Publications

Recent experimental evidence indicates a role for the intermediate filament vimentin in regulating cellular mechanical homeostasis, but its precise contribution remains to be discovered. Mechanical homeostasis requires a balanced bi-directional interplay between the cell's microenvironment and the cellular morphological and mechanical state-this balance being regulated via processes of mechanotransduction and mechanoresponse, commonly referred to as mechanoreciprocity. Here, we systematically analyze vimentin-expressing and vimentin-depleted cells in a swatch of in vitro cellular microenvironments varying in stiffness and/or ECM density. We find that vimentin-expressing cells maintain mechanical homeostasis by adapting cellular morphology and mechanics to micromechanical changes in the microenvironment. However, vimentin-depleted cells lose this mechanoresponse ability on short timescales, only to reacquire it on longer time scales. Indeed, we find that the morphology and mechanics of vimentin-depleted cell in stiffened microenvironmental conditions can get restored to the homeostatic levels of vimentin-expressing cells. Additionally, we observed vimentin-depleted cells increasing collagen matrix synthesis and its crosslinking, a phenomenon which is known to increase matrix stiffness, and which we now hypothesize to be a cellular compensation mechanism for the loss of vimentin. Taken together, our findings provide further insight in the regulating role of intermediate filament vimentin in mediating mechanoreciprocity and mechanical homeostasis.© 2023. The Author(s).

JTD Keywords: contributes, dynamics, focal adhesions, forces, mechanotransduction, migration, motility, organization, tissue, Intermediate-filaments

The term 'mechanosensation' describes the capacity of cells to translate mechanical stimuli into the coordinated regulation of intracellular signals, cellular function, gene expression and epigenetic programming. This capacity is related not only to the sensitivity of the cells to tissue motion, but also to the decryption of tissue geometric arrangement and mechanical properties. The cardiac stroma, composed of fibroblasts, has been historically considered a mechanically passive component of the heart. However, the latest research suggests that the mechanical functions of these cells are an active and necessary component of the developmental biology programme of the heart that is involved in myocardial growth and homeostasis, and a crucial determinant of cardiac repair and disease. In this Review, we discuss the general concept of cell mechanosensation and force generation as potent regulators in heart development and pathology, and describe the integration of mechanical and biohumoral pathways predisposing the heart to fibrosis and failure. Next, we address the use of 3D culture systems to integrate tissue mechanics to mimic cardiac remodelling. Finally, we highlight the potential of mechanotherapeutic strategies, including pharmacological treatment and device-mediated left ventricular unloading, to reverse remodelling in the failing heart.© 2022. Springer Nature Limited.

JTD Keywords: cardiomyocyte proliferation, cross-linking, extracellular-matrix, focal adhesions, gene-expression, mechanical regulation, myocardial-infarction, substrate stiffness affects, t-cells, Ventricular assist device

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

Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration.Copyright © 2022 Elsevier Inc. All rights reserved.

JTD Keywords: activation, c-met, contact inhibition, focal adhesions, heparan-sulfate, mechanical forces, morphogenesis, rho, stress fibers, Collective cell migration, Erk, Feedback regulation, Fret, Growth-factor receptor, Hgf, Lamellipodia, Leader cell specification, Signal transduction, Traction force, Wound healing

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

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

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

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