Ingo Mey

Atomic force microscopy-based microrheology reveals significant differences in the viscoelastic response between malign and benign cell lines.

Mechanical phenotyping of cells by atomic force microscopy (AFM) was proposed as a novel tool in cancer cell research as cancer cells undergo massive structural changes, comprising remodelling of the cytoskeleton and changes of their adhesive properties. In this work, we focused on the mechanical properties of human breast cell lines with different metastatic potential by AFM-based microrheology experiments. Using this technique, we are not only able to quantify the mechanical properties of living cells in the context of malignancy, but we also obtain a descriptor, namely the loss tangent, which provides model-independent information about the metastatic potential of the cell line. Including also other cell lines from different organs shows that the loss tangent (G″/G′) increases generally with the metastatic potential from MCF-10A representing benign cells to highly malignant MDA-MB-231 cells.

Local Membrane Mechanics of Pore-Spanning Bilayers

The mechanical behavior of lipid bilayers spanning the pores of highly ordered porous silicon substrates was scrutinized by local indentation experiments as a function of surface functionalization, lipid composition, solvent content, indentation velocity, and pore radius. Solvent-containing nano black lipid membranes (nano-BLMs) as well as solvent-free pore-spanning bilayers were imaged by fluorescence and atomic force microscopy prior to force curve acquisition, which allows distinguishing between membrane-covered and uncovered pores. Force indentation curves on pore-spanning bilayers attached to functionalized hydrophobic porous silicon substrates reveal a predominately linear response that is mainly attributed to prestress in the membranes. This is in agreement with the observation that indentation leads to membrane lysis well below 5% area dilatation. However, membrane bending and lateral tension dominate over prestress and stretching if solvent-free supported membranes obtained from spreading giant liposomes on hydrophilic porous silicon are indented. An elastic regime diagram is presented that readily allows determining the dominant contribution to the mechanical response upon indentation as a function of load and pore radius.

Mechanically interlocked calix[4]arene dimers display reversible bond breakage under force

The physics of nanoscopic systems is strongly governed by thermal fluctuations that produce significant deviations from the behaviour of large ensembles. Stretching experiments of single molecules offer a unique way to study fundamental theories of statistical mechanics, as recently shown for the unzipping of RNA hairpins. Here, we report a molecular design based on oligo calix[4]arene catenanes-calixarene dimers held together by 16 hydrogen bridges-in which loops within the molecules limit how far the calixarene nanocapsules can be separated. This mechanically locked structure tunes the energy landscape of dimers, thus permitting the reversible rupture and rejoining of the individual nanocapsules. Experimental evidence, supported by molecular dynamics simulations, reveals the presence of an intermediate state involving the concerted rupture of the 16 hydrogen bridges. Stochastic modelling using a three-well potential under external load allows reconstruction of the energy landscape.

Helical Nanofibers of Self-Assembled Bipolar Phospholipids as Template for Gold Nanoparticles

Bipolar phospholipids (bolalipids) represent an exciting class of amphiphilic molecules as they self-assemble in water to distinct structures of nanoscopic dimensions. Reported here are structural details of helical nanofibers, composed of achiral, symmetrical single-chain bolalipids with phosphocholine headgroups. These nanofibers are used as template for the fixation of gold nanoparticles (AuNPs) without prior functionalization. This realization of a metal array on bolalipid nanofibers is one of the rare examples of one-dimensional AuNP arrangements in solution. The loading and the heat of binding of AuNPs are determined applying transmission electron microscopy and isothermal titration calorimetry.

Benefits and Limitations of Porous Substrates as Biosensors for Protein Adsorption

Porous substrates have gained widespread interest for biosensor applications based on molecular recognition. Thus, there is a great demand to systematically investigate the parameters that limit the transport of molecules toward and within the porous matrix as a function of pore geometry. Finite element simulations (FES) and time-resolved optical waveguide spectroscopy (OWS) experiments were used to systematically study the transport of molecules and their binding on the inner surface of a porous material. OWS allowed us to measure the kinetics of protein adsorption within porous anodic aluminum oxide membranes composed of parallel-aligned, cylindrical pores with pore radii of 10-40 nm and pore depths of 0.8-9.6 μm. FES showed that protein adsorption on the inner surface of a porous matrix is almost exclusively governed by the flux into the pores. The pore-interior surface nearly acts as a perfect sink for the macromolecules. Neither diffusion within the pores nor adsorption on the surface are rate limiting steps, except for very low rate constants of adsorption. While adsorption on the pore walls is mainly governed by the stationary flux into the pores, desorption from the inner pore walls involves the rate constants of desorption and adsorption, essentially representing the protein-surface interaction potential. FES captured the essential features of the OWS experiments such as the initial linear slopes of the adsorption kinetics, which are inversely proportional to the pore depth and linearly proportional to protein concentration. We show that protein adsorption kinetics allows for an accurate determination of protein concentration, while desorption kinetics could be used to capture the interaction potential of the macromolecules with the pore walls.

Elasticity Mapping of Pore‐Suspending Native Cell Membranes

The mechanics of cellular membranes are governed by a non-equilibrium composite framework consisting of the semiflexible filamentous cytoskeleton and extracellular matrix proteins linked to the lipid bilayer. While elasticity information of plasma membranes has mainly been obtained from whole cell analysis, techniques that allow addressing local mechanical properties of cell membranes are desirable to learn how their lipid and protein composition is reflected in the elastic behavior on local length scales. Introduced here is an approach based on basolateral membranes of polar epithelial Madin-Darby canine kidney (MDCK) II cells, prepared on a highly ordered porous substrate that allows elastic mapping on a submicrometer-length scale. A strong correlation between the density of actin filaments and the measured membrane elasticity is found. Spatially resolved indentation experiments carried out with atomic force and fluorescence microscope permit relation of the supramolecular structure to the elasticity of cellular membranes. It is shown that the elastic response of the pore spanning cell membranes is governed by local bending modules rather than lateral tension.

Elastic properties of cells in the context of confluent cell monolayers: impact of tension and surface area regulation

Epithelial cells usually form a dense continuous cobblestone-like sheet that is frequently exposed to a variety of mechanical challenges encompassing osmotic stress and external forces. The response to external forces was investigated and the question of how individual polar epithelial cells organized in confluent monolayers respond to pharmaceutical stimuli targeting the key players of cellular mechanics was answered. In particular, we ask how epithelial cells respond to changes in cortical and membrane tension by surface area regulation if challenged by diverse chemical and mechanical cues. Here, a tension-based model is used that allows capturing the relevant modes of cell deformation. Together with independent measurements of membrane tension, cortical tension and excess surface area of confluent MDCK II cells it is possible to draw a mechanistic picture of how confluent cells respond to mechanical stimuli in general. Changes in tension are provoked by external stimuli directed towards the contractile actomyosin cortex (cytochalasin D, blebbistatin), and changes in the excess surface area are produced by cholesterol extraction (methyl-β-cyclodextrin) or inhibition of dynamin (dynasore). A combination of AFM-indentation experiments with membrane–tether pulling at the same position allowed us to simultaneously monitor changes in membrane tension, cortical tension and excess surface area. Generally, we observed that removing or producing excess surface area of the plasma membrane readily adjusts membrane tension that is pivotal for the mechanical response of confluent cells. We found that isolated apical membranes from confluent MDCK II monolayers display similar mechanical properties as the apical side of living MDCK II cells in a confluent monolayer confirming that membrane mechanics in conjunction with cytoskeletal adhesion dominates the elastic response of confluent epithelial cells at large strain.

Biomimetic functionalization of porous substrates: towards model systems for cellular membranes

In this review, membrane-functionalized pore arrays and pore-spanning membranes are revisited as versatile tools in biophysical research. Pore-spanning membranes can be generated on different pore arrays such as porous alumina and porous silicon substrates using appropriate surface functionalization strategies. Their mechanical properties can be readily determined by force indentation experiments revealing that the lateral tension of the membranes is strongly governed by the surface modification of the pore rims. Pore-spanning membranes separate two aqueous compartments, which makes them well-suited to study transport processes across these membranes and entrap molecules into the underlying atto- to pico-liter sized compartments. Besides pore-spanning membranes, membranes covering the entire porous area provide a large surface area for molecular recognition events making this system well-suited for biosensor applications and as an extraction unit for protein purification.

Crosstalk of cardiomyocytes and fibroblasts in co-cultures

Electromechanical function of cardiac muscle depends critically on the crosstalk of myocytes with non-myocytes. Upon cardiac fibrosis, fibroblasts translocate into infarcted necrotic tissue and alter their communication capabilities. In the present in vitro study, we determined a multiple parameter space relevant for fibrotic cardiac tissue development comprising the following essential processes: (i) adhesion to substrates with varying elasticity, (ii) dynamics of contractile function, and (iii) electromechanical connectivity. By combining electric cell-substrate impedance sensing (ECIS) with conventional optical microscopy, we could measure the impact of fibroblast–cardiomyocyte ratio on the aforementioned parameters in a non-invasive fashion. Adhesion to electrodes was quantified via spreading rates derived from impedance changes, period analysis allowed us to measure contraction dynamics and modulations of the barrier resistance served as a measure of connectivity. In summary, we claim that: (i) a preferred window for substrate elasticity around 7 kPa for low fibroblast content exists, which is shifted to stiffer substrates with increasing fibroblast fractions. (ii) Beat frequency decreases nonlinearly with increasing fraction of fibroblasts, while (iii) the intercellular resistance increases with a maximal functional connectivity at 75% fibroblasts. For the first time, cardiac cell–cell junction density-dependent connectivity in co-cultures of cardiomyocytes and fibroblasts was quantified using ECIS.

Phosphatidylinositol 4,5-Bisphosphate Alters the Number of Attachment Sites between Ezrin and Actin Filaments A COLLOIDAL PROBE STUDY

Background: Ezrin can establish a dynamic linkage between plasma membrane and cytoskeleton. Results: The individual bond strength between ezrin and F-actin is small, but the number of attachment sites is significantly altered by phosphatidylinositol 4,5-bisphosphate (PIP2). Conclusion: PIP2 activates ezrin to establish multiple weak ezrin/F-actin interactions. Significance: Plasma membrane tension is maintained by ezrin/F-actin interactions. Direct linkage between the plasma membrane and the actin cytoskeleton is controlled by the protein ezrin, a member of the ezrin-radixin-moesin protein family. To function as a membrane-cytoskeleton linker, ezrin needs to be activated in a process that involves binding of ezrin to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphorylation of a conserved threonine residue. Here, we used colloidal probe microscopy to quantitatively analyze the interaction between ezrin and F-actin as a function of these activating factors. We show that the measured individual unbinding forces between ezrin and F-actin are independent of the activating parameters, in the range of approximately 50 piconewtons. However, the cumulative adhesion energy greatly increases in the presence of PIP2 demonstrating that a larger number of bonds between ezrin and F-actin has formed. In contrast, the phosphorylation state, represented by phosphor-mimetic mutants of ezrin, only plays a minor role in the activation process. These results are in line with in vivo experiments demonstrating that an increase in PIP2 concentration recruits more ezrin to the apical plasma membrane of polarized cells and significantly increases the membrane tension serving as a measure of the adhesion sites between the plasma membrane and the F-actin network.