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- ItemIncreasing Antibacterial Efficiency of Cu Surfaces by targeted Surface Functionalization via Ultrashort Pulsed Direct Laser Interference Patterning(Weinheim : Wiley-VCH, 2020) Müller, Daniel W.; Lößlein, Sarah; Terriac, Emmanuel; Brix, Kristina; Siems, Katharina; Moeller, Ralf; Kautenburger, Ralf; Mücklich, FrankCopper (Cu) exhibits great potential for application in the design of antimicrobial contact surfaces aiming to reduce pathogenic contamination in public areas as well as clinically critical environments. However, current application perspectives rely purely on the toxic effect of emitted Cu ions, without considering influences on the interaction of pathogenic microorganisms with the surface to enhance antimicrobial efficiency. In this study, it is investigated on how antibacterial properties of Cu surfaces against Escherichia coli can be increased by tailored functionalization of the substrate surface by means of ultrashort pulsed direct laser interference patterning (USP-DLIP). Surface patterns in the scale range of single bacteria cells are fabricated to purposefully increase bacteria/surface contact area, while parallel modification of the surface chemistry allows to involve the aspect of surface wettability into bacterial attachment and the resulting antibacterial effectivity. The results exhibit a delicate interplay between bacterial adhesion and the expression of antibacterial properties, where a reduction of bacterial cell viability of up to 15-fold can be achieved for E. coli on USP-DLIP surfaces in comparison to smooth Cu surfaces. Thereby, it can be shown how the antimicrobial properties of copper surfaces can be additionally enhanced by targeted surface functionalization. © 2020 The Authors. Advanced Materials Interfaces published by Wiley-VCH GmbH
- ItemVimentin levels and serine 71 phosphorylation in the control of cell-matrix adhesions, migration speed, and shape of transformed human fibroblasts(Basel : MDPI, 2017) Terriac, Emmanuel; Coceano, Giovanna; Mavajian, Zahra; Hageman, Tijmen A.G.; Christ, Andreas F.; Testa, Ilaria; Lautenschläger, Franziska; Gad, Annica K.G.Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30–50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy. We also studied whether wild-type and phospho-deficient or -mimicking mutants of vimentin changed the size and lifetime of focal adhesions (FAs), cell shape, and cell migration, using live-cell total internal reflection imaging and confocal microscopy. We observed that vimentin exists in fragments of different lengths. Short fragments were mostly the size of a unit-length filament and were mainly localized close to small cell-matrix adhesions. Long vimentin filaments were found in the proximity of large FAs. Vimentin expression in these cells caused a reduction in FAs size and an elongated cell shape, but did not affect FA lifetime, or the speed or directionality of cell migration. Expression of a phospho-mimicking mutant (S71D) of vimentin increased the speed of cell migration. Taken together, our results suggest that in highly migratory, transformed mesenchymal cells, vimentin levels control the cell shape and FA size, but not cell migration, which instead is linked to the phosphorylation status of S71 vimentin. These observations are consistent with the possibility that not only levels, but also the assembly status of vimentin control cell migration.
- ItemScanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying(San Francisco, California, US : PLOS, 2021) Schu, Moritz; Terriac, Emmanuel; Koch, Marcus; Paschke, Stephan; Lautenschläger, Franziska; Flormann, Daniel A.D.The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.
- ItemSize control in mammalian cells involves modulation of both growth rate and cell cycle duration(London : Nature Publishing Group, 2018) Cadar, Clotilde; Monnier, Sylvain; Grilli, Jacopo; Sáez, Pablo J.; Srivastava, Nishit; Attia, Rafaele; Terriac, Emmanuel; Baum, Buzz; Cosentino-Lagomarsino, Marco; Piel, MatthieuDespite decades of research, how mammalian cell size is controlled remains unclear because of the difficulty of directly measuring growth at the single-cell level. Here we report direct measurements of single-cell volumes over entire cell cycles on various mammalian cell lines and primary human cells. We find that, in a majority of cell types, the volume added across the cell cycle shows little or no correlation to cell birth size, a homeostatic behavior called “adder”. This behavior involves modulation of G1 or S-G2 duration and modulation of growth rate. The precise combination of these mechanisms depends on the cell type and the growth condition. We have developed a mathematical framework to compare size homeostasis in datasets ranging from bacteria to mammalian cells. This reveals that a near-adder behavior is the most common type of size control and highlights the importance of growth rate modulation to size control in mammalian cells.
- ItemRed blood cell passage of small capillaries is associated with transient Ca2+-mediated adaptations(Lausanne : Frontiers Media, 2017) Danielczok, Jens G.; Terriac, Emmanuel; Hertz, Laura; Petkova-Kirova, Polina; Lautenschläger, Franziska; Laschke, Matthias W.; Kaestner, LarsWhen red blood cells (RBCs) pass constrictions or small capillaries they need to pass apertures falling well below their own cross section size. We used different means of mechanical stimulations (hypoosmotic swelling, local mechanical stimulation, passing through microfluidic constrictions) to observe cellular responses of human RBCs in terms of intracellular Ca2+-signaling by confocal microscopy of Fluo-4 loaded RBCs.We were able to confirm ourin vitro results in a mouse dorsal skinfold chamber model showing a transiently increased intracellular Ca2+ when RBCs were passing through small capillaries in vivo. Furthermore, we performed the above-mentioned in vitro experiments as well as measurements of RBCs filterability under various pharmacological manipulations (GsMTx-4, TRAM-34) to explore the molecular mechanism of the Ca2+-signaling. Based on these experiments we conclude that mechanical stimulation of RBCs activates mechano-sensitive channels most likely Piezo1. This channel activity allows Ca2+ to enter the cell, leading to a transient activation of the Gardos-channel associated with K+, Cl−, and water loss, i.e., with a transient volume adaptation facilitating the passage of the RBCs through the constricti on.
- ItemPossibilities and Limitations of Photoactivatable Cytochalasin D for the Spatiotemporal Regulation of Actin Dynamics(Washington, D.C. : American Chemical Society, 2020) Nair, Roshna V.; Zhao, Shifang; Terriac, Emmanuel; Lautenschläger, Franziska; Hetmanski, Joseph H.R.; Caswell, Patrick T.; del Campo, AranzazuThe study of the actin cytoskeleton and related cellular processes requires tools to specifically interfere with actin dynamics in living cell cultures, ideally with spatiotemporal control and compatible with real time imaging. A phototriggerable derivative of the actin disruptor Cytochalasin D (CytoD) is described and tested here. It includes a nitroveratryloxycarbonyl (Nvoc) photoremovable protecting group (PPG) at the hydroxyl group at C7 of CytoD. The attachment of the PPG renders Nvoc-CytoD temporarily inactive, and enables light-dosed delivery of the active drug CytoD to living cells. This article presents the full structural and physicochemical characterization, the toxicity analysis. It is complemented with biological tests to show the time scales (seconds) and spatial resolution (cellular level) achievable with a UV source in a regular microscopy setup
- ItemAmoeboid Cell Migration through Regular Arrays of Micropillars under Confinement(New York : Cold Spring Harbor Laboratory, 2022) Sadjadi, Zeinab; Vesperini, Doriane; Laurent, Annalena M.; Barnefske, Lena; Terriac, Emmanuel; Lautenschläger, Franziska; Rieger, HeikoMigrating cells often encounter a wide variety of topographic features—including the presence of obstacles—when navigating through crowded biological environments. Unravelling the impact of topography and crowding on the dynamics of cells is key to better understand many essential physiological processes such as the immune response. We study how migration and search efficiency of HL-60 cells differentiated into neutrophils in quasi two-dimensional environments are influenced by the lateral and vertical confinement and spatial arrangement of obstacles. A microfluidic device is designed to track the cells in confining geometries between two parallel plates with distance h, in which identical micropillars are arranged in regular pillar forests. We find that at each cell-pillar contact event, the cell spends a finite time near the pillar surface, which is independent of the height h and the interpillar spacing e. At low pillar density regime, the directional persistence of cells reduces with decreasing h or e, influencing their diffusivity and first-passage properties. The dynamics is strikingly different at high pillar density regime, where the cells are in simultaneous contact with more than one pillar; the cell velocity and persistence are distinctly higher compared to dilute pillar configurations with the same h. Our simulations reveal that the interplay between cell persistence and cell-pillar interactions can dramatically affect cell diffusivity and, thus, its first-passage properties.
- ItemA novel universal algorithm for filament network tracing and cytoskeleton analysis(Hoboken, NJ : Wiley, 2021) Flormann, Daniel A.D.; Schu, Moritz; Terriac, Emmanuel; Thalla, Divyendu; Kainka, Lucina; Koch, Marcus; Gad, Annica K.B.; Lautenschläger, FranziskaThe rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the mesh hole size, and filament length and connectivity (also known as Coordination Number). In addition, the method allows sub-networks to be distinguished in two-dimensional images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of two-dimensional images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software.