Detecting long-range interactions between migrating cells.

Chemotaxis enables cells to systematically approach distant targets that emit a diffusible guiding substance. However, the visual observation of an encounter between a cell and a target does not necessarily indicate the presence of a chemotactic approach mechanism, as even a blindly migrating cell can come across a target by chance. To distinguish between the chemotactic approach and blind migration, we present an objective method that is based on the analysis of time-lapse recorded cell migration trajectories: For each movement step of a cell relative to the position of a potential target, we compute a p value that quantifies the likelihood of the movement direction under the null-hypothesis of blind migration. The resulting distribution of p values, pooled over all recorded cell trajectories, is then compared to an ensemble of reference distributions in which the positions of targets are randomized. First, we validate our method with simulated data, demonstrating that it reliably detects the presence or absence of remote cell-cell interactions. In a second step, we apply the method to data from three-dimensional collagen gels, interspersed with highly migratory natural killer (NK) cells that were derived from two different human donors. We find for one of the donors an attractive interaction between the NK cells, pointing to a cooperative behavior of these immune cells. When adding nearly stationary K562 tumor cells to the system, we find a repulsive interaction between K562 and NK cells for one of the donors. By contrast, we find attractive interactions between NK cells and an IL-15-secreting variant of K562 tumor cells. We therefore speculate that NK cells find wild-type tumor cells only by chance, but are programmed to leave a target quickly after a close encounter. We provide a freely available Python implementation of our p value method that can serve as a general tool for detecting long-range interactions in collective systems of self-driven agents.

[Results of the Dry Eye Research Grant Award 2016]. / Ergebnisse der Sicca-Forschungsförderung 2016.

The Sicca-Förderpreis (Dry Eye Award) supports the development of scientific research on the pathogenesis, diagnostics, and treatment of dry eye and ocular surface diseases. It is awarded after a limited call for proposals in German-speaking countries, written application and selection of the award winner after evaluation by a jury of ophthalmologists working in basic and clinical science. In this article examples of the results of funded projects of the Sicca-Förderpreis 2016 are cursorily described, which were presented at the Ophthalmological Academy of Germany 2019 (Augenärztliche Akademie Deutschland 2019) and therefore provide an insight into current scientific developments. The role of muscarinic receptors and those of urea in the pathogenesis of dry eye as well as the (missing) correlation of tear film stability, viscosity and surface tension are highlighted. A project on the early detection of ocular involvement in graft versus host disease and the idea of treating meibomian gland dysfunction with eyelid surgery techniques are also groundbreaking. The outlined projects represent the potential for further substantial developments in the understanding, diagnostics and treatment of dry eye; however, their long-term clinical relevance still needs to be established.

Phase transitions in huddling emperor penguins.

Emperor penguins (Aptenodytes forsteri) are highly adapted to the harsh conditions of the Antarctic winter: they are able to fast for up to 134 days during breeding. To conserve energy, emperor penguins form tight groups (huddles), which is key for their reproductive success. The effect of different meteorological factors on the huddling behaviour, however, is not well understood. Using time-lapse image recordings of an emperor penguin colony, we show that huddling can be described as a phase transition from a fluid to a solid state. We use the colony density as order parameter, and an apparent temperature that is perceived by the penguins as the thermodynamic variable. We approximate the apparent temperature as a linear combination of four meteorological parameters: ambient temperature, wind speed, global radiation and relative humidity. We find a wind chill factor of -2.9 °C/(ms -1), a humidity chill factor of -0.5°C/% rel. humidity, and a solar radiation heating factor of 0.3 °C//(Wm 2). In the absence of wind, humidity and solar radiation, the phase transition temperature (50% huddling probability) is -48.2°C for the investigated time period (May 2014). We propose that higher phase transition temperatures indicate a shrinking thermal insulation and thus can serve as a proxy for lower energy reserves of the colony, integrating pre-breeding foraging success at sea and energy expenditure at land due to environmental conditions. As current global change is predicted to have strong detrimental effects on emperor penguins within the next decades, our approach may thus contribute towards an urgently needed long-term monitoring system for assessing colony health.

Traction Force Microscopy in 3-Dimensional Extracellular Matrix Networks.

Cell migration through a three-dimensional (3-D) matrix depends strongly on the ability of cells to generate traction forces. To overcome the steric hindrance of the matrix, cells need to generate sufficiently high traction forces but also need to distribute these forces spatially in a migration-promoting way. This unit describes a protocol to measure spatial maps of cell traction forces in 3-D biopolymer networks such as collagen, fibrin, or Matrigel. Traction forces are computed from the relationship between measured force-induced matrix deformations surrounding the cell and the known mechanical properties of the matrix. The method does not rely on knowledge of the cell surface coordinates and takes nonlinear mechanical properties of the matrix into account. © 2017 by John Wiley & Sons, Inc.

N-cadherin-functionalized polymer-tethered multi-bilayer: a cell surface-mimicking substrate to probe cellular mechanosensitivity.

Fate and function of anchorage-dependent cells depend on a variety of environmental cues, including those of mechanical nature. Previous progress in the understanding of cellular mechanosensitivity has been closely linked to the availability of artificial cell substrates of adjustable viscoelasticity, allowing for a direct correlation between substrate stiffness and cell response. Exemplary, polymeric gel substrates with polymer-conjugated cell-substrate linkers provided valuable insight into the role of mechanical signals during cell migration in an extracellular matrix environment. In contrast, less is known about the role of external mechanical signals across cell-cell interfaces, in part, due to the limitations of traditional polymeric substrates to mimic the remarkable dynamics of cell-cell linkages. To overcome this shortcoming, we introduce a cell surface-mimicking cell substrate of adjustable stiffness, which is comprised of a polymer-tethered lipid multi-bilayer stack with N-cadherin linkers. Unlike traditional polymeric cell substrates with polymer-conjugated linkers, this novel artificial cell substrate is able to replicate the dynamic assembly/disassembly of cadherin linkers into linker clusters and the long-range movements of cadherin-based cell-substrate linkages observed at cell-cell interfaces. Moreover, substrate stiffness can be changed by adjusting the number of bilayers in the multi-bilayer stack, thus enabling the analysis of cellular mechanosensitivity in the presence of artificial cell-cell linkages. The presented biomembrane-mimicking cell substrate provides a valuable tool to explore the functional role of mechanical cues from neighboring cells.

The IsoStretcher: An isotropic cell stretch device to study mechanical biosensor pathways in living cells.

Mechanosensation in many organs (e.g. lungs, heart, gut) is mediated by biosensors (like mechanosensitive ion channels), which convert mechanical stimuli into electrical and/or biochemical signals. To study those pathways, technical devices are needed that apply strain profiles to cells, and ideally allow simultaneous live-cell microscopy analysis. Strain profiles in organs can be complex and multiaxial, e.g. in hollow organs. Most devices in mechanobiology apply longitudinal uniaxial stretch to adhered cells using elastomeric membranes to study mechanical biosensors. Recent approaches in biomedical engineering have employed intelligent systems to apply biaxial or multiaxial stretch to cells. Here, we present an isotropic cell stretch system (IsoStretcher) that overcomes some previous limitations. Our system uses a rotational swivel mechanism that translates into a radial displacement of hooks attached to small circular silicone membranes. Isotropicity and focus stability are demonstrated with fluorescent beads, and transmission efficiency of elastomer membrane stretch to cellular area change in HeLa/HEK cells. Applying our system to lamin-A overexpressing fibrosarcoma cells, we found a markedly reduced stretch of cell area, indicative of a stiffer cytoskeleton. We also investigated stretch-activated Ca(2+) entry into atrial HL-1 myocytes. 10% isotropic stretch induced robust oscillating increases in intracellular Fluo-4 Ca(2+) fluorescence. Store-operated Ca(2+) entry was not detected in these cells. The Isostretcher provides a useful versatile tool for mechanobiology.

Corrosion of Mg alloy AZ91D in the presence of living cells.

Mg and Mg alloys are of interest for biodegradable implants as they readily corrode in biological fluids, and dissolved Mg ions are nontoxic. Even though it is well known that Mg dissolution leads to pH increase in the surroundings, the effect of the corrosion-induced alkalization on the biological environment has not been studied in detail. We therefore explored the interactions between corrosion-induced pH increase and cell growth on Mg alloy AZ91D surface. Cell adhesion and spreading on the alloy surface is unimpeded initially. However, with time a large fraction of cells de-adhere. We attribute this to the observed increase of the pH in the cell culture medium in the process of alloy dissolution. Cytotoxicity tests with HeLa cells grown on glass surfaces confirm that cell death increases with increasing alkalinity of the cell culture medium. We also show that a the cells that adhere on the Mg alloy surface act as a corrosion-blocking surface layer. In consequence, a slower pH increase in the medium takes place when the alloy surface is covered with cells. Electrochemical impedance spectroscopy measurements (EIS) verify that a cell layer slows down the corrosion process.

Fluctuations of cytoskeleton-bound microbeads--the effect of bead-receptor binding dynamics.

The cytoskeleton (CSK) of living cells is a crosslinked fiber network, subject to ongoing biochemical remodeling processes that can be visualized by tracking the spontaneous motion of CSK-bound microbeads. The bead motion is characterized by anomalous diffusion with a power-law time evolution of the mean square displacement (MSD), and can be described as a stochastic transport process with apparent diffusivity D and power-law exponent ß: MSD ∼ D (t/t(0))(ß). Here we studied whether D and ß change with the time that has passed after the initial bead-cell contact, and whether they are sensitive to bead coating (fibronectin, integrin antibodies, poly-L-lysine, albumin) and bead size (0.5-4.5 µm). The measurements are interpreted in the framework of a simple model that describes the bead as an overdamped particle coupled to the fluctuating CSK network by an elastic spring. The viscous damping coefficient characterizes the degree of bead internalization into the cell, and the spring constant characterizes the strength of the binding of the bead to the CSK. The model predicts distinctive signatures of the MSD that change with time as the bead couples more tightly to the CSK and becomes internalized. Experimental data show that the transition from the unbound to the tightly bound state occurs in an all-or-nothing manner. The time point of this transition shows considerable variability between individual cells (2-30 min) and depends on the bead size and bead coating. On average, this transition occurs later for smaller beads and beads coated with ligands that trigger the formation of adhesion complexes (fibronectin, integrin antibodies). Once the bead is linked to the CSK, however, the ligand type and bead size have little effect on the MSD. On longer timescales of several hours after bead addition, smaller beads are internalized into the cell more readily, leading to characteristic changes in the MSD that are consistent with increased viscous damping by the cytoplasm and reduced binding strength.

Noise and critical phenomena in biochemical signaling cycles at small molecule numbers.

Biochemical reaction networks in living cells usually involve reversible covalent modification of signaling molecules, such as protein phosphorylation. Under conditions of small molecule numbers, as is frequently the case in living cells, mass-action theory fails to describe the dynamics of such systems. Instead, the biochemical reactions must be treated as stochastic processes that intrinsically generate concentration fluctuations of the chemicals. We investigate the stochastic reaction kinetics of covalent modification cycles (CMCs) by analytical modeling and numerically exact Monte Carlo simulation of the temporally fluctuating concentration. Depending on the parameter regime, we find for the probability density of the concentration qualitatively distinct classes of distribution functions including power-law distributions with a fractional and tunable exponent. These findings challenge the traditional view of biochemical control networks as deterministic computational systems and suggest that CMCs in cells can function as versatile and tunable noise generators.

Filamin A is essential for active cell stiffening but not passive stiffening under external force.

The material properties of a cell determine how mechanical forces are transmitted through and sensed by that cell. Some types of cells stiffen passively under large external forces, but they can also alter their own stiffness in response to the local mechanical environment or biochemical cues. Here we show that the actin-binding protein filamin A is essential for the active stiffening of cells plated on collagen-coated substrates. This appears to be due to a diminished capability to build up large internal contractile stresses in the absence of filamin A. To show this, we compare the material properties and contractility of two human melanoma cell lines that differ in filamin A expression. The filamin A-deficient M2 cells are softer than the filamin A-replete A7 cells, and exert much smaller contractile stresses on the substratum, even though the M2 cells have similar levels of phosphorylated myosin II light chain and only somewhat diminished adhesion strength. In contrast to A7 cells, the stiffness and contractility of M2 cells are insensitive to either myosin-inhibiting drugs or the stiffness of the substratum. Surprisingly, however, filamin A is not required for passive stiffening under large external forces.

Simple model of cytoskeletal fluctuations.

The spontaneous motion of microbeads bound to the cytoskeleton of living cells is not an ordinary random walk. Unlike Brownian motion, the mean-square displacement undergoes a transition from subdiffusive to superdiffusive behavior with time. This transition is associated with characteristic changes of the turning angle distribution. Recent experimental data demonstrated that force fluctuations measured in an elastic hydrogel matrix beneath the cell correlate with the bead motion [C. Raupach, Phys. Rev. E 76, 011918 (2007)]. These data indicate that the bead trajectory is driven by motor forces originating from the actomyosin network and that cytoskeletal remodeling processes with short- and long-time dynamics are mainly responsible for the non-Brownian behavior. We show that the essential statistical properties of the spontaneous bead motion can be reproduced by a particle diffusing in a potential well with a slowly drifting minimum position. Based on this simple model, which can be solved analytically, we develop a biologically plausible numerical model of a tensed and continuously remodeling actomyosin network that accounts quantitatively for the measured data.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Linearity and time-scale invariance of the creep function in living cells.

We report here the creep function measured in three cell types, after a variety of interventions, and over three time decades (from 3 ms to 3.2 s). In each case the response conformed to a power law, implying that no distinct molecular relaxation times or time constants could characterize the response. These results add to a growing body of evidence that stands in contrast to widely used viscoelastic models featuring at most a few time constants. We show instead that the ability of the matrix to deform is time-scale invariant and characterized by only one parameter: the power law exponent that controls the transition between solid-like and liquid-like behaviour. Moreover, we validate linearity by comparison of measurements in the time and frequency domains.

Scaling the microrheology of living cells.

We report a scaling law that governs both the elastic and frictional properties of a wide variety of living cell types, over a wide range of time scales and under a variety of biological interventions. This scaling identifies these cells as soft glassy materials existing close to a glass transition, and implies that cytoskeletal proteins may regulate cell mechanical properties mainly by modulating the effective noise temperature of the matrix. The practical implications are that the effective noise temperature is an easily quantified measure of the ability of the cytoskeleton to deform, flow, and reorganize.

Selected contribution: time course and heterogeneity of contractile responses in cultured human airway smooth muscle cells.

We measured the time course and heterogeneity of responses to contractile and relaxing agonists in individual human airway smooth muscle (HASM) cells in culture. To this end, we developed a microrheometer based on magnetic twisting cytometry adapted with a novel optical detection system. Ferromagnetic beads (4.5 microm) coated with Arg-Gly-Asp peptide were bound to integrins on the cell surface. The beads were twisted in a sinusoidally varying magnetic field at 0.75 Hz. Oscillatory bead displacements were recorded using a phase-synchronized video camera. The storage modulus (cell stiffness; G'), loss modulus (friction; G"), and hysteresivity (eta; ratio of G" to G') could be determined with a time resolution of 1.3 s. Within 5 s after addition of histamine (100 microM), G' increased by 2.2-fold, G" increased by 3.0-fold, and eta increased transiently from 0.27 to 0.34. By 20 s, eta decreased to 0.25, whereas G' and G" remained above baseline. Comparable results were obtained with bradykinin (1 microM). These changes in G', G", and eta measured in cells were similar to but smaller than those reported for intact muscle strips. When we ablated baseline tone by adding the relaxing agonist dibutyryl cAMP (1 mM), G' decreased within 5 min by 3.3-fold. With relaxing and contracting agonists, G' could be manipulated through a contractile range of 7.3-fold. Cell populations exhibited a log-normal distribution of baseline stiffness (geometric SD = 2.8) and a heterogeneous response to both contractile and relaxing agonists, partly attributable to variability of baseline tone between cells. The total contractile range of the cells (from maximally relaxed to maximally stimulated), however, was independent of baseline stiffness. We conclude that HASM cells in culture exhibit a clear, although heterogeneous, response to contractile and relaxing agonists and express the essential mechanical features characteristic of the contractile response observed at the tissue level.

Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells.

A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

Analysis of cell mechanics in single vinculin-deficient cells using a magnetic tweezer.

A magnetic tweezer was constructed to apply controlled tensional forces (10 pN to greater than 1 nN) to transmembrane receptors via bound ligand-coated microbeadswhile optically measuring lateral bead displacements within individual cells. Use of this system with wild-type F9 embryonic carcinoma cells and cells from a vinculin knockout mouse F9 Vin (-/-) revealed much larger differences in the stiffness of the transmembrane integrin linkages to the cytoskeleton than previously reported using related techniques that measured average mechanical properties of large cell populations. The mechanical properties measured varied widely among cells, exhibiting an approximately log-normal distribution. The median lateral bead displacement was 2-fold larger in F9 Vin (-/-) cells compared to wild-type cells whereas the arithmetic mean displacement only increased by 37%. We conclude that vinculin serves a greater mechanical role in cells than previously reported and that this magnetic tweezer device may be useful for probing the molecular basis of cell mechanics within single cells.

Mechanical properties of cultured human airway smooth muscle cells from 0.05 to 0.4 Hz.

We investigated the rheological properties of living human airway smooth muscle cells in culture and monitored the changes in rheological properties induced by exogenous stimuli. We oscillated small magnetic microbeads bound specifically to integrin receptors and computed the storage modulus (G') and loss modulus (G") from the applied torque and the resulting rotational motion of the beads as determined from their remanent magnetic field. Under baseline conditions, G' increased weakly with frequency, whereas G" was independent of the frequency. The cell was predominantly elastic, with the ratio of G" to G' (defined as eta) being approximately 0. 35 at all frequencies. G' and G" increased together after contractile activation and decreased together after deactivation, whereas eta remained unaltered in each case. Thus elastic and dissipative stresses were coupled during changes in contractile activation. G' and G" decreased with disruption of the actin fibers by cytochalasin D, but eta increased. These results imply that the mechanisms for frictional energy loss and elastic energy storage in the living cell are coupled and reside within the cytoskeleton.

Mechanical control of cyclic AMP signalling and gene transcription through integrins.

This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.