51 research outputs found

    Membrane mechanics governs cell mechanics in epithelial cell: how surface area regulation ensures tension homeostasis

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    Die Plasmamembranspannung von eukaryotischen Zellen soll maßgeblich zur Regulation von zellulären Prozessen wie der Zellmigration, Mitose, Endo- und Exozytose, Membranreparatur, Osmoregulierung und Zellspreiten beitragen, welche zu einer Veränderung der Membranfläche und ihrer Deformation führt. In dieser Arbeit wurde die epitheliale Zelllinie MDCK II (Madin-Darby Canine Kidney) benutzt, um spannungsgesteuerte Oberflächenregulierung zu untersuchen. Indentationsexperimente kombiniert mit dem Herausziehen von Membrannanoröhren wurden mit Hilfe des Rasterkraftmikroskops (Atomic Force Microskope, AFM) durchgeführt, um lokale Variationen in der Membranspannung und überschüssiger Membranfläche als Funktion von äußeren Reizen abzuschätzen. Die verwendeten externen Stimuli beinhalten eine Veränderung der Funktionalität des Actomyosin-Cortexes durch die Wirkung von Blebbistatin und Cytochalasin D, sowie die Manipulation der Zytoskelett-Membran Adhäsionspunkte durch Einzel-Mikroinjektion. Die Injektion von Neomycin verhindert die Anbindung von ERM-Proteinen an das Lipid Phosphatidylinositol-(4,5)-bisphosphat (PIP2) und bewirkt somit die Abkopplung des Zytoskeletts von der Plasmamembran. Als Gegenexperiment diente die Injektion des Lipids PIP2 selbst, welches zur Erhöhung der Anzahl der Zytoskelett-Membran Adhäsionspunkte führte. Weiterhin wurden die als Membranreservoire dienenden Mikrovilli durch den Entzug von Cholesterol entfernt. Auswirkung auf das Vorhandensein von Membranreservoiren hat ebenfalls die Veränderung des osmotischen Drucks innerhalb der Zellen. Zusätzlich wurden die elastischen Eigenschaften von apikalen Zellmembran-Fragmenten von konfluenten MDCK II Zellen untersucht, welche Aufschluss über die intrinsischen Membraneigenschaften ohne den Einfluss des Zytosols und Zytoskeletts geben konnten. Abschließend wurde die Mechanik von adhärierenden und spreitenden Zellen untersucht. Zusammenfassend kann gesagt werden, dass die Plasmamembran, bestehend aus einer Phospholipiddoppelschicht, lateral schwer ausdehnbar ist aufgrund ihrer flüssig-kristallinen Natur. Durch das Vorhandensein von dynamischen Membranreservoiren wie Mikrovilli, die schnell auf Veränderungen der Membranspannung durch Membranhomöostase reagieren, werden zellulare Prozesse wie die Zellmotilität oder die Anpassung an osmotischen Stress ermöglicht. In der vorliegenden Arbeit gelang es gleichzeitig, die Membranspannung und die Verfügbarkeit von Membranfläche von adhärenten konfluenten als auch von adhärierenden und spreiten Zellen zu messen. Die durchgeführten Experimente ergaben ein detailliertes Bild wie sich die zelluläre Oberflächenregulierung in der Membranmechanik widerspiegelt

    Looking at cell mechanics with atomic force microscopy: Experiment and theory

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    This review reports on the use of the atomic force microscopy in the investigation of the mechanical properties of cells. It is shown that the technique is able to deliver information about the cell surface properties (e.g., topography), the Young modulus, the viscosity, and the cell the relaxation times. Another aspect that this short review points out is the utilization of the atomic force microscope to investigate basic questions related to materials physics, biology, and medicine. The review is written in a chronological way to offer an overview of phenomenological facts and quantitative results to the reader. The final section discusses in detail the advantages and disadvantages of the Hertz and JKR models. A new implementation of the JKR model derived by Dufresne is presented

    Entropic Forces Drive Clustering and Spatial Localization of Influenza A M2 During Viral Budding

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    The influenza A matrix 2 (M2) transmembrane protein facilitates virion release from the infected host cell. In particular, M2 plays a role in the induction of membrane curvature and/or in the scission process whereby the envelope is cut upon virion release. Here we show using coarse-grained computer simulations that various M2 assembly geometries emerge due to an entropic driving force, resulting in compact clusters or linearly extended aggregates as a direct consequence of the lateral membrane stresses. Conditions under which these protein assemblies will cause the lipid membrane to curve are explored and we predict that a critical cluster size is required for this to happen. We go on to demonstrate that under the stress conditions taking place in the cellular membrane as it undergoes large-scale membrane remodeling, the M2 protein will in principle be able to both contribute to curvature induction and sense curvature in order to line up in manifolds where local membrane line tension is high. M2 is found to exhibit linactant behavior in liquid-disordered/liquid-ordered phase-separated lipid mixtures and to be excluded from the liquid-ordered phase, in near-quantitative agreement with experimental observations. Our findings support a role for M2 in membrane remodeling during influenza viral budding both as an inducer and a sensor of membrane curvature, and they suggest a mechanism by which localization of M2 can occur as the virion assembles and releases from the host cell, independent of how the membrane curvature is produced

    The effect of surface charge on nonspecific uptake and cytotoxicity of CdSe/ZnS core/shell quantum dots

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    In this work, cytotoxicity and cellular impedance response was compared for CdSe/ZnS core/shell quantum dots (QDs) with positively charged cysteamine–QDs, negatively charged dihydrolipoic acid–QDs and zwitterionic D-penicillamine–QDs exposed to canine kidney MDCKII cells. Pretreatment of cells with pharmacological inhibitors suggested that the uptake of nanoparticles was largely due to receptor-independent pathways or spontaneous entry for carboxylated and zwitterionic QDs, while for amine-functionalized particles involvement of cholesterol-enriched membrane domains is conceivable. Cysteamine–QDs were found to be the least cytotoxic, while D-penicillamine–QDs reduced the mitochondrial activity of MDCKII by 20–25%. Although the cell vitality appeared unaffected (assessed from the changes in mitochondrial activity using a classical MTS assay after 24 h of exposure), the binding of QDs to the cellular interior and their movement across cytoskeletal filaments (captured and characterized by single-particle tracking), was shown to compromise the integrity of the cytoskeletal and plasma membrane dynamics, as evidenced by electric cell–substrate impedance sensing

    Mechanical properties of MDCK II cells exposed to gold nanorods

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    Background: The impact of gold nanoparticles on cell viability has been extensively studied in the past. Size, shape and surface functionalization including opsonization of gold particles ranging from a few nanometers to hundreds of nanometers are among the most crucial parameters that have been focussed on. Cytoxicity of nanomaterial has been assessed by common cytotoxicity assays targeting enzymatic activity such as LDH, MTT and ECIS. So far, however, less attention has been paid to the mechanical parameters of cells exposed to gold particles, which is an important reporter on the cellular response to external stimuli. Results: Mechanical properties of confluent MDCK II cells exposed to gold nanorods as a function of surface functionalization and concentration have been explored by atomic force microscopy and quartz crystal microbalance measurements in combination with fluorescence and dark-field microscopy. Conclusion: We found that cells exposed to CTAB coated gold nanorods display a concentration-dependent stiffening that cannot be explained by the presence of CTAB alone. The stiffening results presumably from endocytosis of particles removing excess membrane area from the cell�s surface. Another aspect could be the collapse of the plasma membrane on the actin cortex. Particles coated with PEG do not show a significant change in elastic properties. This observation is consistent with QCM measurements that show a considerable drop in frequency upon administration of CTAB coated rods suggesting an increase in acoustic load corresponding to a larger stiffness (storage modulus)

    Tension Monitoring during Epithelial-to-Mesenchymal Transition Links the Switch of Phenotype to Expression of Moesin and Cadherins in NMuMG Cells

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    <div><p>Structural alterations during epithelial-to-mesenchymal transition (EMT) pose a substantial challenge to the mechanical response of cells and are supposed to be key parameters for an increased malignancy during metastasis. Herein, we report that during EMT, apical tension of the epithelial cell line NMuMG is controlled by cell-cell contacts and the architecture of the underlying actin structures reflecting the mechanistic interplay between cellular structure and mechanics. Using force spectroscopy we find that tension in NMuMG cells slightly increases 24 h after EMT induction, whereas upon reaching the final <i>mesenchymal-like</i> state characterized by a complete loss of intercellular junctions and a concerted down-regulation of the adherens junction protein E-cadherin, the overall tension becomes similar to that of solitary adherent cells and fibroblasts. Interestingly, the contribution of the actin cytoskeleton on apical tension increases significantly upon EMT induction, most likely due to the formation of stable and highly contractile stress fibers which dominate the elastic properties of the cells after the transition. The structural alterations lead to the formation of single, highly motile cells rendering apical tension a good indicator for the cellular state during phenotype switching. In summary, our study paves the way towards a more profound understanding of cellular mechanics governing fundamental morphological programs such as the EMT.</p></div
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