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Alterations in the organization of the cytoskeleton precede the escape of adherent cells from the framework of cell–cell and cell‐matrix interactions into suspension. With cytoskeletal dynamics being linked to cell mechanical properties, many studies elucidated this relationship under either native adherent or suspended conditions. In contrast, tethered cells that mimic the transition between both states have not been the focus of recent research. Using human embryonic kidney 293 T cells we investigated all three conditions in the light of alterations in cellular shape, volume, as well as mechanical properties and relate these findings to the level, structure, and intracellular localization of filamentous actin (F‐actin). For cells adhered to a substrate, our data shows that seeding density affects cell size but does not alter their elastic properties. Removing surface contacts leads to cell stiffening that is accompanied by changes in cell shape, and a reduction in cellular volume but no alterations in F‐actin density. Instead, we observe changes in the organization of F‐actin indicated by the appearance of blebs in the semi‐adherent state. In summary, our work reveals an interplay between molecular and mechanical alterations when cells detach from a surface that is mainly dominated by cell morphology.
Titin is a multifunctional filamentous protein anchored in the M-band, a hexagonally organized supramolecular lattice in the middle of the muscle sarcomere. Functionally, the M-band is a framework that cross-links myosin thick filaments, organizes associated proteins, and maintains sarcomeric symmetry via its structural and putative mechanical properties. Part of the M-band appears at the C-terminal end of isolated titin molecules in the form of a globular head, named here the “M-complex”, which also serves as the point of head-to-head attachment of titin. We used high-resolution atomic force microscopy and nanosurgical manipulation to investigate the topographical and internal structure and local mechanical properties of the M-complex and its associated titin molecules. We find that the M-complex is a stable structure that corresponds to the transverse unit of the M-band organized around the myosin thick filament. M-complexes may be interlinked into an M-complex array that reflects the local structural and mechanical status of the transversal M-band lattice. Local segments of titin and the M-complex could be nanosurgically manipulated to achieve extension and domain unfolding. Long threads could be pulled out of the M-complex, suggesting that it is a compact supramolecular reservoir of extensible filaments. Nanosurgery evoked an unexpected volume increment in the M-complex, which may be related to its function as a mechanical spacer. The M-complex thus displays both elastic and plastic properties which support the idea that the M-band may be involved in mechanical functions within the muscle sarcomere.
Free radicals are known to induce significant structural and functional modifications to the cell membrane and its components. Biophysical quantification of such changes using single molecule studies highlight the role of these individual biomolecules. In this PhD work, we focus on nitric oxide radical and try to understand how they influence interaction of different biomolecules with lipid membranes by using biomimetic systems. In specific we try to answer how cell membrane permeability and bilayer thickness would be influenced by the nitric oxide radical with different phospholipids compositions (i.e. on planar supported lipid bilayers). Later we tested, interaction of transmembrane protein integrin αiibβ3 incorporated into the bilayer (i.e. nanodiscs) with nitric oxide. Finally, how to overcome the negative effects encountered by the phospholipids and proteins using biopolymer coated gold nanoparticles as delivery system. The study involved use of atomic force microscopy and quartz-crystal microbalance with dissipation as primary investigation tools complemented with other relevant biophysical and biochemical techniques.
Direct monitoring of drug‐induced mechanical response of individual cells by atomic force microscopy
(2020)
Abstract
Mechanical characteristics of individual cells play a vital role in many biological processes and are considered as indicators of the cells’ states. Disturbances including methyl‐β‐cyclodextrin (MβCD) and cytochalasin D (cytoD) are known to significantly affect the state of cells, but little is known about the real‐time response of single cells to these drugs in their physiological condition. Here, nanoindentation‐based atomic force microscopy (AFM) was used to measure the elasticity of human embryonic kidney cells in the presence and absence of these pharmaceuticals. The results showed that depletion of cholesterol in the plasma membrane with MβCD resulted in cell stiffening whereas depolymerization of the actin cytoskeleton by cytoD resulted in cell softening. Using AFM for real‐time measurements, we observed that cells mechanically responded right after these drugs were added. In more detail, the cell´s elasticity suddenly increased with increasing instability upon cholesterol extraction while it is rapidly decreased without changing cellular stability upon depolymerizing actin cytoskeleton. These results demonstrated that actin cytoskeleton and cholesterol contributed differently to the cell mechanical characteristics.
Together with endothelial cells and the glomerular basement membrane, podocytes form the size-specific filtration barrier of the glomerulus with their interdigitating foot processes. Since glomerulopathies are associated with so-called foot process effacement—a severe change of well-formed foot processes into flat and broadened processes—visualization of the three-dimensional podocyte morphology is a crucial part for diagnosis of nephrotic diseases. However, interdigitating podocyte foot processes are too narrow to be resolved by classic light microscopy due to Ernst Abbe's law making electron microscopy necessary. Although three dimensional electron microscopy approaches like serial block face and focused ion beam scanning electron microscopy and electron tomography allow volumetric reconstruction of podocytes, these techniques are very time-consuming and too specialized for routine use or screening purposes. During the last few years, different super-resolution microscopic techniques were developed to overcome the optical resolution limit enabling new insights into podocyte morphology. Super-resolution microscopy approaches like three dimensional structured illumination microscopy (3D-SIM), stimulated emission depletion microscopy (STED) and localization microscopy [stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM)] reach resolutions down to 80–20 nm and can be used to image and further quantify podocyte foot process morphology. Furthermore, in vivo imaging of podocytes is essential to study the behavior of these cells in situ. Therefore, multiphoton laser microscopy was a breakthrough for in vivo studies of podocytes in transgenic animal models like rodents and zebrafish larvae because it allows imaging structures up to several hundred micrometer in depth within the tissue. Additionally, along with multiphoton microscopy, lightsheet microscopy is currently used to visualize larger tissue volumes and therefore image complete glomeruli in their native tissue context. Alongside plain visualization of cellular structures, atomic force microscopy has been used to study the change of mechanical properties of podocytes in diseased states which has been shown to be a culprit in podocyte maintenance. This review discusses recent advances in the field of microscopic imaging and demonstrates their currently used and other possible applications for podocyte research.
Die dilatative Kardiomyopathie (DCM) ist eine Herzmuskelerkrankung, die durch eine Einschränkung der kardialen Funktion bei gleichzeitiger Erweiterung eines (zumeist des linken) oder beider Ventrikel charakterisiert ist. Der Krankheit können verschiedene Ursachen zugrunde liegen. Störungen in der humoralen Immunität, mit der Bildung von Autoantikörpern, stehen im Zusammenhang mit der Entwicklung der DCM. Es konnten bereits verschiedenste kardiale Autoantikörper im Plasma von DCM Patienten nachgewiesen werden. Diese Autoantikörper können mit ihrem Fab-Teil an das Antigen binden und gleichzeitig, so wird angenommen, mit ihrem Fc-Teil den FcγRII, dessen Expression auf Rattenkardiomyozyten gezeigt wurde, quervernetzen, wodurch es zur Induktion eines negativ inotropen Effektes kommt. In der vorliegenden Arbeit wurde die Interaktion von FcγRII-Antikörpern mit Rattenkardiomyozyten untersucht und wie sich diese auf die mechano-dynamischen Eigenschaften der Zellen auswirkt. Im ersten Schritt wurde mit Hilfe der Quarz-Kristall-Mikrowaage mit Dissipationsaufzeichnung (QCM-D) untersucht, ob anti-FcγRII-Antikörper an isolierte Kardiomyozyten von Ratten binden. Zunächst wurden Bindungsstudien mit dem zu untersuchenden anti-FcγRII-Antikörper und transfizierten humanen embryonalen Nierenzellen durchgeführt, die den FcγRII in hoher Zelldichte auf der Zelloberfläche exprimieren. Nach erfolgreicher Etablierung des Assays konnte im Anschluss gezeigt werden, dass die anti-FcγRII-Antikörper auch an Rattenkardiomyozyten binden. Dies weist darauf hin, dass Kardiomyozyten möglicherweise den FcγRII auf ihrer Zelloberfläche exprimieren, wie bereits 2007 von Staudt et al. beschrieben wurde. In einem zweiten Ansatz, wurde die Mechanodynamik (Steifheit und Schlagfrequenz) von Kardiomyozyten in Gegenwart von anti-FcγRII-Antikörpern mittels des Rasterkraftmikroskops (AFM) untersucht. Die Inkubation von Rattenkardiomyozyten mit anti-FcγRII-Antikörpern führte zu einer signifikanten Versteifung der Zellen. Zudem zeigte sich, dass die anti-FcγRII-Antikörper nach der Blockierung der spezifischen Bindung an Kardiomyozyten durch Kontroll-IgG keinen Einfluss mehr auf die Zellversteifung haben. Eine biologisch relevante Änderung in der Schlagfrequenz konnte jedoch nach Inkubation der Kardiomyozyten mit den anti-FcγRII-Antikörpern nicht nachgewiesen werden. In der vorliegenden Arbeit konnte erstmals mittels QCM-D nachgewiesen werden, dass anti-FcγRII-Antikörper möglicherweise über eigenständige Rezeptoren an Kardiomyozyten binden. Die Bindung dieser Antikörper führt zu einer Zunahme der Zellsteifigkeit isolierter Kardiomyozyten. Dieser Befund könnte auf einen neuen pathophysiologisch relevanten Wirkungsmechanismus kardialer Autoantikörper hinweisen, der für die Pathogenese der DCM von Bedeutung ist.
The Atomic Force Microscope (AFM) has become an important tool for probing the mechanical properties of cells and microparticles by force-indentation experiments. In this thesis optimized AFM approaches for these experiments are developed and applied to three types of living human cells in order to answer biologically relevant questions about their mechanics. These microscopic investigations are then interpreted with respect to nanoscopic and macroscopic biologic parameters, such as the function of cell surface receptors or the size of human heart ventricles. This thesis comprises two physical/technical chapters and three medical/biological chapters. The physical/technical chapters discuss the measurement process itself, aiming for its improvement with respect to a proper data analysis and contact model (for spherical cells). The medical/biological chapters investigate the elasticity of cells by the use of optimized AFM approaches, with respect to the used data analysis.