Auswahl der wissenschaftlichen Literatur zum Thema „Cell mechanics, mechanical properties, biophysics, physiology“

The mechanical properties of tissues and cells including renal glomeruli are important determinants of their differentiated state, function, and responses to injury but are not well characterized or understood. Understanding glomerular mechanics is important for understanding renal diseases attributable to abnormal expression or assembly of structural proteins and abnormal hemodynamics. We use atomic force microscopy (AFM) and a new technique, capillary micromechanics, to measure the elastic properties of rat glomeruli. The Young's modulus of glomeruli was 2,500 Pa, and it was reduced to 1,100 Pa by cytochalasin and latunculin, and to 1,400 Pa by blebbistatin. Cytochalasin or latrunculin reduced the F/G actin ratios of glomeruli but did not disrupt their architecture. To assess glomerular biomechanics in disease, we measured the Young's moduli of glomeruli from two mouse models of primary glomerular disease, Col4a3−/− mice (Alport model) and Tg26HIV/nl mice (HIV-associated nephropathy model), at stages where glomerular injury was minimal by histopathology. Col4a3−/− mice express abnormal glomerular basement membrane proteins, and Tg26HIV/nl mouse podocytes have multiple abnormalities in morphology, adhesion, and cytoskeletal structure. In both models, the Young's modulus of the glomeruli was reduced by 30%. We find that glomeruli have specific and quantifiable biomechanical properties that are dependent on the state of the actin cytoskeleton and nonmuscle myosins. These properties may be altered early in disease and represent an important early component of disease. This increased deformability of glomeruli could directly contribute to disease by permitting increased distension with hemodynamic force or represent a mechanically inhospitable environment for glomerular cells.

Plasticity and functional diversity of macrophages play an important role in resisting pathogens invasion, tumor progression and tissue repair. At present, nanodrug formulations are becoming increasingly important to induce and control the functional diversity of macrophages. In this framework, the internalization process of nanodrugs is co-regulated by a complex interplay of biochemistry, cell physiology and cell mechanics. From a biophysical perspective, little is known about cellular mechanics’ modulation induced by the nanodrug carrier’s internalization. In this study, we used the polylactic-co-glycolic acid (PLGA)–polyethylene glycol (PEG) nanofibers as a model drug carrier, and we investigated their influence on macrophage mechanics. Interestingly, the nanofibers internalized in macrophages induced a local increase of stiffness detected by atomic force microscopy (AFM) nanomechanical investigation. Confocal laser scanning microscopy revealed a thickening of actin filaments around nanofibers during the internalization process. Following geometry and mechanical properties by AFM, indentation experiments are virtualized in a finite element model simulation. It turned out that it is necessary to include an additional actin wrapping layer around nanofiber in order to achieve similar reaction force of AFM experiments, consistent with confocal observation. The quantitative investigation of actin reconfiguration around internalized nanofibers can be exploited to develop novel strategies for drug delivery.

To understand the complex biochemistry and biophysics of biological systems, one needs to be able to monitor local concentrations of molecules, physical properties of macromolecular assemblies and activation status of signaling pathways, in real time, within single cells, and at high spatio-temporal resolution. Here we look at the tools that have been / are being / need to be provided by chemical biology to address these challenges. In particular, we highlight the utility of molecular probes that help to better measure mechanical forces and flux through key signalling pathways. Chemical biology can be used to both build biosensors to visualize, but also actuators to perturb biological processes. An emergent theme is the possibility to multiplex measurements of multiple cellular processes. Advances in microscopy automation now allow us to acquire datasets for 1000’s of cells. This produces high dimensional datasets that require computer vision approaches that automate image analysis. The high dimensionality of these datasets are often not immediately accessible to human intuition, and, similarly to ‘omics technologies, require statistical approaches for their exploitation. The field of biosensor imaging is therefore experiencing a multidisciplinary transition that will enable it to realize its full potential as a tool to provide a deeper appreciation of cell physiology.

To withstand physiological loading over a lifetime, human synovial joints are covered and protected by articular cartilage, a layer of low-friction, load-bearing tissue. The unique mechanical function of articular cartilage largely depends on the composition and structural integrity of the cartilage matrix. The matrix is produced by highly specialized resident cells called chondrocytes. Under physiological loading, chondrocytes maintain the balance between degradation and synthesis of matrix macromolecules. Under excessive loading or injury, however, degradation exceeds synthesis, causing joint degeneration and, eventually, osteoarthritis (OA). Hence, the mechanoresponses of chondrocytes play an important role in the development of OA. Despite its clear importance, the mechanobiology of articular chondrocytes is not well understood. To summarize our current understanding, here we review studies of the effect of mechanical forces on mechanical and biological properties of articular chondrocytes. First, we present the viscoelastic properties of the cell nucleus, chondrocyte, pericellular matrix, and chondron. Then we discuss how these properties change in OA. Finally, we discuss the responses of normal and osteoarthritic chondrocytes to a variety of mechanical stimuli. Studies reviewed here may provide novel insights into the pathogenesis of OA and may help in development of effective biophysical treatment.

Abstract After treatment with glucocorticoids (e.g. dexamethasone) or catecholamines (e.g. epinephrine), the white blood cell (WBC) count substantially increases. This is primarily due to WBCs shifting from the marginated to circulating pools (Nakagawa et al., Circulation, 2008) and is traditionally attributed to down-regulation of adhesion molecule expression (Weber et al., J Leukoc Biol, 2004).Recent research has described how mechanical properties determine the radial position of blood cells within the intravascular space (Reasor et. al, Ann Biomed Eng., 2013). In addition, because WBC demargination occurs rapidly (e.g.,<15 min after IV epinephrine infusion (Dimitrov et al., J Immunol. 2010)) on a timescale that may be shorter than that expected for alterations in gene expression, we hypothesized that alterations in WBC mechanical properties upon exposure to glucocorticoids or catecholamines mediate demargination. To that end, we developed an in vitro microfluidic system as a simplified microvasculature model (Fig 1A), which our laboratory has expertise in (Tsai et al., J Clin Invest., 2008 and Rosenbluth et al., Biophys J. , 2006). In the absence of confounding factors such as WBC release from bone marrow or endothelial interactions, this type of assay is ideally suited to determine the role of glucocorticoid and catecholamine treatment on the demargination of WBCs. By flowing whole blood into similar non-functionalized microfluidic devices, other groups have demonstrated that non-activated WBCs marginate to the microfluidic channel wall, which is likely due to their mechanical properties (Jain et al., PLoS One, 2009). Human whole blood was incubated at 37° C with acridine orange (WBC stain) and either dexamethasone or epinephrine at physiologically relevant concentrations. The blood was then flowed through our microfluidics at physiologic shear rates while confocal videomicroscopy was used to image the center plane of the channel. We developed custom analysis software that extracts the position of individual WBCs from a series of confocal images and plots histograms of their locations, tracking over 10,000 WBCs per experiment (Fig 1B). Overall, we found that both dexamethasone and epinephrine (to a slightly lesser extent) cause WBCs to demarginate from the walls of the vessel compared to control conditions (Fig 1C). This glucocorticoid and catecholamine-induced movement of WBCs toward the microchannel center mimics in vivo demargination and our reductionist microfluidic approach strongly suggests that alterations in WBC mechanics play a key role in this process. Indeed, using computational modeling, we confirmed that a reduction in the mechanical stiffness of WBCs is sufficient by itself to explain the observed demargination (Fig 2A) (Kumar et al., Phys Rev Lett., 2012). Using a range of WBC stiffnesses, our simulations revealed that decreases in WBC stiffness correlated with the degree of demargination. To corroborate our microfluidic data, we also directly measured WBC stiffness using atomic force microscopy. WBCs treated with dexamethasone were significantly softer (p< 0.0002) than control WBCs (Fig 2B), supporting our hypothesis that the demargination phenomenon is related to the biophysical changes in WBCs. Experiments measuring the stiffness of epinephrine-treated cells as well as experiments evaluating how these drugs affect the actin cytoskeleton are currently underway. Overall, our data suggest that WBC mechanics play a major role in glucocorticoid- and catecholamine-induced demargination and that the underlying mechanisms may, at least in part, be biophysical in nature. This novel finding may have important implications in other hematologic processes such as WBC margination and recruitment during inflammatory responses or hematopoietic stem cell mobilization and homing. Disclosures: No relevant conflicts of interest to declare.

Embryonic and pluripotent stem cells hold great promise in generating β-cells for both replacing medicine and novel therapeutic discoveries in diabetes mellitus. However, their differentiation in vitro is still inefficient, and functional studies reveal that most of these β-like cells still fail to fully mirror the adult β-cell physiology. For their proper growth and functioning, β-cells require a very specific environment, the islet niche, which provides a myriad of chemical and physical signals. While the nature and effects of chemical stimuli have been widely characterized, less is known about the mechanical signals. We here review the current status of knowledge of biophysical cues provided by the niche where β-cells normally live and differentiate, and we underline the possible machinery designated for mechanotransduction in β-cells. Although the regulatory mechanisms remain poorly understood, the analysis reveals that β-cells are equipped with all mechanosensors and signaling proteins actively involved in mechanotransduction in other cell types, and they respond to mechanical cues by changing their behavior. By engineering microenvironments mirroring the biophysical niche properties it is possible to elucidate the β-cell mechanotransductive-regulatory mechanisms and to harness them for the promotion of β-cell differentiation capacity in vitro.

The topic of this thesis focuses on studying the mechanical properties of the living cell plasma membrane and the mechanical associations of the plasma membrane with the underlying cytoskeleton. The mechanical properties of the cell components, cell plasma membrane and cytoskeleton, as well as membrane-cytoskeleton associations, determine the mechanical properties of the whole cell, important for cellular shape changing behavior and mechanical signal transduction in living cells. Examples of biological processes involving cellular shape changes are deformation of erythrocytes in capillaries, cell division, phagocytosis, pseudopodium and dendritic spine formation, and electromotility of the outer hair cells. The study of cell membrane mechanics accomplished during my PhD activity is based on the use of two most advanced technological approaches to investigate both local membrane deformation by dual laser optical tweezers (DLOT, (Bianco et al., 2011)) and global cell deformation by real-time florescence deformability cytometry (RT-FDC). This dissertation is divided into two main parts. In the first part I present my contribution to the development and application of a system able to study the mechanical properties of plasma membrane of the Human Embryonic Kidney (HEK293) cells in the adherent state. The DLOT was first applied to investigate the dynamics of the formation/retraction of membrane nanotubes (tethers). In physiological conditions, several parameters of plasma membrane and its interaction with the underlying cytoskeleton (tether formation and elongation, tether radius, Threshold force for the membrane tether elongation and its viscoelastic nature, the tether diameter, the bending modulus, the membrane-cytoskeleton adhesion energy) were determined and compared with those present in literature. The information on cell membrane mechanics was integrated with indentation measurements by using the DLOT for applying local deformations in force feedback in the range experienced by the membrane of macrophages during phagocytosis.The mean value of the elastic modulus (Young’s modulus) derived from indentation experiments was 32 ± 8 Pa (mean ± SD), significantly lower than that obtained with AFM measurements. The difference confirms data found in previous work (Coceano et al., 2016) and indicates that my approach, though limited by its intrinsic compliance to lower time resolution, is unique in resolving the load – dependent dynamics of the cytoskeletal rearrangement triggered by a force step. In the second part I present experiments conducted during the one-year Ph-D period spent at the University of Greifswald, under the supervision of Dr. Oliver Otto. The experiments were aimed at characterizing the mechanical properties of the HEK293 cells in suspension and how they are influenced by hypoxic stress. Mechanical parameters from control experiments under physiological condition are compared to those obtained in the presence of an increased subpopulation of cells in apoptotic/necrotic state using a high-throughput system, Real-Time Deformability Cytometry (RT-DC (Otto et al., 2015)), which allows to study a high number of cells in short time (1000 cells/s). In combination with fluorescence-based flow cytometry (RT-FDC, (Rosendahl et al., 2018)), this technology permits to analyze treated fluorescent cells and discriminate between subpopulations. The mechanical parameters considered are cell size, elastic modulus and cell deformation. Hypoxia stressed cells were studied at different incubation times and the dependence from hypoxia of the mechanical parameters were determined. After 12h of oxygen deprivation (12h-hyp) cell stiffness is increased and cell deformation is reduced. An overall increase in concentration of the main cytoskeletal proteins (ß-actin, α/-tubulin, vinculin and talin-1), determined by Western blots, was found to accompany mechanical – structural modification by hypoxia. The successful application of the protocols developed here for the definition of local and global mechanical properties of the cell membrane and the associated underlying cytoskeleton of HEK293 line, opens the possibility of new investigations on the effects on the relevant cell mechanical parameters of different physical and chemical interventions (temperature, pH and buffer ionic composition), different physiological conditions (metabolic stress as hypoxia) modification of the membrane composition, as cholesterol content, or effect of disruption or mutation of membrane-linked cytoskeleton proteins. In this respect the methodology established in this thesis represents a new powerful tool for the investigation o membrane-cytoskeleton structure-function in health and disease.

The effect of mechanical shear on brewing yeast was investigated with a focus on losses incurred through cell rupture and viability loss. The influence of various environmental conditions was studied with regards to the influence on Saccharomyces cerevisiae's ability to resist mechanical shear. Further investigation was performed in order to locate a structure within the yeast cell that contributes to mechanical shear resistance.
It was found that yeast cells grown anaerobically in limited glucose media were more prone to losses in cell viability than cells grown aerobically in the same media, when subjected to mechanical shear. Cells grown anaerobically in high glucose concentrations and allowed to ferment the media to exhaustion were slightly more resistant to mechanical shear compared to cells grown anaerobically without fermentation in minimal glucose media. Higher ethanol concentrations lead to marginally decreased resistance to mechanical shear.
Cell walls of S. cerevisiae were partially digested or extracted using enzymatic treatment or chemical attack. It was found that while the outer mannoprotein layer does not contribute significantly, the inner beta-(1 → 3)-glucan structure plays a significant role in resistance to mechanical shear.

The final stage of the cell cycle is cell division by cytokinesis, when the cell physically separates into two daughter cells. Improper timing or location of the division site results in incorrect segregation of chromosomes and thus genetically unstable aneuploid cells, which is associated with tumorigenesis. Cytokinesis in animal, fungal and amoeboid cells occurs through the assembly and constriction of an actomyosin contractile ring, a mechanism that dates back about one billion years in the common ancestor of these organisms. However, it is not well understood how the ring generates tension or how the rate of ring constriction is set. Long ago a sliding filament mechanism similar to skeletal muscle was proposed, but definitive evidence for muscle-like sarcomeric order in the ring is lacking. Here we build mathematical models of cytokinesis in the fission yeast Schizosaccharomyces pombe, where the most complete inventory of more than 150 cytokinesis genes have been documented. The models explicitly represent proteins in the contractile ring such as formin, myosin, actin, α-actinin, etc. and implements their quantities, biomechanical properties and organizations from the best available experimental information. At the same time, the models adopt coarse-grain approaches that are able to describe the collective behaviors of thousands of ring components, which include tension production, constriction, and disassembly of the ring. In the first part of this thesis, we modeled the extraordinarily rapid constriction of the partially unanchored ring in fission yeast cell ghosts. Experiments on isolated fission yeast rings showed sections of ring unanchoring from the membrane and shortening ~30-fold faster than normal (1). We demonstrated that anchoring of actin to the plasma membrane generates tension in the fission yeast cytokinetic ring by showing (1) unanchored segments in these experiments were tensionless, and (2) only a barbed-end anchoring of actin can generate tension in the normally anchored ring, and can explain the extraordinary behavior of unanchored segments. Molecularly explicit simulations accurately reproduced experimental constriction rates, and showed a novel non-contractile reeling-in mechanism by which the unanchored segment shortens, despite being tensionless. In the second part of this thesis, we built a highly coarse-grained model to study how ring tension is generated and how structural stability is maintained. Recently, a super-resolution microscopy study of the fission yeast ring revealed that myosins and formins that nucleate actin filaments colocalize in plasma membrane-anchored complexes called nodes in the constricting ring (2). The nodes move bidirectionally around the ring. Here we construct and analyze a coarse-grained mathematical model of the fission yeast ring to explore essential consequences of the recently discovered ring ultrastructure. The model reproduces experimentally measured values of ring tension, explains why nodes move bidirectionally and shows that tension is generated by myosin pulling on barbed-end-anchored actin filaments in a stochastic sliding-filament mechanism. This mechanism is not based on an ordered sarcomeric organization. We show that the ring is vulnerable to intrinsic contractile instabilities, and protection from these instabilities and organizational homeostasis require both component turnover and anchoring of components to the plasma membrane. In the third part of this thesis, we measured ring tension in fission yeast protoplasts. We found ~650 pN tension in wild type cells, ~65% the normal tension in myp2 deletion mutants and ~40% normal tension in myo2-E1 mutant cells with negligible ATPase activity and reduced actin binding. To understand the relation between organization and tension, we developed a molecularly explicit simulation of the fission yeast ring with the above organization. Our simulations revealed a clear division of labor between the 2 myosin-II isoforms, which maintains organization and maximal tension. (1) Myo2 anchors the ring to the plasma membrane, and transmits ring tension to the membrane. (2) Myo2, extending ~100 nm away from the membrane, bundles half (~25) of the actin filaments in the cross-section due to filament packing constraints, as only ~25 filaments are within reach. (3) To increase tension requires that the ring be thickened, as tensions in the ~25 membrane-proximal filaments are close to fracture. (4) Unanchored Myp2 indeed enables thickening, by bundling an additional ~25 filaments and doubling tension. Anchoring of these filaments to the membrane is indirect, via filaments shared with the anchored Myo2. (5) In simulated myo2-E1 rings ~20% of the actin filaments peeled away from the ring and formed Myp2-dressed bridges, as observed experimentally in myo2-E1 cells. (6) The organization in simulated Δmyp2 rings was highly disrupted, with ~ 50% of the actin filaments unbundled. Therefore, beyond their widely recognized job to pull actin and generate tension, myosin-II isoforms are vital crosslinking organizational elements of the ring. Two isoforms in the ring cooperate to organize the ring for maximal actomyosin interaction and tension.

Bacteria adhere despite severe mechanical perturbations. In Gram-positive bacteria, this adhesion is dependent upon a set of extracellular proteins, most notably pili, that have a unique abundance of internal disulfide, isopeptide, and thioester bonds. How these cell adhesion proteins manage to withstand such mechanical assaults, and what role these internal covalent bonds play to that end, remain open questions. Herein, we apply single-molecule force spectroscopy to delve into the mechanical behavior of three Gram-positive pilus proteins. We find that structural components of the Actinomyces oris and Corynebacterium diphtheriae pili have isopeptide-delimited extensions at extreme mechanical forces. This behavior enables efficient energy dissipation under high mechanical loads. Meanwhile, the pilus tip adhesin of Streptococcus pyogenes can covalently bind to targets via its internal thioester bond. We find that reactions with this internal thioester bond are reversible, and that both the nucleophilic bond cleavage and its spontaneous reformation are negatively force-dependent, inhibited at forces above ~30 pN and above ~7 pN, respectively. Based on these observations, we propose a model of shear-enhanced covalent adhesion for Gram-positive bacteria. Finally, we move from single-molecule characterization to application as we explore the potential of a peptide competitors to modulate the folding and function of bacterial virulence factors.