by Keyword: Focal adhesion

Beedle, AEM, Garcia-Manyes, S, (2022). The role of single-protein elasticity in mechanobiology Nature Reviews Materials

Mechanical force modulates the conformation and function of individual proteins, and this underpins many mechanically driven cellular processes. This Review addresses single-molecule force spectroscopy experiments conducted on proteins with a known role in mechanosensing and mechanotransduction in eukaryotic cells.; In addition to biochemical signals and genetic considerations, mechanical forces are rapidly emerging as a master regulator of human physiology. However, the molecular mechanisms that regulate force-induced functionalities across a wide range of scales, encompassing the cell, tissue or organ levels, are not well understood in comparison. With the advent, development and refining of single-molecule nanomechanical techniques that enable the conformational dynamics of individual proteins under the effect of a calibrated force to be probed, we have begun to acquire a comprehensive knowledge of the diverse physicochemical principles that regulate the elasticity of single proteins. Here, we review the major advances underpinning our current understanding of how the elasticity of single proteins regulates mechanosensing and mechanotransduction. We discuss the present limitations and future challenges of this prolific and burgeoning field.

JTD Keywords: Cadherin adhesion, Energy landscape, Extracellular-matrix protein, Focal adhesion kinase, Mechanical stability, Molecule force spectroscopy, Muscle protein, N2b element, Stranded-dna, Structural basis

Casanellas, Ignasi, Lagunas, Anna, Vida, Yolanda, Pérez-Inestrosa, Ezequiel, Andrades, José A., Becerra, José, Samitier, Josep, (2019). Matrix nanopatterning regulates mesenchymal differentiation through focal adhesion size and distribution according to cell fate Biomimetics Biomimetic Nanotechnology for Biomedical Applications (NanoBio&Med 2018) , MDPI (Barcelona, Spain) 4, (2), 43

Extracellular matrix remodeling plays a pivotal role during mesenchyme patterning into different lineages. Tension exerted from cell membrane receptors bound to extracellular matrix ligands is transmitted by the cytoskeleton to the cell nucleus inducing gene expression. Here, we used dendrimer-based arginine–glycine–aspartic acid (RGD) uneven nanopatterns, which allow the control of local surface adhesiveness at the nanoscale, to unveil the adhesive requirements of mesenchymal tenogenic and osteogenic commitments. Cell response was found to depend on the tension resulting from cell–substrate interactions, which affects nuclear morphology and is regulated by focal adhesion size and distribution.

JTD Keywords: Arginine–glycine–aspartic acid (RGD), Nanopattern, Mesenchymal stem cells, Tenogenesis, Osteogenesis, Cell nuclei, Focal adhesions

Neri, L., Lasa, M., Elosegui-Artola, A., D'Avola, D., Carte, B., Gazquez, C., Alve, S., Roca-Cusachs, P., Iñarrairaegui, M., Herrero, J., Prieto, J., Sangro, B., Aldabe, R., (2017). NatB-mediated protein N-α-terminal acetylation is a potential therapeutic target in hepatocellular carcinoma Oncotarget 8, (25), 40967-40981

The identification of new targets for systemic therapy of hepatocellular carcinoma (HCC) is an urgent medical need. Recently, we showed that hNatB catalyzes the N-α-terminal acetylation of 15% of the human proteome and that this action is necessary for proper actin cytoskeleton structure and function. In tumors, cytoskeletal changes influence motility, invasion, survival, cell growth and tumor progression, making the cytoskeleton a very attractive antitumor target. Here, we show that hNatB subunits are upregulated in in over 59% HCC tumors compared to non-tumor tissue and that this upregulation is associated with microscopic vascular invasion. We found that hNatB silencing blocks proliferation and tumor formation in HCC cell lines in association with hampered DNA synthesis and impaired progression through the S and the G2/M phases. Growth inhibition is mediated by the degradation of two hNatB substrates, tropomyosin and CDK2, which occurs when these proteins lack N-α-terminal acetylation. In addition, hNatB inhibition disrupts the actin cytoskeleton, focal adhesions and tight/adherens junctions, abrogating two proliferative signaling pathways, Hippo/YAP and ERK1/2. Therefore, inhibition of NatB activity represents an interesting new approach to treating HCC by blocking cell proliferation and disrupting actin cytoskeleton function.

JTD Keywords: CDK2, Cell cycle arrest, Cell-cell junctions, Focal adhesions, Tropomyosin

Crosas-Molist, E., Meirelles, T., López-Luque, J., Serra-Peinado, C., Selva, J., Caja, L., Gorbenko Del Blanco, D., Uriarte, J. J., Bertran, E., Mendizábal, Y., Hernández, V., García-Calero, C., Busnadiego, O., Condom, E., Toral, D., Castellà, M., Forteza, A., Navajas, D., Sarri, E., Rodríguez-Pascual, F., Dietz, H. C., Fabregat, I., Egea, G., (2015). Vascular smooth muscle cell phenotypic changes in patients with marfan syndrome Arteriosclerosis, Thrombosis, and Vascular Biology , 35, (4), 960-972

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

Mrkonji, Garcia-Elias, A., Pardo-Pastor, C., Bazellières, E., Trepat, X., Vriens, J., Ghosh, D., Voets, T., Vicente, R., Valverde, M. A., (2015). TRPV4 participates in the establishment of trailing adhesions and directional persistence of migrating cells Pflugers Archiv European Journal of Physiology , 467, (10), 2107-2119

Calcium signaling participates in different cellular processes leading to cell migration. TRPV4, a non-selective cation channel that responds to mechano-osmotic stimulation and heat, is also involved in cell migration. However, the mechanistic involvement of TRPV4 in cell migration is currently unknown. We now report that expression of the mutant channel TRPV4-121AAWAA (lacking the phosphoinositide-binding site 121KRWRK125 and the response to physiological stimuli) altered HEK293 cell migration. Altered migration patterns included periods of fast and persistent motion followed by periods of stalling and turning, and the extension of multiple long cellular protrusions. TRPV4-WT overexpressing cells showed almost complete loss of directionality with frequent turns, no progression, and absence of long protrusions. Traction microscopy revealed higher tractions forces in the tail of TRPV4-121AAWAA than in TRPV4-WT expressing cells. These results are consistent with a defective and augmented tail retraction in TRPV4-121AAWAA- and TRPV4-WT-expressing cells, respectively. The activity of calpain, a protease implicated in focal adhesion (FA) disassembly, was decreased in TRPV4-121AAWAA compared with TRPV4-WT-expressing cells. Consistently, larger focal adhesions were seen in TRPV4-121AAWAA compared with TRPV4-WT-expressing HEK293 cells, a result that was also reproduced in T47D and U87 cells. Similarly, overexpression of the pore-dead mutant TRPV4-M680D resumed the TRPV4-121AAWAA phenotype presenting larger FA. The migratory phenotype obtained in HEK293 cells overexpressing TRPV4-121AAWAA was mimicked by knocking-down TRPC1, a cationic channel that participates in cell migration. Together, our results point to the participation of TRPV4 in the dynamics of trailing adhesions, a function that may require the interplay of TRPV4 with other cation channels or proteins present at the FA sites.

JTD Keywords: Calcium, Calpain, Focal adhesion, Migration, Traction forces, TRPV4

Estévez, M., Martínez, Elena, Yarwood, S. J., Dalby, M. J., Samitier, J., (2015). Adhesion and migration of cells responding to microtopography Journal of Biomedical Materials Research - Part A , 103, (5), 1659-1668

It is known that cells respond strongly to microtopography. However, cellular mechanisms of response are unclear. Here, we study wild-type fibroblasts responding to 25 μm2 posts and compare their response to that of FAK-/- fibroblasts and fibroblasts with PMA treatment to stimulate protein kinase C (PKC) and the small g-protein Rac. FAK knockout cells modulated adhesion number and size in a similar way to cells on topography; that is, they used more, smaller adhesions, but migration was almost completely stalled demonstrating the importance of FAK signaling in contact guidance and adhesion turnover. Little similarity, however, was observed to PKC stimulated cells and cells on the topography. Interestingly, with PKC stimulation the cell nuclei became highly deformable bringing focus on these surfaces to the study of metastasis. Surfaces that aid the study of cellular migration are important in developing understanding of mechanisms of wound healing and repair in aligned tissues such as ligament and tendon.

JTD Keywords: Adhesion, Cell migration, Cell morphology, Focal adhesion kinase, Microstructures

Lagunas, A., Garcia, A., Artés, J. M., Vida, Y., Collado, D., Pérez-Inestrosa, E., Gorostiza, P., Claros, S., Andrades, J. A., Samitier, J., (2014). Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycine-aspartic acid (RGD) density effects on cell adhesion Nano Research , 7, (3), 399-409

Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix (ECM), being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid (RGD) density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy (AFM), scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS), showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout: Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions (FAs). Therefore, dendrimer nanopatterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells.

JTD Keywords: Arginine-glycine-aspartic acid, Atomic force microscopy, Cell adhesion, Dendrimer, Focal adhesions, Scanning tunneling microscopy

Moore, S. W., Roca-Cusachs, P., Sheetz, M. P., (2010). Stretchy proteins on stretchy substrates: The important elements of integrin-mediated rigidity sensing Developmental Cell 19, (2), 194-206

Matrix and tissue rigidity guides many cellular processes, including the differentiation of stem cells and the migration of cells in health and disease. Cells actively and transiently test rigidity using mechanisms limited by inherent physical parameters that include the strength of extracellular attachments, the pulling capacity on these attachments, and the sensitivity of the mechanotransduction system. Here, we focus on rigidity sensing mediated through the integrin family of extracellular matrix receptors and linked proteins and discuss the evidence supporting these proteins as mechanosensors.

JTD Keywords: Focal adhesion kinase, Atomic Force Microscopy, Smooth-muscle cells, Traction forces, Living cells, Mechanical force, Locomoting cells

Estevez, M., Fernandez-Ulibarri, I., Martinez, E., Egea, G., Samitier, J., (2010). Changes in the internal organization of the cell by microstructured substrates Soft Matter 6, (3), 582-590

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

Trepat, X., Wasserman, M. R., Angelini, T. E., Millet, E., Weitz, D. A., Butler, J. P., Fredberg, J. J., (2009). Physical forces during collective cell migration Nature Physics 5, (6), 426-430

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