Distribution of sialic acids on mucins and gels: a defense mechanism

Moist mucosal epithelial interfaces that are exposed to external environments are dominated by sugar epitopes, some of which (e.g., sialic acids) are involved in host defense. In this study, we determined the abundance and distribution of two sialic acids to assess differences in their availability to an exogenous probe in isolated mucins and mucous gels. We used atomic force microscopy to obtain force maps of human preocular mucous and purified ocular mucins by probing and locating the interactions between tip-tethered lectins Maackia amurensis and Sambucus nigra and their respective receptors, α-2,3 and α-2,6 N-acetylneuraminic (sialic) acids. The rupture force distributions were not affected by neighboring sugar-bearing molecules. Energy contours for both lectin-sugar bonds were fitted to a two-barrier model, suggesting a conformational change before dissociation. In contrast to data from purified mucin molecules, the preocular gels presented numerous large clusters (19,000 ± 4000 nm2) of α-2,6 sialic acids, but very few small clusters (2000 ± 500 nm2) of α-2,3 epitopes. This indicates that mucins, which are rich in α-2,3 sialic acids, are only partially exposed at the surface of the mucous gel. Microorganisms that recognize α-2,3 sialic acids will encounter only isolated ligands, and the adhesion of other microorganisms will be enhanced by large islands of neighboring α-2,6 sialic acids. We have unveiled an additional level of mucosal surface heterogeneity, specifically in the distribution of pro- and antiadhesive sialic acids that protect underlying epithelia from viruses and bacteria

A beginner's guide to atomic force microscopy probing for cell mechanics

European Commission . Grant Number: CIG14-2013-631011 Dunhill Medical Trust . Grant Number: R454/111

Atomic force microscopy-based mechanobiology

Mechanobiology emerges at the crossroads of medicine, biology, biophysics and engineering and describes how the responses of proteins, cells, tissues and organs to mechanical cues contribute to development, differentiation, physiology and disease. The grand challenge in mechanobiology is to quantify how biological systems sense, transduce, respond and apply mechanical signals. Over the past three decades, atomic force microscopy (AFM) has emerged as a key platform enabling the simultaneous morphological and mechanical characterization of living biological systems. In this Review, we survey the basic principles, advantages and limitations of the most common AFM modalities used to map the dynamic mechanical properties of complex biological samples to their morphology. We discuss how mechanical properties can be directly linked to function, which has remained a poorly addressed issue. We outline the potential of combining AFM with complementary techniques, including optical microscopy and spectroscopy of mechanosensitive fluorescent constructs, super-resolution microscopy, the patch clamp technique and the use of microstructured and fluidic devices to characterize the 3D distribution of mechanical responses within biological systems and to track their morphology and functional state.Peer ReviewedPostprint (published version

Investigation of effect of fullerenol on viscoelasticity properties of human hepatocellular carcinoma by AFM-Based creep tests

Cellular elasticity is frequently measured to investigate the biomechanical effects of drug treatment, diseases and aging. In light of cellular viscosity property exhibited by filament actin networks, this study investigates the viscoelasticity alterations of human hepatocellular carcinoma (SMMC-7721) cell subjected to fullerenol treatment by means of creep tests realized by AFM indentation. An SMMC-7721 cell was first modeled as a sphere and then a flattened layer with finite thickness. Both Sneddon’s solutions and Dimitriadis model have been modified to adapt for viscoelastic situation, which are used to fit the same indentation depth – time curves obtained by creep tests. We find that the SMMC-7721 cell’s creep behavior is well described by the two modified models, and the divergence of parameters determined by the two models is justified. By fullerenol treatment, the SMMC-7721 cell exhibits a significant decrease of elastic modulus and viscosity, which is presumably due to the disruption of actin filaments. This work represents a new attempt to understand the alternation of the viscoelastic properties of cancerous cells under the treatment of fullerenol, which has the significance of comprehensively elucidating the biomechanical effects of anticancer agents (such as fullerenol) on cancer cells

Determination of cellular strains by combined atomic force microscopy and finite element modeling

Many organs adapt to their mechanical environment as a result of physiological change or disease. Cells are both the detectors and effectors of this process. Though many studies have been performed in vitro to investigate the mechanisms of detection and adaptation to mechanical strains, the cellular strains remain unknown and results from different stimulation techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by atomic force microscopy with finite element modeling and computational fluid dynamics, we report the cellular strain distributions exerted by common whole-cell straining techniques and from micromanipulation techniques, hence enabling their comparison. Using data from our own analyses and experiments performed by others, we examine the threshold of activation for different signal transduction processes and the strain components that they may detect. We show that modulating cell elasticity, by increasing the F-actin content of the cytoskeleton, or cellular Poisson ratio are good strategies to resist fluid shear or hydrostatic pressure. We report that stray fluid flow in some substrate-stretch systems elicits significant cellular strains. In conclusion, this technique shows promise in furthering our understanding of the interplay among mechanical forces, strain detection, gene expression, and cellular adaptation in physiology and disease

Application and Development of Mechanoresponsive Polymer Structures

Mechanoresponsive Systeme antworten auf mechanische Reize mit einer Eigenschaftsänderung. Diese Dissertation umfasst die Arbeiten mit zwei mechanoresponsiven Systemen, die optisch auf mechanische Reize antworten. Sie basieren auf polymeren Strukturen, einer Polymerbürste und einem Hydrogelnetzwerk. Ihr optischer Antwortmechanismus ermöglicht die Beobachtung wirkender Kräfte als ein Ansatz zur in situ-Kraftmessung. Im ersten Teil wird ein existierendes, mechanoresponsives System zur Anwendung gebracht, das auf einer mit Fluoreszenzfarbstoff markierten Polyelektrolytbürste basiert. Die Ladungen des Polyelektrolyts können die Fluoreszenz des Farbstoffs unterdrücken, sodass lokale Kompression und Zugspannung über die Fluoreszenzintensität unterschieden werden können. Die mechanoresponsive Polymerbürste wurde als mechanosensitive Oberflächenbeschichtung angewandt, um Unterschiede in der Kontaktspannungsverteilung von Gecko-inspirierten adhäsiven Mikrostempelstrukturen aufzuklären. Die erarbeiteten Ergebnisse und daraus abgeleiteten Ablösemechanismen der Mikrostempeltypen deckten sich qualitativ mit Vorhersagen aus theoretischen Ansätzen. Aufgrund geometrischer Einschränkungen einer planaren Oberflächenbeschichtung zielt der zweite Teil darauf ab, dieses mechanoresponsive Prinzip in ein dreidimensionales Netzwerk zu überführen und ein mechanoresponsives Hydrogelnetzwerk als Plattform zur Kraftmessung zu entwickeln. Konzeptionell besitzt ein homogenes Netzwerk vorhersagbare mechanische Eigenschaften, sodass lokale optische Antworten auf mechanische Kräfte ermöglichen könnten, die wirkenden Kräfte zu lokalisieren und quantifizieren. Basierend auf einer Gestaltung nach der Flory-Rehner-Theorie wurden Präkursoren mit vordefinierter Größe und Architektur für die Hydrogelherstellung eingesetzt, um auf ein homogenes Netzwerk abzuzielen. Zu diesem Zweck wurde das Mischungsvolumen durch Tropfenmikrofluidik reduziert. Für den optischen Antwortmechanismus wurden die Hydrogelnetzwerk-Präkursoren mit zwei verschiedenen Fluorophoren markiert, die sich durch abstandsabhängige Emission über Förster-Resonanzenergietransfer auszeichnen. Die Funktionalität des optischen Antwortmechanismus wurde auf globaler Ebene durch Kollabieren und kontrolliertes Quellen des Netzwerks, dann auf lokalisierter Ebene durch definierte mechanische Belastung mit Rasterkraftmikroskopie gezeigt. Durch ihre Anpassbarkeit könnte die Hydrogelplattform zukünftig verschiedenste Anwendungen im Bereich intrisischer Kraftmessung weicher Materie bedienen.Mechanoresponsive systems respond to mechanical triggers by changes in a certain property. This thesis covers the work conducted with two mechanoresponsive systems that respond optically to mechanical triggers. These two systems are based on polymer structures, a polymer brush and a hydrogel network. Thus, the optical response mechanism allows observing acting forces as an approach to force sensing in situ. In the first part, an existing mechanoresponsive system based on a polyelectrolyte brush labeled with a fluorescent dye is engaged in application. The charges of the polyelectrolyte are able to quench the fluorescence of the dye so that local compression or tension can be distinguished from the local fluorescence intensity. The mechanoresponsive polymer brush was applied as mechanosensitive surface coating to elucidate differences in the contact stress distributions of gecko-inspired adhesive micropillar structures. The determined results and the derived detachment mechanisms of the micropillar types were in qualitative accordance with predictions from theoretical approaches. Overcoming the geometrical limitations of a planar surface coating, the second part aims at translating the mechanoresponse principle to a three-dimensional network and developing a mechanoresponsive hydrogel as a platform for force sensing. Conceptually, a homogeneous network allows to predict mechanical properties so that localized optical mechanoresponses could enable locating and quantifying acting forces. Based on network design principles from the Flory-Rehner theory, precursors with predefined size and architecture were utilized in hydrogel preparation, aiming for a homogeneous network. Further in this regard, the mixing volume was reduced by employing droplet microfluidics. As optical response mechanism, the hydrogel network precursors were labeled with two kinds of fluorophore, featuring distance-dependent emission from Förster Resonance Energy Transfer. The functionality of the optical response mechanism was demonstrated on global level by collapsing and controlled swelling of the network, and on a localized level by defined mechanical stress, applied with Atomic Force Microscopy. Owing to its adjustability, the hydrogel platform might be employed in various applications that require intrinsic force sensing of soft matter in future

Probing nanomechanical properties from biomolecules to living cells

Atomic force microscopy is being increasingly used to explore the physical properties of biological structures. This technique involves the application of a force to the sample and a monitoring of the ensuing deformation process. The available experimental setups can be broadly divided into two categories, one of which involves a stretching and the other an indentation of the organic materials. In this review, we will focus on the indentation technique and will illustrate its application to biological materials with examples that range from single molecules to living cell

Mechanical cell-matrix feedback explains pairwise and collective endothelial cell behavior in vitro

In vitro cultures of endothelial cells are a widely used model system of the collective behavior of endothelial cells during vasculogenesis and angiogenesis. When seeded in an extracellular matrix, endothelial cells can form blood vessel-like structures, including vascular networks and sprouts. Endothelial morphogenesis depends on a large number of chemical and mechanical factors, including the compliancy of the extracellular matrix, the available growth factors, the adhesion of cells to the extracellular matrix, cell-cell signaling, etc. Although various computational models have been proposed to explain the role of each of these biochemical and biomechanical effects, the understanding of the mechanisms underlying in vitro angiogenesis is still incomplete. Most explanations focus on predicting the whole vascular network or sprout from the underlying cell behavior, and do not check if the same model also correctly captures the intermediate scale: the pairwise cell-cell interactions or single cell responses to ECM mechanics. Here we show, using a hybrid cellular Potts and finite element computational model, that a single set of biologically plausible rules describing (a) the contractile forces that endothelial cells exert on the ECM, (b) the resulting strains in the extracellular matrix, and (c) the cellular response to the strains, suffices for reproducing the behavior of individual endothelial cells and the interactions of endothelial cell pairs in compliant matrices. With the same set of rules, the model also reproduces network formation from scattered cells, and sprouting from endothelial spheroids. Combining the present mechanical model with aspects of previously proposed mechanical and chemical models may lead to a more complete understanding of in vitro angiogenesis.Comment: 25 pages, 6 figures, accepted for publication in PLoS Computational Biolog