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AbstractWe developed an original computational model for cell deformation and migration capable of accounting for the cell sensitivity to the environment and its appropriate adaptation. This cell model is ultimately intended to be used to address tissue morphogenesis. Hence it has been designed to comply with four requirements: (1) the cell should be able to probe and sense its environment and respond accordingly; (2) the model should be easy to parametrize to adapt to different cell types; (3) the model should be able to extend to 3D cases; (4) simulations should be fast enough to integrate many interacting cells. The simulations carried out focused on two aspects: first, the general behaviour of the cell on a homogeneous substrate, as observed experimentally, for model validation. This enabled us to decipher the mechanisms by which the cell can migrate, highlighting respective influences of the adhesions lifetimes and their sensitivity to traction; second, it predicts the sensitivity of the cell to an anisotropic patterned substrate, in agreement with recently published experiments. The results show that mechanosensors simulated by the model make it possible to reproduce such experiments in terms of migration bias generated by the substrate anisotropy. Here again, the model provides a biomechanical explanation of this phenomenon, depending on cell-matrix interactions and adhesion maturation rate. [ABSTRACT FROM AUTHOR]

Microtubule‐depolymerizing agents can selectively disrupt tumor vessels via inducing endothelial membrane blebbing. However, the mechanism regulating blebbing is largely unknown. IMB5046 is a newly discovered microtubule‐depolymerizing agent. Here, the functions of focal adhesion kinase (FAK) during IMB5046‐induced blebbing and the relevant mechanism are studied. We found that IMB5046 induced membrane blebbing and reassembly of focal adhesions in human vascular endothelial cells. Both FAK inhibitor and knock‐down expression of FAK inhibited IMB5046‐induced blebbing. Mechanism study revealed that IMB5046 induced the activation of FAK via GEF‐H1/ Rho/ ROCK/ MLC2 pathway. cRGD peptide, a ligand of integrin, also blocked IMB5046‐induced blebbing. After activation, FAK further promoted the phosphorylation of MLC2. This positive feedback loop caused more intensive actomyosin contraction and continuous membrane blebbing. FAK inhibitor blocked membrane blebbing via inhibiting actomyosin contraction, and stimulated stress fibre formation via promoting the phosphorylation of HSP27. Conclusively, these results demonstrate that FAK is a molecular switch controlling endothelial blebbing and stress fibre formation. Our study provides a new molecular mechanism for microtubule‐depolymerizing agents to be used as vascular disrupting agents. [ABSTRACT FROM AUTHOR]

The physiological function of actin filaments is challenging to dissect because of the pleiotropic impact of global disruption of the actin cytoskeleton. Tropomyosin isoforms have provided a unique opportunity to address this issue. A substantial fraction of actin filaments in animal cells consist of co-polymers of actin with specific tropomyosin isoforms which determine the functional capacity of the filament. Genetic manipulation of the tropomyosins has revealed isoform specific roles and identified the physiological function of the different actin filament types based on their tropomyosin isoform composition. Surprisingly, there is remarkably little redundancy between the tropomyosins resulting in highly penetrant impacts of both ectopic overexpression and knockout of isoforms. The physiological roles of the tropomyosins cover a broad range from development and morphogenesis to cell migration and specialised tissue function and human diseases. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Polymeric surfaces with varying roughness are manufactured from a blend of a PS and an ABS dispersion. • HDF cell proliferation on the surfaces is correlated to some roughness parameters of the surfaces obtained with AFM. • Cells grown on surfaces with a moderate roughness were observed to form less stress fibres. Abstract In this study hierarchically-structured latex polymer coatings and self-supporting films were characterised and their suitability for cell growth studies was tested with Human Dermal Fibroblasts (HDF). Latex can be coated or printed on rigid or flexible substrates thus enabling high-throughput fabrication. Here, coverslip glass substrates were coated with blends of two different aqueous latex dispersions: hydrophobic polystyrene (PS) and hydrophilic carboxylated acrylonitrile butadiene styrene (ABS). The nanostructured morphology and topography of the latex films was controlled by varying the mixing ratio of the components in the latex blend. Thin latex-coatings retain high transparency on glass allowing optical and high resolution imaging of cell growth and morphology. Compared to coverslip glass surfaces and commercial well-plates HDF cell growth was enhanced up to 150–250 % on latex surfaces with specific nanostructure. Growth rates were correlated with selected roughness parameters such as effective surface area (S q), RMS-roughness (S dr) and correlation length (S cl37). High-resolution confocal microscopy clearly indicated less actin stress-fibre development in cells on the latex surface compared to coverslip glass. The results show that surface nanotopography can, by itself, passively modulate HDF cell proliferation and cytoskeletal architecture. [ABSTRACT FROM AUTHOR]

Myosin II filaments form ordered superstructures in both cross-striated muscle and non-muscle cells. In cross-striated muscle, myosin II (thick) filaments, actin (thin) filaments and elastic titin filaments comprise the stereotypical contractile units of muscles called sarcomeres. Linear chains of sarcomeres, called myofibrils, are aligned laterally in registry to form cross-striated muscle cells. The experimentally observed dependence of the registered organization of myofibrils on extracellular matrix elasticity has been proposed to arise from the interactions of sarcomeric contractile elements (considered as force dipoles) through the matrix. Non-muscle cells form small bipolar filaments built of less than 30myosin II molecules. These filaments are associated in registry forming superstructures ('stacks') orthogonal to actin filament bundles. Formation of myosin II filament stacks requires the myosin II ATPase activity and function of the actin filament crosslinking, polymerizing and depolymerizing proteins. We propose that the myosin II filaments embedded into elastic, intervening actin network (IVN) function as force dipoles that interact attractively through the IVN. This is in analogy with the theoretical picture developed for myofibrils where the elastic medium is now the actin cytoskeleton itself. Myosin stack formation in non-muscle cells provides a novel mechanism for the self-organization of the actin cytoskeleton at the level of the entire cell. This article is part of the theme issue 'Self-organization in cell biology'. [ABSTRACT FROM AUTHOR]

The key role of electro-chemical signals in cellular processes had been known for many years, but more recently the interplay with mechanics has been put in evidence and attracted substantial research interests. Indeed, the sensitivity of cells to mechanical stimuli coming from the microenvironment turns out to be relevant in many biological and physiological circumstances. In particular, experimental evidence demonstrated that cells on elastic planar substrates undergoing periodic stretches, mimicking native cyclic strains in the tissue where they reside, actively reorient their cytoskeletal stress fibres. At the end of the realignment process, the cell axis forms a certain angle with the main stretching direction. Due to the importance of a deeper understanding of mechanotransduction, such a phenomenon was studied both from the experimental and the mathematical modelling point of view. The aim of this review is to collect and discuss both the experimental results on cell reorientation and the fundamental features of the mathematical models that have been proposed in the literature. • Cells respond to mechanical stimuli by reorienting their cytoskeleton. • A review of experiments and mathematical models of cell orientation under stretch. • Collection of experimental data highlighting the relevant mechanical factors. • Summary of mathematical models proposed to describe cell realignment. [ABSTRACT FROM AUTHOR]

Substance P (SP) plays a role in vasodilatation and tissue integrity through its receptor, neurokinin 1 (NK1R). However, its specific effect on blood-brain barrier (BBB) remains unknown. The impact of SP on the integrity/function of human BBB model in vitro, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, was assessed by measurements of transendothelial electrical resistance and paracellular flux of sodium fluorescein (NaF), respectively in the absence/presence of specific inhibitors targeting NK1R (CP96345), Rho-associated protein kinase (ROCK; Y27632) and nitric oxide synthase (NOS; N(G)-nitro- L -arginine methyl ester). Sodium nitroprusside (SNP), a NO donor, was employed as a positive control. The levels of tight junction proteins, zonula occludens-1, occludin and claudin-5 alongside RhoA/ROCK/myosin regulatory light chain-2 (MLC2) and extracellular signal‑regulated protein kinase (Erk1/2) proteins were detected by western analyses. Subcellular localisations of F-actin and tight junction proteins were visualized by immunocytochemistry. Flow cytometry was used to detect transient calcium release. Exposure to SP increased RhoA, ROCK2 and phosphorylated serine-19 MLC2 protein levels and Erk1/2 phosphorylation in BMECs which were abolished by CP96345. These increases were independent of the changes in intracellular calcium availability. SP perturbed BBB in a time-dependent fashion through induction of stress fibres. Changes in tight junction protein dissolution or relocalisation were not involved in SP-mediated BBB breakdown. Inhibition of NOS, ROCK and NK1R mitigated the effect of SP on BBB characteristics and stress fibre formation. SP promoted a reversible decline in BBB integrity independent of tight junction proteins expression or localisation. • Substance P promoted a transient disruption in blood-brain barrier (BBB) integrity and function in in vitro settings. • Substance P evokes RhoA/ROCK2/MLC2 and Erk1/2 pathways in brain microvascular endothelial cells via neurokinin 1 receptor. • Formation of actin stress fibres and induction of nitric oxide synthase played a role in substance P-evoked BBB damage. • Substance P-induced BBB damage was independent of its effects on the expression and localisation of tight junction proteins. [ABSTRACT FROM AUTHOR]

Finite element (FE) simulations of contractile responses of vascular muscular thin films (vMTFs) and endothelial cells resting on an array of microposts under stimulation of soluble factors were conducted in comparison with experimental measurements reported in the literature. Two types of constitutive models were employed in the simulations, i.e. smooth muscle cell type and non-smooth muscle cell type. The time histories of the effects of soluble factors were obtained via calibration against experimental measurements of contractile responses of tissues or cells. The numerical results for vMTFs with micropatterned tissues suggest that the radius of curvature of vMTFs under stimulation of soluble factors is sensitive to width of the micropatterned tissue, i.e. the radius of curvature increases as the tissue width decreases. However, as the tissue response is essentially isometric, the time history of the maximum principal stress of the micropatterned tissues is not sensitive to tissue width. Good agreement has been achieved for predictions of the vasoconstrictor endothelin-1-induced contraction stress between the FE numerical simulation and the experiment-based approach of Alford (Integr Biol 3:1063-1070, 2011) for the vMTFs with 40, 60, 80 and 100 $$\upmu \hbox {m}$$ width patterns. This may suggest the contraction stress is weakly sensitive to the tissue width for these patterns. However, for 20 $$\upmu \hbox {m}$$ width tissue patterning, the numerical simulation result for contraction stress is less than the average value of experimental measurements, which may suggest the thinner and more elongated spindle-like cells within the 20 $$\upmu \hbox {m}$$ width tissue patterning have higher contractile output. The constitutive model for non-smooth muscle cells was used to simulate the contractile response of the endothelial cells. The substrate was treated as an effective continuum. For agonists such as lysophosphatidic acid and vascular endothelial growth factor, the deformation of the cell diminishes from edge to centre and the central part of the cell is essentially under isometric state. Numerical studies demonstrated the scenarios that cell polarity can be triggered via manipulation of the effective stiffness and Possion's ratio of the substrate. [ABSTRACT FROM AUTHOR]

Ischaemic stroke, accompanied by neuroinflammation, impairs blood–brain barrier integrity through a complex mechanism involving both protein kinase C (PKC) and urokinase. Using an in vitro model of human blood–brain barrier (BBB) composed of brain microvascular endothelial cells (HBMEC) and astrocytes, this study assessed the putative roles of these elements in BBB damage evoked by enhanced availability of pro-inflammatory cytokine, TNF-α. Treatment of HBMEC with TNF-α significantly increased the mRNA and protein expressions of all plasminogen–plasmin system (PPS) components, namely tissue plasminogen activator, urokinase, urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 and also the activities of urokinase, total PKC and extracellular MMP-2. Inhibition of urokinase by amiloride abated the effects of TNF-α on BBB integrity and MMP-2 activity without affecting that of total PKC. Conversely, pharmacological inhibition of conventional PKC isoforms dramatically suppressed TNF-α-induced overactivation of urokinase. Knockdown of PKC-α gene via specific siRNA in HBMEC suppressed the stimulatory effects of TNF-α on protein expression of all PPS components, MMP-2 activity, DNA fragmentation rates and pro-apoptotic caspase-3/7 activities. Establishment of co-cultures with BMEC transfected with PKC-α siRNA attenuated the disruptive effects of TNF-α on BBB integrity and function. This was partly due to elevations observed in expression of a tight junction protein, claudin-5 and partly to prevention of stress fibre formation. In conclusion, specific inhibition of PKC-α in cerebral conditions associated with exaggerated release of pro-inflammatory cytokines, notably TNF-α may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane. [ABSTRACT FROM AUTHOR]

Cancer metastasis occurs via a progress involving abnormal cell migration. Cell migration, a dynamic physical process, is controlled by the cytoskeletal system, which includes the dynamics of actin organization and cellular adhesive organelles, focal adhesions ( FAs). However, it is not known whether the organization of actin cytoskeletal system has a regulatory role in the physiologically relevant aspects of cancer metastasis. In the present studies, it was found that lung adenocarcinoma cells isolated from the secondary lung cancer of the lymph nodes, H1299 cells, show specific dynamics in terms of the actin cytoskeleton and FAs. This results in a higher level of mobility and this is regulated by an immature FA component, β- PIX ( PAK-interacting exchange factor-β). In H1299 cells, β- PIX's activity was found not to be down-regulated by sequestration onto stress fibres, as the cells did not bundle actin filaments into stress fibres. Thus, β- PIX mainly remained localized at FAs, which allowed maturation of nascent adhesions into focal complexes; this resulted in actin polymerization, increased actin network integrity, changes in the intracellular microrheology at the peripheral of the cell, and cell polarity, which in turn regulated cell migration. Perturbation of β- PIX caused an inhibition of cell migration, including migration velocity, accumulated distance and directional persistence. Our results demonstrate the importance of β- PIX to the regulation of high mobility of lung adenocarcinoma cell line H1299 and that this occurs via regulation of FA dynamics, changes in actin cytoskeleton organization and cell polarity. [ABSTRACT FROM AUTHOR]

Different tissues have specific mechanical properties and cells of different geometries, such as elongated muscle cells and polygonal endothelial cells, which are precisely regulated during embryo development. However, the mechanisms that underlie these processes are not clear. Here, we built an in vitro model to mimic the cellular microenvironment of muscle by combining both mechanical stretch and geometrical control. We found that mechanical stretch was a key factor that determined the optimal geometry of myoblast C2C12 cells under stretch, whereas vascular endothelial cells and fibroblasts had no such dependency. We presented the first experimental evidence that can explain why myoblasts are destined to take the elongated geometry so as to survive and maintain parallel actin filaments along the stretching direction. The study is not only meaningful for the research on myogenesis but also has potential application in regenerative medicine. [ABSTRACT FROM AUTHOR]

Cell migration requires the fine spatiotemporal integration of many proteins that regulate the fundamental processes that drive cell movement. Focal adhesion (FA) dynamics is a continuous process involving coordination between FA and actin cytoskeleton, which is essential for cell migration. We studied the spatiotemporal relationship between the dynamics of focal adhesion kinase (FAK) and paxillin at FAs in the protrusion of living endothelial cells. Concurrent dual-color imaging showed that FAK was assembled at FA first, which was followed by paxillin recruitment to the FA. By tracking and quantifying FAK and paxillin in migrating cells, the normalized FAK/Paxillin fluorescence intensity (FI) ratio is > 1 (≈4 fold) at cell front, ≈1 at cell center, and, < at cell rear. The significantly higher FAK FI than paxillin FI at cell front indicates that the assembly of FAK-FAs occurs ahead of paxillin at cell front. To determine the time difference between the assemblies of FAK and paxillin at nascent FAs, FAs containing both FAK and paxillin were quantified by image analysis and time correlation. The results show that FAK assembles at the nascent FAs earlier than paxillin in the protrusions at cell front. [ABSTRACT FROM AUTHOR]

Cell migration relies on traction forces in order to propel a cell. Several computational models have been developed that help explain the trajectory that cells take during migration, but little attention has been placed on traction forces during this process. Here, we investigated the spatiotemporal dynamics of cell migration by using a bio-chemical-mechanical contractility model that incorporates the first steps of cell migration on an array of posts. In the model, formation of a new adhesion causes a reactivation of stress fibre assembly within a cell. The model was able to predict the spatial distribution of traction forces observed with previous experiments. Moreover, the model found that the strain energy exerted by the traction forces of a migrating cell underwent a cyclic relationship that rose with the formation of a new adhesion and fell with the release of an adhesion at its rear. [ABSTRACT FROM AUTHOR]

In this paper, we propose a computational model to investigate the coupling between cell’s adhesions and actin fibres and how this coupling affects cell shape and stability. To accomplish that, we take into account the successive stages of adhesion maturation from adhesion precursors to focal complexes and ultimately to focal adhesions, as well as the actin fibres evolution from growing filaments, to bundles and finally contractile stress fibres.We use substrates with discrete patterns of adhesive patches, whose inter-patches distance can be modulated in order to control the location of the adhesions and the resulting fibres architecture. We then investigate the emergence of stable cell morphologies as a function of the inter-patches distance, for two different cell phenotypes generated from the model. Force generated by the stress fibres on the focal adhesions and specifically the influence of the cell contractility are also investigated.Our results suggest that adhesion lifetime and fibre growing rate are the key parameters in the emergence of stable cell morphologies and the limiting factors for the magnitude of the mean tension force from the fibres on the focal adhesions. [ABSTRACT FROM PUBLISHER]

The ROCK-I serine/threonine protein kinase mediates the effects of RhoA to promote the formation of actin stress fibres and integrin-based focal adhesions. ROCK-I phosphorylates the unconventional G-protein RhoE on multiple N- and C-terminal sites. These phosphorylation events stabilise RhoE, which functions to antagonise RhoA-induced stress fibre assembly. Here, we provide a molecular explanation for multi-site phosphorylation of RhoE from the crystal structure of RhoE in complex with the ROCK-I kinase domain. RhoE interacts with the C-lobe αG helix of ROCK-I by means of a novel binding site remote from its effector region, positioning its N and C termini proximal to the ROCK-I catalytic site. Disruption of the ROCK-I:RhoE interface abolishes RhoE phosphorylation, but has no effect on the ability of RhoE to disassemble stress fibres. In contrast, mutation of the RhoE effector region attenuates RhoE-mediated disruption of the actin cytoskeleton, indicating that RhoE exerts its inhibitory effects on ROCK-I through protein(s) binding to its effector region. We propose that ROCK-I phosphorylation of RhoE forms part of a feedback loop to regulate RhoA signalling. [ABSTRACT FROM AUTHOR]

Stress fibres are contractile acto-myosin structures found from many types of non-muscle cells, where they are involved in adhesion, motility and morphogenesis. Stress fibres typically display a periodic α-actinin–myosin II pattern and are thus suggested to resemble the sarcomeric actin filament structures of muscle cells. Mammalian cells contain three categories of stress fibres: ventral stress fibres that are attached to focal adhesions at both ends, dorsal stress fibres that are attached to focal adhesions typically at one end and transverse arcs that are curved acto-myosin bundles, which do not directly attach to focal adhesions. In this review, we discuss the definition of stress fibres, organization of actin filaments and other components within these contractile structures, and the mechanisms of stress fibre assembly. [ABSTRACT FROM AUTHOR]

In confluent primary cultures of rat hepatocytes,hypertonic stress leads to cell shrinkage and activates non-selective cation channels as the main mechanism of regulatory cell volume increase. The process is found to employ the exocytotic insertion of channels into the plasma membrane and (in addition to PKC) PLC, tyrosine kinases and G proteins, but not PI 3-kinase are part of the signalling network. Furthermore, hypertonic stress leads to the formation of stress fibres and significantly alters the activity of RhoA, Rac and Cdc42. These latter effects, however, are likely to reflect the restoration of cell shape rather than the regulation of cell volume, both most probably converging at the level of focal adhesions and integrins. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: α-Actinin is an evolutionarily conserved actin filament crosslinking protein with functions in both muscle and non-muscle cells. In non-muscle cells, interactions between α-actinin and its many binding partners regulate cell adhesion and motility. In Drosophila, one non-muscle and two muscle-specific α-actinin isoforms are produced by alternative splicing of a single gene. In wild-type ovaries, α-actinin is ubiquitously expressed. The non-muscle α-actinin mutant Actn Δ233 , which is viable and fertile, lacks α-actinin expression in ovarian germline cells, while somatic follicle cells express α-actinin at late oogenesis. Here we show that this latter population of α-actinin, termed FC-α-actinin, is absent from the dorsoanterior follicle cells, and we present evidence that this is the result of a negative regulation by combined Epidermal growth factor receptor (EGFR) and Decapentaplegic signalling. Furthermore, EGFR signalling increased the F-actin bundling activity of ectopically expressed muscle-specific α-actinin. We also describe a novel morphogenetic event in the follicle cells that occurs during egg elongation. This event involves a transient repolarisation of the basal actin fibres and the assembly of a posterior β-integrin-dependent adhesion site accumulating α-actinin and Enabled. Clonal analysis using Actn null alleles demonstrated that although α-actinin was not necessary for actin fibre formation or maintenance, the cytoskeletal remodelling was perturbed, and Enabled did not localise in the posterior adhesion site. Nevertheless, epithelial morphogenesis proceeded normally. This work provides the first evidence that α-actinin is involved in the organisation of the cytoskeleton in a non-muscle tissue in Drosophila. [Copyright &y& Elsevier]

Abstract: We have shown that protein kinase C (PKC)ε, independently of the catalytic domain, induces outgrowth of cellular processes via its regulatory domain in both neural cells and fibroblasts. This was accompanied by stress fibre loss. Here, we have examined the role of the small GTPases, Rac1, and Cdc42, in these PKC-mediated morphological and cytoskeletal changes. Both constitutively active and dominant negative Rac1 and Cdc42 attenuated the PKC-mediated outgrowth of processes. The suppression was larger for Cdc42 than for Rac1. The PKC-mediated dismantling of the stress fibres in both HiB5 and fibroblasts was inhibited by the expression of the Cdc42 mutants whereas they had smaller effects on the stress fibre dismantling induced by the ROCK inhibitor, Y-27632, indicating a more crucial role for Cdc42 in the PKC-mediated pathway. We conclude that Cdc42 is an important downstream factor in the pathway through which PKC mediates morphological and cytoskeletal effects. [Copyright &y& Elsevier]

Platelets are crucial at the site of vascular injury, adhering to the sub-endothelial matrix through receptors on their surface, leading to cell activation and aggregation to form a haemostatic plug. Platelets display focal adhesions as well as stress fibres to contract and facilitate expulsion of growth and pro-coagulant factors contained in the granules and to constrict the clot. The interaction of F-actin with different actin-binding proteins determines the properties and composition of the focal adhesions. Recently, we demonstrated the presence of dystrophin-associated protein complex corresponding to short dystrophin isoforms (Dp71d and Dp71 ) and the uthophin gene family (Up400 and Up71), which promote shape change, adhesion, aggregation, and granule centralisation. To elucidate participation of both complexes during the platelet adhesion process, their potential association with integrin β-1 fraction and the focal adhesion system ( α-actinin, vinculin and talin) was evaluated by immunofluorescence and immunoprecipitation assays. It was shown that the short dystrophin-associated protein complex participated in stress fibre assembly and in centralisation of cytoplasmic granules, while the utrophin-associated protein complex assembled and regulated focal adhesions. The simultaneous presence of dystrophin and utrophin complexes indicates complementary structural and signalling mechanisms to the actin network, improving the platelet haemostatic role. [ABSTRACT FROM AUTHOR]

Cyclic AMP (cAMP)-raising agents induce astrocytes grownin vitroto adopt a stellate morphology resembling theirin vivoappearance, through the depolymerization of actomyosin stress fibres. The signalling pathways responsible for cAMP-induced astrocyte stellation have thus far remained largely elusive. We showed in this study that the neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) mimicked the effect of forskolin, a direct activator of adenylate cyclase, on the actin cytoskeleton of primary rat astrocytes. The depolymerization of stress fibres induced by PACAP or forskolin was prevented by the expression of a constitutively active mutant of RhoA, but not by a protein kinase A (PKA) blocker, indicating that cAMP-raising agents act upstream of RhoA, in a PKA-independent manner. In addition, PACAP and forskolin inhibited basal Akt phosphorylation, and basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3-kinase (PI 3-K) activities. Incubation with a PI 3-K blocker resulted in the depolymerization of stress fibres. This effect was blocked by the expression of a constitutively active mutant of RhoA, indicating that PI 3-K inhibition acted upstream of RhoA. Together, these data demonstrate for the first time that depolymerization of stress fibres, and the resulting astrocyte stellation, induced by stimulation of cAMP production involves the inhibition of the PI 3-K–RhoA pathway. [ABSTRACT FROM AUTHOR]

Astrocytes have a variety of roles in maintaining neural tissue physiology, including energetic support, uptake and metabolism of glutamate and secretion of neurotrophic factors. Glutamate toxicity has been implicated in neurodegenerative disorders associated with conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. Glucose is the main energetic substrate for brain cells but, in some circumstances, the ketone bodies are used as a supplementary source and have been suggested to be neuroprotective agents against seizure disorders. Here, we investigate some possible biochemical changes in astrocyte cultures induced by β-hydroxy-butyrate, the predominant blood ketone body. Its effect upon S100B secretion, astrocyte morphology and glutamate uptake was particularly investigated. S100B, a calcium-binding protein expressed and secreted by astrocytes, has neurotrophic activity and a possible role in epileptogenesis. Cell morphology was investigated by phase-contrast microscopy and immunocytochemistry for actin, GFAP and S100B. Our data show that β-hydroxy-butyrate induces dramatic changes in astrocyte morphology and, independent of this, causes changes in the extracellular content of S100B. We observed an increment in S100B 1h after β-hydroxy-butyrate addition and a decrease 24h later. No changes were observed in glutamate uptake. These astrocytic modifications may be associated with reduced neuronal excitability observed in the ketogenic condition. [Copyright &y& Elsevier]

We have shown that protein kinase C (PKC) &epsiv;, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKC&epsiv; resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKC&epsiv;-overexpressing cells and led to neurite induction in cells overexpressing PKCδ. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCδ induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCδ-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCδ can be counteracted by PKCδ kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth. [Copyright &y& Elsevier]

Background/Aims: Cytoskeletal reorganization plays an important role in the regulation of different cell functions, such as proliferation and migration. Since platelet-derived growth factor (PDGF) stimulates both proliferation and chemotaxis of hepatic stellate cells (HSC), we investigated the effects of this cytokine on cytoskeletal components of cultured rat HSC.Methods/Results: Exposure of HSC to PDGF induced the formation of stress fibres and of a ruffled configuration of the plasma membrane, evaluated by both fluorescence and electron microscopy. These modifications were also induced by exposure to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) and abolished by pretreatment with the PKC inhibitor calphostin C, with the Rho inhibitor C3 exoenzyme and with the intracellular calcium chelator MAPTAM, but not with the PI-3 kinase inhibitor wortmannin or with the mitogen-activated protein kinase kinase inhibitor PD 98059. PDGF induced a translocation of Rho from the cytosol to the membrane which was inhibited by C3 exoenzyme and by calpostin C, and which was also induced by PMA. Moreover, PDGF induced a rearrangement of vinculin which was prevented by C3 exoenzyme and calphostin C.Conclusions: PDGF-induced cytoskeletal reorganization in HSC is dependent on PKC and Rho, thus suggesting that these two pathways may play an important role in the response of liver to injury. [Copyright &y& Elsevier]

Tendons consist of parallel longitudinal rows of cells separated by collagen fibres. The cells are in intimate contact longitudinally within rows, and laterally via sheet-like lateral cell processes between rows. At points of contact, they are linked by gap junctions. Since tendons stretch under load, such cell contacts require protection. Here we describe the organisation of the actin cytoskeleton and actin-based cell–cell interactions in vivo and examine the effect of cyclic tensile loading on tendon cells in vitro. Cells within longitudinal rows contained short longitudinally running actin stress fibres. Each fibre was aligned with similar fibres in the cells longitudinally on either side, and fibres appeared to be linked via adherens junctions. Overall, these formed long oriented rows of stress fibres running along the rows of tendon cells. In culture, junctional components n-cadherin and vinculin and the stress fibre component tropomyosin increased in strained cultures, whereas actin levels remained constant. These results suggest that: (1) cells are linked via actin-associated adherens junctions along the line of principal strain; and (2) under load, cells appear to attach themselves more strongly together, and assemble more of their cytoplasmic actin into stress fibres with tropomyosin. Taken together, this suggests that cell–cell contacts are protected during stretch, and also that the stress fibres, which are contractile, may provide an active mechanism for recovery from stretch. In addition, stress fibres are ideally oriented to monitor tensile load and thus may be important in mechanotransduction and the generation of signals passed via the gap junction network. [Copyright &y& Elsevier]

Clostridium botulinum C2 toxin (C2 toxin) and purified ADP-ribosylated-α-actin (ADP-r-α-actin) cause specific actin depolymerisation in living cells. This effect was used to investigate the actin microfilament system with particular emphasis on cell-cell adhesion and plasma membrane integrity in endothelial cells. C2 toxin caused time- and dose-dependent (15–100 ng/ml) changes in endothelial surface morphology (investigated by atomic force microscopy), intercellular gap formation and cell detachment under shear stress. Low concentrations of C2 toxin (1.5 ng/ml), however, did not induce cell detachment but inhibited shear stress-dependent cell alignment. Gap formation as well as cell loss under shear stress was also observed in cells microinjected with purified ADP-r-α-actin. Intercellular gap formation was mediated by increased α-catenin solubility (40%) due to actin filament depolymerisation. Disintegration of plasma membranes (measured by LDH release) and cell fragmentation during simultaneous exposure to shear stress and C2 toxin were due to a loss of more than 50% of membrane-associated actin. These data show that small disturbances in actin dynamics inhibit shear stress-dependent cell alignment; that depolymerisation of actin filaments increases the solubility of α-catenin, thus resulting in cell dissociation and that actin filaments of the membrane cytoskeleton are required to protect the cells from haemodynamic injury such as shear stress. Together, the study shows a heterogeneous regulation of actin filament dynamics at subcellular locations. Junction-associated actin filaments displayed the highest sensitivity whereas stress fibres were far more stable. [ABSTRACT FROM AUTHOR]

The discovery of the green fluorescent protein (GFP) and its use as a marker for proteins in cells revolutionised cell biology. Among its applications are the intracellular localisation of proteins and the investigation of the organisation, regulation and dynamics of the cytoskeleton. GFP itself is considered to be an inert protein, homogeneously distributed within the cytoplasm. Here we investigated the intracellular distribution of GFP in an amphibian and in various mammalian cell lines (XTH2, CHO-K1, HaCaT, MDCK, NIH-3T3) by confocal laser scanning microscopy. After paraformaldehyde fixation GFP became associated with microfilaments in all the cell lines investigated. This interaction was not impaired by detergent treatment (1% Brij 58 for 10 min). In contrast to the F-actin binding of GFP in fixed cells, association of GFP with stress fibres was not detectable in living cells. The actin-binding property of GFP might contribute also to the interaction of fusion proteins with microfilaments. Thus, careful controls are unavoidable in investigating (weak) actin-binding proteins in fixed cells. Because no association of GFP with microfilaments was detectable in living cells, it is recommended to monitor the intracellular distribution of GFP-tagged proteins in vivo. [ABSTRACT FROM AUTHOR]

The effect of externally applied mechanical stress was investigated by thin section electron microscopy of the microvessels in the unaffected palmar fascia in the carpal tunnel syndrome and in patients with Dupuytren's contracture before and after application of a continuous elongation device. In the unaffected palmar fascia the microfilaments of the endothelial cells were connected to a few adherens junctions and focal contacts; stress fibres were absent. In the cord of Dupuytren's disease the microfilaments were increased in quantity. The length ratios of the connections with the lateral and basal cell membrane were significantly higher than in the control group and increased to an even greater extent in the continuously extended fascia. Stress fibres appeared in the endothelial cells of postcapillary venules in the nonextended cord and in the endothelium of both arterioles and venules after extension elongation. The numerous intermediate filaments and the rare microtubules remained unchanged in the endothelial cells of all palmar fasciae analysed. In the endothelial cells of the microvessels the mechanical stress applied from outside mainly affected the contractile component of the cytoskeleton. [ABSTRACT FROM AUTHOR]

The sensitivity of cells to alterations in the microenvironment and in particular to external mechanical stimuli is significant in many biological and physiological circumstances. In this regard, experimental assays demonstrated that, when a monolayer of cells cultured on an elastic substrate is subject to an external cyclic stretch with a sufficiently high frequency, a reorganization of actin stress fibres and focal adhesions happens in order to reach a stable equilibrium orientation, characterized by a precise angle between the cell major axis and the largest strain direction. To examine the frequency effect on the orientation dynamics, we propose a linear viscoelastic model that describes the coupled evolution of the cellular stress and the orientation angle. We find that cell orientation oscillates tending to an angle that is predicted by the minimization of a very general orthotropic elastic energy, as confirmed by a bifurcation analysis. Moreover, simulations show that the speed of convergence towards the predicted equilibrium orientation presents a changeover related to the viscous-elastic transition for viscoelastic materials. In particular, when the imposed oscillation period is lower than the characteristic turnover rate of the cytoskeleton and of adhesion molecules such as integrins, reorientation is significantly faster., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)