Publications

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-beta 1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (alpha- SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

JTD Keywords: Adhesio, Cardiac fibrosis modelling, Decellularized extracellular matrix, Differentiation, Expression, Fibroblast activation, Fibronectin, Heart, Induced pluripotent stem cells, Ipsc-derived-cardiac fibroblasts, Ipsc-derived-cardiomyocyte, Myocardial-infarction, Neonatal cardiomyocytes, Smooth muscle actin, Substrate stiffness

Mesenchymal stem cells (MSCs) interact with their surroundings via integrins, which link to the actin cytoskeleton and translate physical cues into biochemical signals through mechanotransduction. N-cadherins enable cell-cell communication and are also linked to the cytoskeleton. This crosstalk between integrins and cadherins modulates MSC mechanotransduction and fate. Here we show the role of this crosstalk in the mechanosensing of viscosity using supported lipid bilayers as substrates of varying viscosity. We functionalize these lipid bilayers with adhesion peptides for integrins (RGD) and N-cadherins (HAVDI), to demonstrate that integrins and cadherins compete for the actin cytoskeleton, leading to an altered MSC mechanosensing response. This response is characterised by a weaker integrin adhesion to the environment when cadherin ligation occurs. We model this competition via a modified molecular clutch model, which drives the integrin/cadherin crosstalk in response to surface viscosity, ultimately controlling MSC lineage commitment. The crosstalk between cell-cell and cell-matrix adhesions regulates stem cell fate. Here, the authors reveal a critical role for matrix viscosity in controlling this crosstalk, which they explain via a modified molecular clutch model.

JTD Keywords: Actin cytoskeleton, Adhesion, Animals, Arginyl-glycyl-aspartic acid, Cadherins, Cell adhesion, Cell communication, Fibronectin, Force transmission, Humans, Hydrogel, Integrins, Lipid bilayers, Matrix, Mechanotransduction, Mechanotransduction, cellular, Mesenchymal stem cells, Mobility, Oligopeptides, Osteogenic differentiation, Substrate stiffness, Vinculin, Viscosity

[No abstract available]

JTD Keywords: Alpha synuclein, Animal cell, Article, Astrocyte, Brain blood flow, Capillary endothelial cell, Cardiovascular system, Cell interaction, Coculture, Degenerative disease, Differential expression analysis, Endothelium cell, Entactin, Extracellular matrix, Fibronectin, Gene expression, Human, Human cell, Huntington chorea, Hydroxyapatite, In vitro study, Induced pluripotent stem cell, Laminin, Macrophage, Maturity, Microglia, Nervous system, Nervous system inflammation, Neuroprotection, Neurotoxicity, Nonhuman, Parkinson disease, Pericyte, Perivascular space, Personalized medicine, Shear stress, Smooth muscle cell, Three dimensional printing

The cell nucleus plays a key role in cellular mechanoresponses. 3D genome organisation, gene expression, and cell behaviour, in general, are affected by mechanical force application to the nucleus, which is transmitted from the cellular environment via a network of interconnected cytoskeletal components. To effectively regulate cell responses, these cytoskeletal components must not only exert forces but also withstand external forces when necessary. This review delves into the latest research concerning how the cytoskeleton safeguards the nucleus from mechanical perturbations. Spe-cifically, we focus on the three primary cytoskeletal polymers: actin, intermediate filaments, and microtubules, as well as their interactions with the cell nucleus. We discuss how the cyto-skeleton acts as a protective shield for the nucleus, ensuring structural integrity and conveying context-specific mechanoresponses.

JTD Keywords: Actin, Architecture, Cytoskeleton, Envelope, F-actin, Filaments, Force, Genome, Intermediate filaments, Lamin, Mechanotransduction, Membrane protein, Microtubules, Nesprin-1, Nucleus

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111-unlike fibronectin or collagen I-impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin β4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that β4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.© 2023. The Author(s).

JTD Keywords: actin, cell migration, filaments, force transmission, localization, membrane, motility, proteins, yap, Cell adhesion, Cytoskeleton, Extracellular matrix, Fibronectins, Integrin alpha-6-beta-4, Integrins, Keratins, Laminin

Clathrin-mediated endocytosis (CME) is an essential cellular process, conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of specific pharmacological agents to interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor targeting the AP2-β-adaptin/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP, demonstrating that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts. This is despite the β-adaptin/β-arrestin interaction not being conserved in yeast. Our data indicate that the AP2 α-adaptin is the functional target of activated TL2. We identified as interacting partners for the α-appendage, the Eps15 and epsin homologues Ede1 and Ent1. This demonstrates that endocytic cargo loading and sensing can be executed by conserved molecular interfaces, regardless of the proteins involved.© 2023 The Author(s).

JTD Keywords: adapters, alpha-appendage, azobenzene, cross-linker, mechanism, peptides, proteins, receptor, trafficking, Actin polymerization, Biochemistry, Biological sciences, Cell biology, Molecular biology, Natural sciences

As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nanoscale topography. Here, we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nanoscale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by I-BAR proteins, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.© 2023, Quiroga et al.

JTD Keywords: arp2/3 complex, bar, bar proteins, cdc42, cells, domain, human, irsp53, membrane biophysics, mouse, proteins, rac, tension, Actin polymerization, Actins, Bar proteins, Cell biology, Cell membrane, Homeostasis, Human, Mechanobiology, Membrane biophysics, Mouse, Physics of living systems

Live cell actin visualization is fundamental for exploring cellular motility, cytokinesis, intracellular transport, and other correlated functions. The current imaging techniques that allow imaging of actin in its native environment are optical and electron microscopy. Such imaging techniques offer high enough resolution to investigate the ultrastructure of actin however they come at the expense of actin integrity. Inspired by the lack of suitable probes that preserve actin's integrity, we designed a cyclometalated Ir (III) complex that interacts with live cells and displays light switch behaviour upon specific actin binding. The exceptional photophysical properties of the proposed probe allow unprecedented resolution of cytoskeleton ultrastructures under stimulated emission depletion (STED) super-resolution nanoscopy. Moreover, the Ir complex enables the capability of visualizing actin polymers and periodicity under correlative light electron microscopy (CLEM) and liquid-phase electron microscopy (LPEM) at similar to 8 nm resolution.

JTD Keywords: Actin dynamics, Actin targeting, Adhesion, Cells, Clem, Fluorescent, Iridium (iii) complex, Lead, Light, Lpem, Super-resolution ultrastructures

JTD Keywords: actin cytoskeleton, extracellular vesicles, vesicular transport, virulence factors, Actin cytoskeleton, Extracellular vesicles, Host-parasite interactions, Vesicular transport, Virulence factors

JTD Keywords: alignment, bioresorbable stents, cells, design, electrospinning, everolimus, impact, in-vitro, poly(l-lactic-co-e-caprolactone), proliferation, release, sirolimus, Scaffold topography, Solvent-cast direct-writing

Topographical patterns are a powerful tool to study directional migration. Grooved substrates have been extensively used as in vitro models of aligned extracellular matrix fibers because they induce cell elongation, alignment, and migration through a phenomenon known as contact guidance. This process, which involves the orientation of focal adhesions, F-actin, and microtubule cytoskeleton along the direction of the grooves, has been primarily studied on hard materials of non-physiological stiffness. But how it unfolds when the stiffness of the grooves varies within the physiological range is less known. Here we show that substrate stiffness modulates the cellular response to topographical contact guidance. We find that for fibroblasts, while focal adhesions and actin respond to topography independently of the stiffness, microtubules show a stiffness-dependent response that regulates contact guidance. On the other hand, both clusters and single breast carcinoma epithelial cells display stiffness-dependent contact guidance, leading to more directional and efficient migration when increasing substrate stiffness. These results suggest that both matrix stiffening and alignment of extracellular matrix fibers cooperate during directional cell migration, and that the outcome differs between cell types depending on how they organize their cytoskeletons.© 2023 The Authors.

JTD Keywords: actin, behavior, cell migration, contact guidance, cytoskeleton, fibroblasts, focal adhesions, matrix, microtubules, stiffness, stress fibers, topography, transduction, Contact guidance, Substrate stiffness, Topography

Several factors present in the extracellular environment regulate epithelial cell adhesion and dynamics. Among them, growth factors such as EGF, upon binding to their receptors at the cell surface, get internalized and directly activate the acto-myosin machinery. In this study we present the effects of EGF on the contractility of epithelial cancer cell colonies in confined geometry of different sizes. We show that the extent to which EGF triggers contractility scales with the cluster size and thus the number of cells. Moreover, the collective contractility results in a radial distribution of traction forces, which are dependent on integrin β1 peripheral adhesions and transmitted to neighboring cells through adherens junctions. Taken together, EGF-induced contractility acts on the mechanical crosstalk and linkage between the cell-cell and cell-matrix compartments, regulating collective responses.Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.

JTD Keywords: actin, activation, actomyosin, adherens junctions, adhesion, e-cadherin, egf, maturation, mechanical regulation, micropatterning, migration, traction forces, transduction, transmission, Actomyosin, Adherens junctions, Cell adhesion, Cell membrane, Collective contractility, Egf, Epidermal growth factor, Epidermal-growth-factor, Epithelial cells, Micropatterning, Myosins, Traction forces

While chemokines were originally described for their ability to induce cell migration, many studies show how these proteins also take part in many other cell functions, acting as adaptable messengers in the communication between a diversity of cell types. In the nervous system, chemokines participate both in physiological and pathological processes, and while their expression is often described on glial and immune cells, growing evidence describes the expression of chemokines and their receptors in neurons, highlighting their potential in auto- and paracrine signalling. In this study we analysed the role of nociception in the neuronal chemokinome, and in turn their role in axonal growth. We found that stimulating TRPV1(+) nociceptors induces a transient increase in CCL21. Interestingly we also found that CCL21 enhances neurite growth of large diameter proprioceptors in vitro. Consistent with this, we show that proprioceptors express the CCL21 receptor CCR7, and a CCR7 neutralizing antibody dose-dependently attenuates CCL21-induced neurite outgrowth. Mechanistically, we found that CCL21 binds locally to its receptor CCR7 at the growth cone, activating the downstream MEK-ERK pathway, that in turn activates N-WASP, triggering actin filament ramification in the growth cone, resulting in increased axonal growth.

JTD Keywords: axonal growth, ccl21, ccr7, mek-erk, Actin dynamics, Axonal growth, Ccl21, Ccr7, Cell-migration, Central-nervous-system, Chemokine, Ligands, Mek-erk, Microglia, Neurons, Neuropathic pain, Nociception, Phosphorylation, Regeneration

Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid were used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conducted on the nanopatterns. Cohesion and GJIC were evaluated in cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

JTD Keywords: arginine-glycine-aspartic acid, arginine–glycine–aspartic acid, cell adhesion, condensation, dendrimer-based nanopatterning, gap junction intercellular communication, Actin, Adhesion, Arginine-glycine-aspartic acid, Cell adhesion, Collagen, Condensation, Connexin-43, Dendrimer-based nanopatterning, Dynamics, Extracellular-matrix, Fibronectin, Gap junction intercellular communication, Mesenchymal stem cells, Permeability, Phenotype, Vinculin

JTD Keywords: augmentation, calcium-phosphate, expansion, fabrication, hydroxyapatite, made artificial bones, osteogenesis, reconstruction, regeneration, 3-d printing, 3d printers, 3d printing, 3d-printing, Abstracting, Additives, Biomaterial, Bone, Bone regeneration, Bone substitution, Bone tissue engineering, Ceramic, Ceramics, Clinic, Controlled drug delivery, Personalized medicines, Surgical planning, Targeted drug delivery, Three-dimensional-printing, Tricalcium phosphate scaffolds

Synaptic plasticity involves structural modifications in dendritic spines that are modulated by local protein synthesis and actin remodeling. Here, we investigated the molecular mechanisms that connect synaptic stimulation to these processes. We found that the phosphorylation of isoform-specific sites in eEF1A2-an essential translation elongation factor in neurons-is a key modulator of structural plasticity in dendritic spines. Expression of a nonphosphorylatable eEF1A2 mutant stimulated mRNA translation but reduced actin dynamics and spine density. By contrast, a phosphomimetic eEF1A2 mutant exhibited decreased association with F-actin and was inactive as a translation elongation factor. Activation of metabotropic glutamate receptor signaling triggered transient dissociation of eEF1A2 from its regulatory guanine exchange factor (GEF) protein in dendritic spines in a phosphorylation-dependent manner. We propose that eEF1A2 establishes a cross-talk mechanism that coordinates translation and actin dynamics during spine remodeling.

JTD Keywords: cytoskeleton, expression, f-actin, factor 1-alpha, factor 1a, messenger-rna, nucleotide exchange, protein-synthesis, synaptic plasticity, Aminoacyl-transfer-rna

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat

Purpose: This paper aims to explore how the cross-fertilization of knowledge and technologies in EU-funded research projects, including serious games and gamification, is influenced by the following variables: multidisciplinarity, knowledge base and organizations (number and diversity). The interrelation of actors and projects form a network of innovation. The largest contribution to cross-fertilization comes from the multidisciplinary nature of projects and the previous knowledge and technology of actors. The analysis draws on the understanding of how consortia perform as an innovation network, what their outcomes are and what capabilities are needed to reap value. Design/methodology/approach: All the research projects including serious games and/or gamification, funded by the EU-Horizon 2020 work programme, have been analyzed to test the hypotheses in this paper. The study sample covers the period between 2014 and 2016 (June), selecting the 87 research projects that comprised 519 organizations as coordinators and participants, and 597 observations – because more organizations participate in more than one project. The data were complemented by documentary and external database analysis. Findings: To create cross-fertilization of knowledge and technologies, the following emphasis should be placed on projects: partners concern various disciplines; partners have an extensive knowledge base for generating novel combinations and added-value technologies; there is a diverse typology of partners with unique knowledge and skills; and there is a limited number of organizations not too closely connected to provide cross-fertilization. Research limitations/implications: First, the database sample covers a period of 30 months. The authors’ attention was focused on this period because H2020 prioritized for the first time the serious games and gamification with two specific calls (ICT-21–14 and ICT-24–16) and, second, for the explosion of projects including these technologies in the past years (Adkins, 2017). These facts can be understood as a way to push the research to higher technology readiness levels (TRLs) and introducing the end-user in the co-creation and co-development along the value chain. Second, an additional limitation makes reference to the European focus of the projects, missing strong regional initiatives not identified and studied. Originality/value: This paper has attempted to explore and define theoretically and empirically the characteristics found in the cross-fertilization of collaborative research projects, emphasizing which variables, and how, need to be stimulated to benefit more multidisciplinary consortia and accelerate the process of innovation. © 2021, Manel González-Piñero, Cristina Páez-Avilés, Esteve Juanola-Feliu and Josep Samitier.

JTD Keywords: absorptive-capacity, business model, cross-fertilization of knowledge, diversity, front-end, impact, innovation systems, knowledge management, management research, science, social networks, team, technology, Cross-fertilization of knowledge, Innovation, Knowledge management, Management research, Research-and-development, Technology

The actin cytoskeleton is central to many cellular processes involving changes in cell shape, migration, and adhesiveness. Therefore, there is a great interest in the identification of the signaling pathways leading to the regulation of actin polymerization and assembly into stress fibers (SFs). However, to date it is not well understood how the mechanical interactions between cells and their environment activate the assembly of SFs. Here, we demonstrate that the mechanosensitive Piezo2 channel is required to sense physical cues from the environment to generate a calcium signal that maintains RhoA active and the formation and orientation of SFs and focal adhesions. Besides, this Piezo2-initiated signaling pathway has implications for different hallmarks of cancer invasion and metastasis.

JTD Keywords: Mechanotransduction, Calcium signaling, RhoA, Actin stress fibers, Cancer

Objective - Marfan's syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan's syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Approach and Results - Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-β pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. Conclusions - In Marfan VSMC, both in tissue and in culture, there are variable TGF-β-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan's syndrome aneurysm formation.

JTD Keywords: Actin, Aortic aneurysms, Aortic stiffness, Extracellular matrix, Focal adhesion, Myocardin, RhoA, TGF-β

Several computational models based on experimental techniques and theories have been proposed to describe cytoskeleton (CSK) mechanics. Tensegrity is a prominent model for force generation, but it cannot predict mechanics of individual CSK components, nor explain the discrepancies from the different single cell stimulating techniques studies combined with cytoskeleton-disruptors. A new numerical concept that defines a multi-structural 3D finite element (FE) model of a single-adherent cell is proposed to investigate the biophysical and biochemical differences of the mechanical role of each cytoskeleton component under loading. The model includes prestressed actin bundles and microtubule within cytoplasm and nucleus surrounded by the actin cortex. We performed numerical simulations of atomic force microscopy (AFM) experiments by subjecting the cell model to compressive loads. The numerical role of the CSK components was corroborated with AFM force measurements on U2OS-osteosarcoma cells and NIH-3T3 fibroblasts exposed to different cytoskeleton-disrupting drugs. Computational simulation showed that actin cortex and microtubules are the major components targeted in resisting compression. This is a new numerical tool that explains the specific role of the cortex and overcomes the difficulty of isolating this component from other networks invitro. This illustrates that a combination ofcytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics.

JTD Keywords: Actin bundles, Actin cortex, AFM (atomic force microscopy), Cytoskeleton, Finite element modeling, Microtubules

Biological olfaction outperforms chemical instrumentation in specificity, response time, detection limit, coding capacity, time stability, robustness, size, power consumption, and portability. This biological function provides outstanding performance due, to a large extent, to the unique architecture of the olfactory pathway, which combines a high degree of redundancy, an efficient combinatorial coding along with unmatched chemical information processing mechanisms. The last decade has witnessed important advances in the understanding of the computational primitives underlying the functioning of the olfactory system. EU Funded Project NEUROCHEM (Bio-ICT-FET- 216916) has developed novel computing paradigms and biologically motivated artefacts for chemical sensing taking inspiration from the biological olfactory pathway. To demonstrate this approach, a biomimetic demonstrator has been built featuring a large scale sensor array (65K elements) in conducting polymer technology mimicking the olfactory receptor neuron layer, and abstracted biomimetic algorithms have been implemented in an embedded system that interfaces the chemical sensors. The embedded system integrates computational models of the main anatomic building blocks in the olfactory pathway: The olfactory bulb, and olfactory cortex in vertebrates (alternatively, antennal lobe and mushroom bodies in the insect). For implementation in the embedded processor an abstraction phase has been carried out in which their processing capabilities are captured by algorithmic solutions. Finally, the algorithmic models are tested with an odour robot with navigation capabilities in mixed chemical plumes.

JTD Keywords: Antennal lobes, Artificial olfaction, Computational neuroscience, Olfactory bulbs, Plume tracking, Abstracting, Actuators, Algorithms, Biomimetic processes, Chemical sensors, Conducting polymers, Data processing, Flavors, Odors, Robots, Smart sensors, Embedded systems

Surface features at the micro and nanometre scale have been shown to influence and even determine cell behaviour and cytoskeleton organization through direct mechanotransductive pathways. Much less is known about the function and internal distribution of organelles of cells grown on topographically modified surfaces. In this study, the nanoimprint lithography technique was used to manufacture poly(methyl methacrylate) (PMMA) sheets with a variety of features in the micrometre size range. Normal rat kidney (NRK) fibroblasts were cultured on these substrates and immunofluorescence staining assays were performed to visualize cell adhesion, the organization of the cytoskeleton and the morphology and subcellular positioning of the Golgi complex. The results show that different topographic features at the micrometric scale induce different rearrangements of the cell cytoskeleton, which in turn alter the positioning and morphology of the Golgi complex. Microposts and microholes alter the mechanical stability of the Golgi complex by modifying the actin cytoskeleton organization leading to the compaction of the organelle. These findings prove that physically modified surfaces are a valuable tool with which to study the dynamics of cell cytoskeleton organization and its subsequent repercussion on internal cell organization and associated function.

JTD Keywords: Actin stress fibers, Golgi-complex, Focal adhesions, Cytoskeletal organization, Osteoblast adhesion, Mammalian-cells, Micron-scale, Nanoscale, Dynamics, Rho

Fundamental biological processes including morphogenesis, tissue repair and tumour metastasis require collective cell motions(1-3), and to drive these motions cells exert traction forces on their surroundings(4). Current understanding emphasizes that these traction forces arise mainly in 'leader cells' at the front edge of the advancing cell sheet(5-9). Our data are contrary to that assumption and show for the first time by direct measurement that traction forces driving collective cell migration arise predominately many cell rows behind the leading front edge and extend across enormous distances. Traction fluctuations are anomalous, moreover, exhibiting broad non-Gaussian distributions characterized by exponential tails(10-12). Taken together, these unexpected findings demonstrate that although the leader cell may have a pivotal role in local cell guidance, physical forces that it generates are but a small part of a global tug-of-war involving cells well back from the leading edge.

JTD Keywords: Focal adhesions, Granular matter, Bead packs, Morphogenesis, Sheets, Actin, Fluctuations, Fibroblasts, Microscopy, Diversity

The cytoskeleton is a complex polymer network that regulates the structural stability of living cells. Although the cytoskeleton plays a key role in many important cell functions, the mechanisms that regulate its mechanical behaviour are poorly understood. Potential mechanisms include the entropic elasticity of cytoskeletal filaments, glassy-like inelastic rearrangements of cross-linking proteins and the activity of contractile molecular motors that sets the tensional stress (prestress) borne by the cytoskeleton filaments. The contribution of these mechanisms can be assessed by studying how cell mechanics depends on temperature. The aim of this work was to elucidate the effect of temperature on cell mechanics using atomic force microscopy. We measured the complex shear modulus (G*) of human alveolar epithelial cells over a wide frequency range (0.1-25.6 Hz) at different temperatures (13-37 degrees C). In addition, we probed cell prestress by mapping the contractile forces that cells exert on the substrate by means of traction microscopy. To assess the role of actomyosin contraction in the temperature-induced changes in G* and cell prestress, we inhibited the Rho kinase pathway of the myosin light chain phosphorylation with Y-27632. Our results show that with increasing temperature, cells become stiffer and more solid-like. Cell prestress also increases with temperature. Inhibiting actomyosin contraction attenuated the temperature dependence of G* and prestress. We conclude that the dependence of cell mechanics with temperature is dominated by the contractile activity of molecular motors.

JTD Keywords: Membrane Stress Failure, Frog Skeletal-Muscle, Extracellular-Matrix, Glass-Transition, Energy Landscape, Actin-Filaments, Living Cell, Single, Traction, Cytoskeleton