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.

From defined reactive diblock copolymers to functional HPMA-based self-assembled nanoaggregates.

This paper describes the synthesis of functional amphiphilic poly( N-(2-hydroxypropyl) methacrylamide)-block-poly(lauryl methacrylate) copolymers by RAFT polymerization via the intermediate step of activated ester block copolymers (pentafluoro-phenyl methacrylate). Block copolymers with molecular weights from 12000-28000 g/mol and PDIs of about 1.2 have been obtained. The amphiphilic diblock copolymers form stable super structures (nanoaggregates) by self-organization in aqueous solution. The diameters of these particles are between 100 and 200 nm and depend directly on the molecular weight of the block copolymer. Furthermore, we investigated the impact of these nanoaggregates on cell viability and on the motility of adherent cells. Cytotoxicity was investigated by the MTS test and the fluctuation in cell shape was monitored employing ECIS (electrical cell-substrate impedance sensing). In these investigations, the formed particles are not cell toxic up to a concentration of 2 mg/mL. Thus, our polymeric particles offer potential as polymer therapeutics.

The quartz crystal microbalance as a novel means to study cell-substrate interactions in situ.

The quartz crystal microbalance (QCM) was first introduced as a mass sensor in gas phase and in vacuum. Since oscillator circuits capable of exciting shear vibrations of quartz resonators under liquid loading have been developed, the QCM became accepted as a new, powerful technique to follow adsorption processes at solid-liquid interfaces in chemical and biological research. Lately, the QCM technique has attracted considerable interest as a novel means to monitor cell-substrate interactions of mammalian cells in vitro. Because the establishment and modulation of cell-substrate contacts is important for many physiological processes, and potent techniques to measure these interactions noninvasively are rare, the present review highlights applications of the QCM technique in this field. The suitability of the QCM device to monitor attachment and spreading of mammalian cells in real time has been well established. The QCM response is dependent on the individual cell type that is examined. In order to identify the sources for these cell-type-specific results of QCM readings, and to understand the information content of the signal, attempts have been made to decompose the overall QCM response into subcellular contributions. The aforementioned subjects, together with a condensed introduction into the QCM technology, are included in this article.

Scanning force microscopy of artificial membranes.

Visualization of biological membranes by scanning force microscopy (SFM) has tremendously improved the current understanding of protein-lipid interactions under physiological conditions. SFM is the only tool to directly image processes on surfaces in aqueous solution at molecular resolution. Besides being a supportive means to confirm results on lipid phases and domains obtained from fluorescence spectroscopy, calorimetry, and X-ray crystallography, SFM has contributed distinct aspects on the formation of 2D crystals of various membrane-confined proteins and morphological changes of membranes due to the interaction of peptides and proteins. This review will focus on recent results in SFM imaging of artificial solid-supported membranes, their phase behavior as a response to the environment, and changes in membrane morphology induced by the partitioning of peptides and proteins.

Energy landscapes of ligand-receptor couples probed by dynamic force spectroscopy.

Playing a dominant role in many biochemical processes are the dynamic properties of molecular linkages; examples include cell adhesion, enzyme-catalyzed reactions, and molecular recognition by antibodies. Dynamic force spectroscopy, namely separating molecular bonds under external force ramps has rapidly become a powerful tool to study the rugged energy landscape of noncovalent ligand-receptor bonds. The picture shows a surface and tip-bound pair being pulled apart and the derived potential energy diagram.

Piezoelectric Mass-Sensing Devices as Biosensors-An Alternative to Optical Biosensors?

In the early days of electronic communication-as a result of the limited number of quartz resonators available-frequency adjustment was accomplished by a pencil mark depositing a foreign mass layer on the crystal. In 1959, Sauerbrey showed that the shift in resonance frequency of thickness-shear-mode resonators is proportional to the deposited mass. This was the starting point for the development of a new generation of piezoelectric mass-sensitive devices. However, it was the development of new powerful oscillator circuits that were capable of operating thickness shear mode resonators in fluids that enabled this technique to be introduced into bioanalytic applications. In the last decade adsorption of biomolecules on functionalized surfaces turned in to one of the paramount applications of piezoelectric transducers. These applications include the study of the interaction of DNA and RNA with complementary strands, specific recognition of protein ligands by immobilized receptors, the detection of virus capsids, bacteria, mammalian cells, and last but not least the development of complete immunosensors. Piezoelectric transducers allow a label-free detection of molecules; they are more than mere mass sensors since the sensor response is also influenced by interfacial phenomena, viscoelastic properties of the adhered biomaterial, surface charges of adsorbed molecules, and surface roughness. These new insights have recently been used to investigate the adhesion of cells, liposomes, and proteins onto surfaces, thus allowing the determination of the morphological changes of cells as a response to pharmacological substances and changes in the water content of biopolymers without employing labor-intense techniques. However, the future will show whether the quartz-crystal microbalance will assert itself against established label-free sensor devices such as surface plasmon resonance spectroscopy and interferometry.

Force Spectroscopy of Molecular Systems-Single Molecule Spectroscopy of Polymers and Biomolecules.

How do molecules interact with each other? What happens if a neurotransmitter binds to a ligand-operated ion channel? How do antibodies recognize their antigens? Molecular recognition events play a pivotal role in nature: in enzymatic catalysis and during the replication and transcription of the genome; it is also important for the cohesion of cellular structures and in numerous metabolic reactions that molecules interact with each other in a specific manner. Conventional methods such as calorimetry provide very precise values of binding enthalpies; these are, however, average values obtained from a large ensemble of molecules without knowledge of the dynamics of the molecular recognition event. Which forces occur when a single molecular couple meets and forms a bond? Since the development of the scanning force microscope and force spectroscopy a couple of years ago, tools have now become available for measuring the forces between interfaces with high precision-starting from colloidal forces to the interaction of single molecules. The manipulation of individual molecules using force spectroscopy is also possible. In this way, the mechanical properties on a molecular scale are measurable. The study of single molecules is not an exclusive domain of force spectroscopy; it can also be performed with a surface force apparatus, laser tweezers, or the micropipette technique. Regardless of these techniques, force spectroscopy has been proven as an extraordinary versatile tool. The intention of this review article is to present a critical evaluation of the actual development of static force spectroscopy. The article mainly focuses on experiments dealing with inter- and intramolecular forces-starting with "simple" electrostatic forces, then ligand-receptor systems, and finally the stretching of individual molecules.

Formation of three-dimensional protein-lipid aggregates in monolayer films induced by surfactant protein B.

This study focuses on the structural organization of surfactant protein B (SP-B) containing lipid monolayers. The artificial system is composed of the saturated phospholipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in a molar ratio of 4:1 with 0.2 mol% SP-B. The different "squeeze-out" structures of SP-B were visualized by scanning probe microscopy and compared with structures formed by SP-C. Particularly, the morphology and material properties of mixed monolayers containing 0.2 mol% SP-B in a wide pressure range of 10 to 54 mN/m were investigated revealing that filamentous domain boundaries occur at intermediate surface pressure (15-30 mN/m), while disc-like protrusions prevail at elevated pressure (50-54 mN/m). In contrast, SP-C containing lipid monolayers exhibit large flat protrusions composed of stacked bilayers in the plateau region (app. 52 mN/m) of the pressure-area isotherm. By using different scanning probe techniques (lateral force microscopy, force modulation, phase imaging) it was shown that SP-B is dissolved in the liquid expanded rather than in the liquid condensed phase of the monolayer. Although artificial, the investigation of this system contributes to further understanding of the function of lung surfactant in the alveolus.

Analysis of the composite response of shear wave resonators to the attachment of mammalian cells.

The suitability of the quartz crystal microbalance (QCM) technique for monitoring the attachment and spreading of mammalian cells has recently been established. Different cell species were shown to generate an individual response of the QCM when they make contact with the resonator surface. Little is known, however, about the underlying mechanisms that determine the QCM signal for a particular cell type. Here we describe our results for different experimental approaches designed to probe the particular contributions of various subcellular compartments to the overall QCM signal. Using AC impedance analysis in a frequency range that closely embraces the resonators' fundamental frequency, we have explored the signal contribution of the extracellular matrix, the actin cytoskeleton, the medium that overlays the cell layer, as well as the liquid compartment that is known to exist between the basal plasma membrane and the culture substrate. Results indicate that the QCM technique is only sensitive to those parts of the cellular body that are involved in cell substrate adhesion and are therefore close to the resonator surface. Because of its noninvasive nature, sensitivity, and time resolution, the QCM is a powerful means of quantitatively studying various aspects of cell-substrate interactions.

Membrane partitioning of the cleavage peptide in flock house virus.

Membrane translocation of the ssRNA genome of nodaviruses has been proposed to be mediated by direct lipid-protein interactions between a postassembly autocatalytic cleavage product from the capsomere and the target membrane. We have recently shown that the 21-residue Met-->Nle variant of the N-terminal helical domain (denoted gamma(1)) of the cleavage peptide in flock house nodavirus increases membrane permeability to hydrophilic solutes and can alter both membrane structure and function, suggesting the possibility of peptide-triggered disruption of the endosomal membrane as a prelude to viral uncoating in the host cytoplasm. Elucidation of partitioning energetics would allow an assessment of the likelihood of this mechanism. We report herein complete thermodynamic characterization of the partitioning of gamma(1) to phospholipids by lipid-peptide titrations following changes in ellipticity, fluorescence signature, or calorimetric response. These experiments revealed a partitioning energy comparable to natural membrane-active peptide toxins, suggesting that the proposed mechanism may be possible. Additionally, a novel switch in the balance of partitioning forces was found: when the lipid headgroup was changed from zwitterionic to negatively charged, membrane association of the peptide became completely entropy-driven.

Specific adhesion of vesicles monitored by scanning force microscopy and quartz crystal microbalance.

The specific adhesion of unilamellar vesicles with an average diameter of 100 nm on functionalized surfaces mediated by molecular recognition was investigated in detail. Two complementary techniques, scanning force microscopy (SFM) and quartz crystal microbalance (QCM) were used to study adhesion of liposomes consisting of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine and varying concentrations of N-((6-biotinoyl)amino)hexanoyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-X-DHPE). Monitoring the adhesion of the receptor-doped vesicles to avidin-coated gold surfaces by QCM (f(0) = 5 MHz) revealed an increased shift in resonance frequency with increasing biotin concentration up to 10 mol% biotin-X-DHPE. To address the question of how the morphology of the liposomes changes upon adhesion and how that contributes to the resonator's frequency response, we performed a detailed analysis of the liposome morphology by SFM. We found that, with increasing biotin-concentration, the height of the liposomes decreases considerably up to the point where vesicle rupture occurs. Thus, we conclude that the unexpected high frequency shifts of the quartz crystal (>500 Hz) can be attributed to a firm attachment of the spread bilayers, in which the number of contacts is responsible for the signal. These findings are compared with one of our recent studies on cell adhesion monitored by QCM.

Micropatterned solid-supported membranes formed by micromolding in capillaries.

The formation of individually addressable micropatterned solid-supported lipid bilayers has been accomplished by means of micromolding in capillaries. Small unilamellar vesicles were spread on glass slides to form planar supported membranes along microscopic capillaries molded as trenches into a polydimethylsiloxane (PDMS) elastomer. PDMS provides an elastic and transparent carrier for microcapillaries molded from silicon wafers displaying the desired inverse trenches. The so-called master structure has been conventionally etched into silicon by photolithography. The cured PDMS elastomer was briefly exposed to an oxygen plasma, rendering the surface hydrophilic, and subsequently attached to a glass surface in order to form hydrophilic capillaries equipped with flow-promoting pads on either side. One flowpad acts as a reservoir to be filled with the vesicle suspension, while the other one serves as a collector to ensure a sufficient capillary flow to cover the substrate completely. Formation of planar lipid bilayers on the glass slide along the capillaries was followed by imaging the flow and spreading of fluorescently labeled DMPC liposomes with confocal laser scanning microscopy. By means of scanning force microscopy in aqueous solution the formed lipid structures were identified and the height of the lipid bilayers was accurately determined. With both techniques, it was shown that the patterned bilayers remain separated and persist for several hours on the substrate in aqueous solution.

A highly membrane-active peptide in Flock House virus: implications for the mechanism of nodavirus infection.

BACKGROUND: Nodaviruses are among the simplest animal viruses, and are therefore attractive systems for deconvoluting core viral processes such as assembly, infection and uncoating. Membrane translocation of the single-stranded RNA genome of nodaviruses has been proposed to be mediated by direct lipid-protein interactions between a post-assembly autocatalytic cleavage product from the capsomere and the target membrane. To probe the validity of this hypothesis, we have synthesized a 21-residue Met-->Nle (norleucine) variant of the amino-terminal helical domain (denoted here as gamma1) of the cleavage peptide in Flock House nodavirus (FHV) and studied its ability to alter membrane structure and function. RESULTS: The synthetic peptide gamma1 increases membrane permeability to hydrophilic solutes, as judged by fluorescence experiments with liposome-encapsulated dyes and ion-conductance measurements. Furthermore, peptide orientation and location within lipid bilayers was determined using tryptophan-fluorescence-quenching experiments and attenuated total reflectance infrared spectroscopy. CONCLUSIONS: The helical domain of the FHV cleavage product partitions spontaneously into lipid bilayers and increases membrane permeability, consistent with the postulated mechanism for viral genome translocation. The existence of a membrane-binding domain in the FHV cleavage sequence suggests peptide-triggered disruption of the endosomal membrane as a prelude to viral uncoating in the host cytoplasm. A model for this interaction is proposed.

An animal virus-derived peptide switches membrane morphology: possible relevance to nodaviral transfection processes.

The N-terminal domain of the capsid protein cleavage product of the flock house virus (FHV) consists of 21 residues and forms an amphipathic alpha-helix, which is thought to play a crucial role in permeabilizing biological membranes for RNA translocation in the host cell. We have found that the Met --> Nle variant of this domain (denoted here as gamma1) efficiently induces the formation of the interdigitated gel phase (LbetaI) of 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers. In situ scanning force microscopy of solid supported bilayers and fluorescence spectroscopy of peptide-treated DPPC vesicles provide evidence for the formation of acyl chain interdigitated lipid domains. It could be shown by fluorescence spectroscopy that the peptide inserts in the DPPC matrix above the main transition temperature of the lipid, while the formation of domains with decreased thickness occurs after the sample is cooled to 25 degrees C. The orientation and secondary structure of the peptide in lipid bilayers were investigated using attenuated total reflectance infrared (ATR-IR) and circular dichroism (CD) spectroscopy. These results enabled us to formulate a mechanistic model for the peptide-mediated induction of interdigitation in DPPC bilayers. Moreover, the membrane activity of gamma1 with gel phase lipids established in this study may have further implications for the infection strategy adopted by simple RNA viruses.

Cell adhesion monitoring using a quartz crystal microbalance: comparative analysis of different mammalian cell lines.

The quartz crystal microbalance (QCM) has been widely accepted as a sensitive technique to follow adsorption processes in gas as well as in liquid environments. However, there are only a few reports about the use of this technique to monitor the attachment and spreading of mammalian cells onto a solid support in culture. Using a QCM-setup we investigated the time course of cell attachment and spreading as a function of seeding density for three widespread and frequently used cell lines (MDCK strains I and II and Swiss 3T3-fibroblasts). Results were found to be in good agreement with the geometrical properties of the individual cell types. The shifts of the resonance frequency associated with confluent cell layers on top of the quartz resonators were found to be dependent on the cell species [MDCK-I: (320 +/- 20) Hz; MDCK-II: (530 +/- 25) Hz; 3T3: (240 +/- 15) Hz] reflecting their individual influence on the shear oscillation of the resonator. These findings are discussed with respect to the basic models of materials in contact with an oscillating quartz resonator. We furthermore showed by inhibition-assays using soluble RGD-related peptides, that only specific, integrin mediated cell adhesion is detected using this QCM approach, whereas the sole presence of the cellular body in close vicinity to the resonator surface is barely detectable.

Quantification of the Interaction between Charged Guest Molecules and Chemisorbed Monothiolated ß-Cyclodextrins.

The quantification of small molecules in aqueous solution by surface bound supramolecular host molecules is an important goal in the research field of chemo- and biosensor development. In this paper we present an attempt to quantify the interaction of different charged guest molecules with chemisorbed monothiolated ß-cyclodextrin monolayers by means of impedance spectroscopy in the presence of the redox couple [Fe(CN)(6)](3)(-)/[Fe(CN)(6)](4)(-). Self-assembled monolayers of mercaptopropane-N-mono-6-deoxy-ß-cyclodextrin amide (MPA-CD) on gold surfaces were formed with coverage of 99-100%. The inclusion of charged guest molecules was detected by monitoring the changes in the charge-transfer resistance, which is sensitive to the surface charge density in terms of repulsion or attraction of the redox active ions. Adsorption of positively charged 1-adamantanamino hydrochloride (1-ADHC) led to a considerable increase in the charge-transfer resistance, whereas the inclusion of both negatively charged 1-adamantanecarboxylic acid (1-ADC) and 2-(p-toluidinyl)naphthalene-6-sulfonate (2,6-TNS) caused a decrease. Applying the Frumkin correction to obtain the surface charge density and the Gouy-Chapman-Stern theory to account for the electrochemical double layer, we were able to quantify the binding of the charged guest molecules in terms of binding isotherms. The isotherms display a distinct two step adsorption process probably owing to the presence of two energetically different binding sites on the surface. Complete reversibility of the binding process of the guest molecules could be demonstrated by the addition of ß-cyclodextrin in solution, which allowed the reuse of the functionalized surfaces.

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes.

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides GM1, GM3, GD1a, GD1b, GT1b and asialo-GM1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for GM1 (Ka = 1.8.10(8)M-1), a lower one for asialo-GM1 (Ka = 1.0.10(7)M-1) and no affinity for GM3. The C-fragment of tetanus toxin binds to ganglioside GD1a, GD1b and GT1b containing membranes with similar affinity (Ka approximately 10(6)M-1), while no binding was observed with GM3. Pertussis toxin binds to membranes containing the ganglioside GD1a with a binding constant of Ka = 1.6.10(6)M-1, but only if large amounts (40 mol%) of GD1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to GD1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to GD1a.

Impedance and shear wave resonance analysis of ligand-receptor interactions at functionalized surfaces and of cell monolayers.

The present paper scrutinizes the application of impedance spectroscopy and quartz-crystal microbalance (QCM) measurements in the analysis of composite layers of receptor containing lipid bilayers, and their interaction with external ligands or pore-forming peptides. The formation of supramolecular structures and their analysis will be discussed. Impedance measurement allows one to follow the adsorption of proteins on artificial membranes. This method is even more suitable for quantifying changes in membrane conductivity induced by channel peptides incorporated into the lipid membrane. The QCM is another sophisticated method for analyzing ganglioside-lectin and ganglioside-toxin interactions. A critical comparison between both methods will be given. Moreover, we will demonstrate that the QCM method, especially in combination with impedance analysis, is a completely new approach for determining electrical and viscoelastic properties of epithelial and endothelial cell monolayers that form controlled barriers in vivo.

Impedance analysis of supported lipid bilayer membranes: a scrutiny of different preparation techniques.

One topic of this study is the comparison of different preparation techniques to build up solid supported lipid bilayers onto gold substrates. The deposited lipid bilayers were investigated by a.c. impedance spectroscopy. Three different strategies were applied: (1) The gold surface was initially covered with a chemisorbed monolayer of octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The second monolayer consisting of phospholipids was then deposited onto this hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) from lipid solution in n-decane/isobutanol, (iii) by the lipid/detergent dilution technique or (iv) by fusion of vesicles. (2) Charged molecules carrying thiol-anchors for attachment to the gold surface by chemisorption were used. Negatively charged surfaces of 3-mercaptopropionic acid were found to be excellent substrates that allow the attachment of planar lipid bilayers by applying positively charged dimethyldioctadecylammoniumbromide (DODAB) vesicles or negatively charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol vesicles in the presence of chelating Ca2+-ions. If positively charged first monolayers of mercaptoethylammoniumhydrochloride were used we were able to attach mixed 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol/1,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine vesicles to form planar lipid bilayers via electrostatic interaction. (3) Direct deposition of lipid bilayers is possible from vesicles containing 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). A critical amount of more than 50 mol% of DMPTE was found to be necessary to form a solid supported lipid bilayer. Bilayers obtained with these different preparation techniques were scrutinized with respect to their capacitances, kinetics of formation and their long-term stabilities by impedance spectroscopy. The second feature of this paper is the application of the supported bilayers to study ion transport through channel-forming peptides. We used a DODAB-bilayer for the reconstitution of gramicidin D channels. By circular dichroism measurements we verified that the peptide is in its channel conformation. The ion transport of Cs+-ions through the channels was recorded by impedance analysis.