Receptor-mediated Forces for Cell Sensing of Extracellular Ligands and for Virus Particle Uptake

Mechanical forces between cells ensure organic development and homeostasis, but they are also associated with diseases such as cancer or viral infections. The absolute forces in nature can be as high as 1.5 kN, which allows the mantis shrimp to smash oysters, down to a few pN transduced by single cellular receptors. However, these small forces are strong enough for cells to probe their local environment. While the mechanism, which enables cells to investigate the stiffness of their surrounding, is already well elucidated, information on how cells sense spatial distribution of ligands is missing. In this thesis I established a method, which allows to measure cellular traction forces on elastic substrates with varying nano-spacing of extracellular ligands. In contrast to previous studies on stiff substrates, adhesion complexes and tractions were larger for longer distances between extracellular adhesion sites. This can be theoretically explained by the force load on individual integrin receptors, which has to exceed a certain threshold value to promote adhesion growth through conformational changes in a protein of the “clutch complex”. In order to experimentally access the force load per integrin heterodimer, I combined molecular tension fluorescence microscopy (MTFM) with traction force microscopy (TFM). For the first time, I could assess a homogeneous distribution of forces > 19pN underneath the adhesion area of cells on soft substrates. Simultaneously, macroscopic tractions up to 2.7 kPa were observed at the cell edges. Applying stronger tension probes and analyzing tractions in the zdirection will help to cross-validate the results obtained from these two state-of-the-art methods in biomechanics as a next step. In the second part I investigated the mechanical parameters of virus particle uptake by cells. Many intracellular pathogens, such as mammalian reoviruses as employed in this thesis, mimic extracellular motives to interact with host cells and initiate their internalization. This leads to the assumption that host cells sense this specific ligand presentation, engage the endocytic machinery and generate forces, which are able to overcome the bending and tension energy of their plasma membrane. I demonstrated that these forces exerted on single reovirus particles on the basolateral side of cells are strong enough to break down the biotin-NeutrAvidin bond used for virus immobilization on stiff and soft substrates. I quantified the forces to exceed 40pN by kinetic analysis of the tearing of viruses from these surfaces and single MTFM with covalently immobilized reoviruses. The herein presented methods are powerful tools to study forces exerted by individual receptors as well as on single particles e.g. during endocytosis. The involvement of the actin cytoskeleton, specific receptors or molecules of the endocytic machinery was examined. Inhibition of the ligand-receptor interactions between reoviruses and cells did not significantly change the rate of virus uptake. Interestingly, bare nanoparticles of comparable diameter lacking specific binding sites were torn off at a similar rate and thus with the same forces as viruses. Hence, specific receptors seem to be dispensable for virus particle uptake

Antimicrobial Functionalization of Prolamine–Silica Hybrid Coatings with Fumaric Acid for Food Packaging Materials and Their Biocompatibility

The interest of the food packaging industry in biodegradable, recyclable, and functional materials has steadily increased in recent years. The use of hydrogels in the food sector holds great potential for use in packaging systems or as carriers for bioactive substances. The synthesis of an oxygen barrier coating of prolaminic silica material and antimicrobial functionalization with fumaric acid for packaging materials described here is an elegant way to meet these requirements. The developed material achieved a significant antimicrobial activity against Escherichia coli and Staphylococcus aureus , two common clinical pathogens. Another pre-requisite of such materials is a high biocompatibility, which can be assessed using human cell models, to help ensure consumer safety. The biocompatibility was determined by luminescence adenosine triphosphate and photometric lactate dehydrogenase assays. No cytotoxic effects on human keratinocytes in vitro were found for the test materials

From tattered threads to digital dreams: Unleashing the circular revolution in fashion!

The textile and clothing (T&C) industry has long been associated with unsustainable practices, lacking in recycling and circularity. However, a paradigm shift is underway as the industry recognises the need for change. Here, we explore the transformation of the German T&C industry and assess the role of digital technologies in supporting a more sustainable future

Sustainability, the circular economy and digitalisation in the German textile and clothing industry

The textile and clothing (T&C) industry is not usually viewed as an exemplar of sustainable development and the circular economy (CE), as the industry has hitherto developed its products in a linear fashion, with relatively little recycling of the finished goods. This article examines the industry’s approach to the core sustainability concept and the CE in particular through a review of the available academic literature, evidence from corporate sustainability reports and websites, and feedback from an online survey of industry professionals. More specifically, the article investigates how German T&C companies are addressing sustainability in their corporate strategies and what activities relating to the CE are being pursued in the industry. The role of digital technologies in the transition to sustainability and the CE in the German T&C industry is also explored. The study finds that whilst sustainability is now firmly embedded at the strategic level in the vast majority of the companies studied, attitudes towards the CE are mixed. The use of digital technologies in support of sustainability objectives is also limited at present, but the need to meet compliance requirements and new customer perceptions of sustainability will speed the transition to CE activities, which will be facilitated by the greater exploitation of these technologies. An operational framework for initiating such a transition is developed, and action lists in the key areas of change — organization, products, and processes —are presented. These may be used as a guideline for practitioners, and the findings also make a small contribution to the scarcity of literature in this field of research

Architectural Analysis of Systems Based on the Publisher-Subscriber Style

Architectural styles impose constraints on both the topology and the interaction behavior of involved parties. In this paper, we propose an approach for analyzing implemented systems based on the publisher-subscriber architectural style. From the style definition, we derive a set of reusable questions and show that some of them can be answered statically whereas others are best answered using dynamic analysis. The paper explains how the results of static analysis can be used to orchestrate dynamic analysis. The proposed method was successfully applied on the NASA's Goddard Mission Services Evolution Center (GMSEC) software product line. The results show that the GMSEC has a) a novel reusable vendor-independent middleware abstraction layer that allows the NASA's missions to configure the middleware of interest without changing the publishers' or subscribers' source code, and b) some high priority bugs due to behavioral discrepancies, which were eluded during testing and code reviews, among different implementations of the same APIs for different vendors

Substrate engagement of integrins α5 β1 and αv β3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5 β1 and αv β3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5 β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αv β3 or α5 β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration.Instituto de Investigaciones Fisicoquímicas Teóricas y AplicadasConsejo Nacional de Investigaciones Científicas y Técnica

Substrate engagement of integrins α5 β1 and αv β3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

The interplay between specific integrin-mediated matrix adhesion and directional persistence in cell migration is not well understood. Here, we characterized fibroblast adhesion and migration on the extracellular matrix glycoproteins fibronectin and vitronectin, focusing on the role of α5 β1 and αv β3 integrins. Fibroblasts manifested high directional persistence in migration on fibronectin-, but not vitronectin-coated substrates, in a ligand density-dependent manner. Fibronectin stimulated α5 β1-dependent organization of the actin cytoskeleton into oriented, ventral stress fibers, and assembly of dynamic, polarized protrusions, characterized as regions free of stress fibers and rich in nascent adhesions at their edge. Such protrusions correlated with persistent, local leading edge advancement, but were not sufficient, nor necessary for directional migration over longer times. Selective blocking of αv β3 or α5 β1 integrins using small molecule integrin antagonists reduced directional persistence on fibronectin, indicating integrin cooperativity in maintaining directionality. On the other hand, patterned substrates, designed to selectively engage either integrin, or their combination, were not sufficient to establish directional migration. Overall, our study demonstrates adhesive coating-dependent regulation of directional persistence in fibroblast migration and challenges the generality of the previously suggested role of β1 and β3 integrins in directional migration.Instituto de Investigaciones Fisicoquímicas Teóricas y AplicadasConsejo Nacional de Investigaciones Científicas y Técnica

Forces during cellular uptake of viruses and nanoparticles at the ventral side

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake