TNTs have been predominantly reported among cells in monolayer culture. Using various imaging modalities, including scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM), this work reports the finding of TNTs between cells within human mesenchymal stem cell (MSC) spheroids. TNTs visualized by SEM are consistent in size and geometry with those observed in cellular monolayer culture. LSCM imaging of living spheroids confirms the presence of F-actin filaments within the TNTs, which are known to maintain nanotube integrity. In addition, LSCM revealed the distribution of F-actin fibers across the entire spheroid body instead of being confined within individual cells. Intracellular material transport by TNTs was tested in MSC spheroids treated with cytochalasin D (CytoD), a known actin polymerization inhibitor for disrupting TNT formation. CytoD treatment decreased the transport of cytosolic material by at least four-fold compared to untreated spheroids. To the best of our knowledge, this work represents the first direct observation of TNTs within MSC spheroids. These findings offer new physical insight into cellular interactions within spheroids, providing structural information for increasing interests in spheroid-based cell therapy.

Latrunculin A (LatA), a toxin from the red sea sponge Latrunculia magnifica, is the most widely used reagent to depolymerize actin filaments in experiments on live cells. LatA binds actin monomers and sequesters them from polymerization [1, 2]. Low concentrations of LatA result in rapid (tens of seconds) disassembly of actin filaments in animal [3] and yeast cells [2]. Depolymerization is usually assumed to result from sequestration of actin monomers. Our observations of single-muscle actin filaments by TIRF microscopy showed that LatA bound ATP-actin monomers with a higher affinity (K d  = 0.1 μM) than ADP-P i -actin (K d  = 0.4 μM) or ADP-actin (K d  = 4.7 μM). LatA also slowly severed filaments and increased the depolymerization rate at both ends of filaments freshly assembled from ATP-actin to the rates of ADP-actin. This rate plateaued at LatA concentrations >60 μM. LatA did not change the depolymerization rates of ADP- actin filaments or ADP-P i -actin filaments generated with 160 mM phosphate in the buffer. LatA did not increase the rate of phosphate release from bulk samples of filaments assembled from ATP-actin. Thermodynamic analysis showed that LatA binds weakly to actin filaments with a K d >100 μM. We propose that concentrations of LatA much lower than this K d promote phosphate dissociation only from both ends of filaments, resulting in depolymerization limited by the rate of ADP-actin dissociation. Thus, one must consider both rapid actin depolymerization and severing in addition to sequestering actin monomers when interpreting the effects of LatA on cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

There are reports of fluorochloridone (FLC)-induced male reproductive toxicity, but the underlying toxicological mechanisms remain unknown. In this study, we looked at how FLC exposure affected the integrity of the blood-testis barrier (BTB) and the Sertoli cell barrier and studied the molecular mechanisms. Male rats received gavage administration of FLC (30 mg/kg/d) for 14 consecutive days with sample collection at the 7th and 14th day; and primary cultured Sertoli cells were treated with 0-10 μM FLC in vitro for 24 h. Our in vivo findings revealed that FLC exposure caused time-dependent testicular injuries, sperm quality decrease as well as adverse changes in BTB integrity, F-actin organization, and expressions of claudin-11 and Arp3. In Sertoli cells isolated from FLC-treated rat testis, Sertoli cell barrier tightness was increased. In Sertoli cells in vitro exposed to FLC, abnormal changes in the barrier permeability were also observed, and the protein expressions of occludin, claudin-11, ZO-1, connexin-43, and Arp3 were significantly decreased in a dose- and time-dependent manner. Furthermore, the FLC-induced adverse changes in Sertoli cell barrier and F-actin were partly alleviated by the induction of Arp3 overexpression. In conclusion, our findings revealed that FLC perturbed BTB/Sertoli cell barrier function through Arp3-mediated F-actin disorganization., (Copyright © 2018 Elsevier B.V. All rights reserved.)

It is generally recognised that mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical properties of the substrates. However, the precise biophysical mechanism that enables MSCs to respond to substrate properties remains unclear. In the current study, polydimethylsiloxane (PDMS) substrates with different stiffnesses were fabricated and the way in which the elastic modulus of the substrate regulated differentiation towards osteogenesis and adipogenesis in adipose-derived stromal cells (ASCs) was explored. Initially, a cell morphology change by SEM was observed between the stiff and soft substrates. The cytoskeleton stains including microfilament by F-actin and microtubule by α- and β-tubulin further showed a larger cell spreading area on the stiff substrate. Then the expression of vinculin, in charge for the linkage of adhesion molecules to the actin cytoskeleton, was enhanced on the stiff substrate. This change in focal adhesion plaque further triggered intracellular β-catenin signaling and promoted its nuclear translocation especially on the stiff substrate. The influence of β-catenin signaling on direct differentiation to osteogenic lineages was through direct binding between its downstream protein, Lef-1, and the osteogenic transcriptional factors, Runx2 and Osx, while on differentiation to adipogenic lineages was through modulating the expression of PPARγ. The imbalance of stiffness-induced β-catenin signaling finally induced a stronger osteogenesis and a weaker adipogenesis on the stiff substrate relative to those on the soft substrate. This study indicates the importance of stiffness on ASC differentiation and could help to increase understanding of the mechanism underlying molecular signal transduction from mechanosensing, mechanotransducing to stem cell differentiation., Statement of Significance: Mesenchymal stem cells can differentiate into multiple lineages, such as adipogenesis, myogenesis, neurogenesis, angiogenesis and osteogenesis, through influence of biophysical properties of the extracellular matrix. However, the precise bio-mechanism that triggers stem cell differentiation in response to matrix biophysical properties remains unclear. In the current study, we provide a series of experiments involving the characterization of cell morphology, microfilament, microtubule and adhesion capacity of adipose-derived stromal cells (ASCs) in response to substrate stiffness, and further elucidation of cytoplasmic β-catenin-dependent signal transduction, nuclear translocation and resultant promoter activation of transcriptional factors for osteogenesis and adipogenesis. This study provides an explanation on deeper understanding of bio-mechanism underlying substrate stiffness-triggered β-catenin signal transduction from active mechanosensing, mechanotransducing to stem cell differentiation., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

This paper aimed to observe the protective effect of catalpol on the high glucose induced destruction of tight junctions of rat primary brain microvascular endothelial cells (BMECs). Catalpol co-administrated with high glucose increased BMECs survival, decreased its ET-1 secretion, and improved transmembrane electrical resistance in a time-dependent manner. Furthermore, transmission electron microscopy was used to observe catalpol's protective effect on tight junction. Fluorescence staining displayed that catalpol reversed the rearrangement of the cytoskeleton protein F-actin and up-regulated the tight junction proteins claudin-5 and ZO-1, which were further demonstrated by the mRNA expression levels of claudin-5, occludin, ZO-1, ZO-2, ZO-3, α -actintin, vinculin and cateinins. This study indicated that catalpol reverses the disaggregation of cytoskeleton actin in BMECs and up-regulates the expression of tight junction proteins, such as claudin-5, occludin, and ZO-1, and finally alleviates the increase in high glucose-induced BMECs injury., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Xerostomia and hyposalivation are debilitating side effects for patients treated with ionizing radiation for head and neck cancer. Despite technological advances, collateral damage to the salivary glands remains a significant problem for patients and severely diminishes their quality of life. During the wound healing process, restoration of junctional contacts is necessary to maintain polarity, structural integrity, and orientation cues for secretion. However, little is known about whether these structural molecules are impacted following radiation damage and more importantly, during tissue restoration. We evaluated changes in adherens junctions and cytoskeletal regulators in an injury model where mice were irradiated with 5 Gy and a restoration model where mice injected postradiation with insulin-like growth factor 1 (IGF1) are capable of restoring salivary function. Using coimmunoprecipitation, there is a decrease in epithelial (E)-cadherin bound to β-catenin following damage that is restored to untreated levels with IGF1. Via its adaptor proteins, β-catenin links the cadherins to the cytoskeleton and part of this regulation is mediated through Rho-associated coiled-coil containing kinase (ROCK) signaling. In our radiation model, filamentous (F)-actin organization is fragmented, and there is an induction of ROCK activity. However, a ROCK inhibitor, Y-27632, prevents E-cadherin/β-catenin dissociation following radiation treatment. These findings illustrate that radiation induces a ROCK-dependent disruption of the cadherin-catenin complex and alters F-actin organization at stages of damage when hyposalivation is observed. Understanding the regulation of these components will be critical in the discovery of therapeutics that have the potential to restore function in polarized epithelium.

Clinical and experimental studies demonstrate the potential of low-magnitude high-frequency vibration (LMHFV) to enhance bone formation in the intact skeleton and during fracture healing. Moreover, it was shown that the effects of vibration therapy during fracture healing are highly dependent on the estrogen status of the vibrated individual and that estrogen receptor (ER) α signaling plays a major role in mechanotransduction of LMHFV. Because it is known that LMHFV can directly act on osteogenic cells, we hypothesize that the differential effects of LMHFV in the presence and absence of estrogen are mediated by ERα signaling in osteoblasts. To prove this hypothesis, we subjected preosteoblastic MC3T3-E1 cells and primary osteoblasts to LMHFV in vitro. We found increased Cox2 gene expression, cell metabolic activity and cell proliferation after LMHFV in the absence of estrogen, whereas the effects were contrary in the presence of estrogen. Blocking of ERα signaling by Esr1-siRNA knockdown or adding the selective ERα antagonist MPP dihydrochloride abolished the effects of LMHFV on osteoblast proliferation and Cox2 expression. Furthermore, primary osteoblasts isolated from ERα-knockout mice did not show a response towards LMHFV in the presence of estrogen. Additionally, blocking of actin cytoskeletal remodeling by adding the p160ROCK inhibitor Y-27632 abolished the effects of LMHFV. In contrast, expression of primary cilium was not necessary for mechanotransduction of LMHFV. These results suggest that direct effects of LMHFV on osteoblasts are dependent on ERα signaling and cytoskeletal remodeling., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Alterations in cell morphology involve changes in the actin cytoskeleton and play crucial roles in determining chondrocyte phenotypes. Although the effects of simvastatin (SV) have been demonstrated in various cell types, the mechanisms and effects of SV on chondrocyte differentiation and actin cytoskeletal rearrangement are still unclear. Here, we investigated the roles of actin filament rearrangement on SV-induced differentiation of rabbit articular chondrocytes. Treatment with SV caused actin remodeling in comparison with that in untreated chondrocytes, as determined by immunofluorescence staining. Moreover, treatment with cytochalasin D (CD) and jasplakinolide (JAS), which modulate actin filament formation, resulted in reorganization of the actin cytoskeleton compared with that induced by SV in chondrocytes. In addition, CD synergistically enhanced the SV-induced increase in type II collagen expression, whereas JAS dramatically inhibited SV-induced differentiation. We also found that differentiation via SV-dependent actin cytoskeleton changes was regulated by the extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase pathways. These results demonstrated that actin cytoskeletal rearrangement by SV regulated type II collagen expression and suggested that ERK-1/2 and p38 kinase pathways may play important roles in SV-induced type II collagen expression by altering actin cytoskeletal reorganization in rabbit articular chondrocytes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

CSPGs are components of the extracellular matrix in the nervous system, where they serve as cues for axon guidance during development. After a peripheral nerve injury, CSPGs switch roles and become axon inhibitors and become diffusely distributed at the injury site. To investigate whether the spatial distribution of CSPGs affects their role, we combined in vitro DRG cultures with CSPG stripe or coverage assays to simulate the effect of a patterned substrate or dispersive distribution of CSPGs on growing neurites. We observed neurite steering at linear CSPG interfaces and neurite inhibition when diffused CSPGs covered the distal but not the proximal segment of the neurite. The repellent and inhibitory effects of CSPGs on neurite outgrowth were associated with the disappearance of focal actin filaments on growth cones. The application of an actin polymerization inducer, jasplakinolide, allowed neurites to break through the CSPG boundary and grow on CSPG-coated surfaces. The results of our study collectively reveal a novel mechanism that explains how the spatial distribution of CSPGs determines whether they act as a cue for axon guidance or as an axon-inhibiting factor. Increasing our understanding of this issue may promote the development of novel therapeutic strategies that regulate the spatial distributions of CSPGs to use them as an axon guidance cue., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydrogen peroxide (H 2 O 2 ) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with H 2 O 2 activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of H 2 O 2 to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of H 2 O 2 to increase paracellular permeability. H 2 O 2 treatment did not, however, affect the total cell content or contents of the Triton X-100-soluble and -insoluble fractions for occludin, ZO-1, or ZO-2. H 2 O 2 treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, H 2 O 2 increased ERK 1/2 activation in ZO-1 knockdown and occludin knockdown cells. Inhibition of ERK 1/2 activation blocked the increase in paracellular permeability in occludin knockdown cells. ZO-1 knockdown cell paracellular permeability was regulated by PP1, an src inhibitor, indicating that the loss of response to H 2 O 2 was not a general loss of the ability to regulate the paracellular barrier. Inhibition of myosin ATPase activity with blebbistatin increased paracellular permeability in ZO-1 knockdown cells but not in wild-type MDCK cells. H 2 O 2 treatment sensitized wild-type MDCK cells to inhibition of myosin ATPase. Knockdown of TOCA-1 protein, which promotes formation of local branched actin networks, reproduced the effects of ZO-1 protein knockdown. These results demonstrate that H 2 O 2 increases MDCK cell paracellular permeability through activation of ERK 1/2. This H 2 O 2 action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.

In our previous study, advanced glycosylation end‑product specific receptor (RAGE) was observed to bind to S100A8/A9 and cause epithelial mesenchymal transition (EMT). The results from target gene prediction revealed that microRNA (miR)‑185‑5p had a RAGE binding site. However, the function of miR‑185‑5p in the invasion and migration of breast cancer remains ambiguous. In the present study, the expression of miR‑185‑5p was examined in breast cancer tissues and cells. Clinical features revealed a negative correlation between miR‑185‑5p and tumor size, as well as in tumor differentiation and lymph node metastasis in breast cancer. In addition, miR‑185‑5p was negatively associated with RAGE, and this miRNA reversed the EMT of breast cancer by modulating RAGE in vitro. In addition, miR‑185‑5p inhibited the S100A8/A9‑induced EMT of breast cancer cells by the nuclear factor‑κB/Snail signaling pathway. Notably, miR‑185‑5p upregulation inhibited the F‑actin polymerization induced by S100A8/A9 in breast cancer. Furthermore, overexpression of miR‑185‑5p and reduction of RAGE inhibited lung metastasis node in vivo. Thus, miR‑185‑5p represents a potential therapeutic target in breast cancer by modulating RAGE.

The disturbance of podocyte motility is an essential pathogenic mechanisms of foot process effacement during proteinuric diseases, and myosin light chain (MLC) is a pivotal component in regulating the motility of podocytes. Inflammatory cytokine interleukin-6 (IL-6) has been reported to induce podocyte abnormalities by various mechanisms, however, whether aberrant cell motility contributes to the IL-6-induced podocyte injury remains unknown. Here, by wound healing, transwell, and cell migration assays, we confirmed that IL-6 accelerates the motility of podocyte. Simultaneously, the phosphorylation of MLC is elevated along with perturbed focal adhesion (FAs) and cytoskeleton. Next, via genetic and pharmacologic interruption of MLC or its phosphorylation we revealed that the activation of MLC is implicated in IL-6-mediated podocyte hypermotility as well as the disassembly of FAs and F-actin. By using stattic, an inhibitor for STAT3 phosphorylation, we uncovered that STAT3 activation is the upstream event for MLC phosphorylation and the following aberrant motility of podocytes. Additionally, we found that calcitriol markedly attenuates podocyte hypermotility via blocking STAT3-MLC. In conclusion, our study demonstrated that IL-6 interrupts FAs dynamic, cytoskeleton organization, and eventually leads to podocyte hypermotility via STAT3/MLC, whereas calcitriol exerts its protective role by inhibiting this pathway. These findings enrich the mechanisms accounting for IL-6-mediated podocyte injury from the standpoint of cell motility and provide a novel therapeutic target for podocyte disorders., (© 2018 Wiley Periodicals, Inc.)

Tetraspanins co-emerged with multi-cellular organisms during evolution and are typically localized at the cell–cell interface, [corrected] and form tetraspanin-enriched microdomains (TEMs) by associating with each other and other membrane molecules. Tetraspanins affect various biological functions, but how tetraspanins engage in multi-faceted functions at the cellular level is largely unknown. When cells interact, the membrane microextrusions at the cell-cell interfaces form dynamic, digit-like structures between cells, which we term digitation junctions (DJs). We found that (1) tetraspanins CD9, CD81, and CD82 and (2) TEM-associated molecules integrin α3β1, CD44, EWI2/PGRL, and PI-4P are present in DJs of epithelial, endothelial, and cancer cells. Tetraspanins and their associated molecules also regulate the formation and development of DJs. Moreover, (1) actin cytoskeleton, RhoA, and actomyosin activities and (2) growth factor receptor-Src-MAP kinase signaling, but not PI-3 kinase, regulate DJs. Finally, we showed that DJs consist of various forms in different cells. Thus, DJs are common, interactive structures between cells, and likely affect cell adhesion, migration, and communication. TEMs probably modulate various cell functions through DJs. Our findings highlight that DJ morphogenesis reflects the transition between cell-matrix adhesion and cell-cell adhesion and involves both cell-cell and cell-matrix adhesion molecules.

Our previous studies indicated that α4 was involved in the toxicity of MC-LR on the cytoskeleton via the change of PP2A activity in HEK 293. To explore the role of α4 in MC-LR toxicity via PP2A regulation in different cell lines, the HL7702 cell overexpressing α4 protein was exposed to MC-LR, and the change of PP2A, cytoskeletal structure, and cytoskeleton-related proteins were investigated. The results showed that PP2A activity was decreased, PP2A/C subunit expression and phosphorylation (Tyr307) increased significantly, but methylation (Leu 309)clearly decreased. The structure of the actin filaments and microtubules (MTs) remained unchanged, and the expression and phosphorylation of the cytoskeleton-related proteins showed different changes. In addition, the main components of the MAPK pathway, JNK, P38, and ERK1/2, were activated together. Our results indicated that elevated α4 expression did confer some resistance to MC-LR-induced cytoskeletal changes, but the responses of different cell lines to MC-LR, under the α4-overexpression condition, are not exactly the same., (© 2018 Wiley Periodicals, Inc.)