Cell-based screening for membranal and cytoplasmatic markers using dielectric spectroscopy.

Dielectric spectroscopy (DS) of living biological cells is based on the analysis of the complex dielectric permittivity of cells suspended in a physiological medium. It provides knowledge on the polarization-relaxation response of cells to external electric field as function of the excitation frequency. This response is strongly affected by both structural and molecular properties of cells and therefore, can reveal rare insights on cell physiology and behaviour. This study demonstrates the mapping potential of DS after cytoplasmatic and membranal markers for cell-based screening analysis. The effect of membrane permittivity and cytoplasm conductivity was examined using tagged MBA and MDCK cell lines respectively. Comparing the permittivity spectra of tagged and native cell lines reveals clear differences between the analyzed suspensions. In addition, differences on the matching dielectric properties of cells were obtained. Those findings support the high distinction resolution and sensitivity of DS after fine molecular and cellular changes, and hence, highlight the high potential of DS as non invasive screening tool in cell biology research.

Site localization of membrane-bound proteins on whole cell level using atomic force microscopy.

This study presents molecular recognition method, which is based on specific force measurements between modified AFM (atomic force microscopy) tip and mammalian cell. The presented method allows recognition of specific cell surface proteins and receptor sites by nanometer accuracy level. Here we demonstrate specific recognition of membrane-bound Osteopontin (OPN) sites on preosteogenic cell membrane. By merging specific force detection map of the proteins and topography image of the cell, we create a new image (recognition image), which demonstrates the exact locations of the proteins relative to the cell membrane. The recognition results indicate the strong affinity between the modified tip and the target molecules, therefore, it enables the use of an AFM as a remarkable nanoscale tracking tool on the whole cell level.

Mechano-molecular transduction: Putting the pieces together.

When subjected to extracellular mechanical loads, cells express a variety of responses ranging from differentiation to apoptosis. The transducing mechanism between the physical stimulus and the molecular response is known as mechanotransduction. In this study, a mechano-molecular model is established that facilitates the integration of cellular mechanics with molecular signaling. It reveals a unique coupling mechanism between the physical stimuli, the extracellular mechanical properties, and the Rho signaling pathway. Myosin activation is shown to be correlated with the rate of intracellular activity and found to vary extremely for different extracellular rigidities. These findings can explain and interpret the fundamental observations of mechanotransduction.

A biomimetic gelatin-based platform elicits a pro-differentiation effect on podocytes through mechanotransduction.

Using a gelatin microbial transglutaminase (gelatin-mTG) cell culture platform tuned to exhibit stiffness spanning that of healthy and diseased glomeruli, we demonstrate that kidney podocytes show marked stiffness sensitivity. Podocyte-specific markers that are critical in the formation of the renal filtration barrier are found to be regulated in association with stiffness-mediated cellular behaviors. While podocytes typically de-differentiate in culture and show diminished physiological function in nephropathies characterized by altered tissue stiffness, we show that gelatin-mTG substrates with Young's modulus near that of healthy glomeruli elicit a pro-differentiation and maturation response in podocytes better than substrates either softer or stiffer. The pro-differentiation phenotype is characterized by upregulation of gene and protein expression associated with podocyte function, which is observed for podocytes cultured on gelatin-mTG gels of physiological stiffness independent of extracellular matrix coating type and density. Signaling pathways involved in stiffness-mediated podocyte behaviors are identified, revealing the interdependence of podocyte mechanotransduction and maintenance of their physiological function. This study also highlights the utility of the gelatin-mTG platform as an in vitro system with tunable stiffness over a range relevant for recapitulating mechanical properties of soft tissues, suggesting its potential impact on a wide range of research in cellular biophysics.

Cell shape information is transduced through tension-independent mechanisms.

The shape of a cell within tissues can represent the history of chemical and physical signals that it encounters, but can information from cell shape regulate cellular phenotype independently? Using optimal control theory to constrain reaction-diffusion schemes that are dependent on different surface-to-volume relationships, we find that information from cell shape can be resolved from mechanical signals. We used microfabricated 3-D biomimetic chips to validate predictions that shape-sensing occurs in a tension-independent manner through integrin ß3 signaling pathway in human kidney podocytes and smooth muscle cells. Differential proteomics and functional ablation assays indicate that integrin ß3 is critical in transduction of shape signals through ezrin-radixin-moesin (ERM) family. We used experimentally determined diffusion coefficients and experimentally validated simulations to show that shape sensing is an emergent cellular property enabled by multiple molecular characteristics of integrin ß3. We conclude that 3-D cell shape information, transduced through tension-independent mechanisms, can regulate phenotype.

Dielectric screening of early differentiation patterns in mesenchymal stem cells induced by steroid hormones.

In the framework of this study, target identification and localization of differentiation patterns by means of dielectric spectroscopy is presented. Here, a primary pre-osteoblastic bone marrow-derived MBA-15 cellular system was used to study the variations in the dielectric properties of mesenchymal stem cells while exposed to differentiation regulators. Using the fundamentals of mixed dielectric theories combined with finite numerical tools, the permittivity spectra of MBA-15 cell suspensions have been uniquely analyzed after being activated by steroid hormones to express osteogenic phenotypes. Following the spectral analysis, significant variations were revealed in the dielectric properties of the activated cells in comparison to the untreated populations. Based on the differentiation patterns of MBA-15, the electrical modifications were found to be highly correlated with the activation of specific cellular mechanisms which directly react to the hormonal inductions. In addition, by describing the dielectric dispersion in terms of transfer functions, it is shown that the spectral perturbations are well adapted to variations in the electrical characteristics of the cells. The reported findings vastly emphasize the tight correlation between the cellular and electrical state of the differentiated cells. It therefore emphasizes the vast abilities of impedance-based techniques as potential screening tools for stem cell analysis.

Theoretical examination of aggregation effect on the dielectric characteristics of spherical cellular suspension.

Dielectric dispersion analysis of cellular suspension is generally based on the analogy to equivalent periodic material made up of identical inclusions. However, under true physiological conditions, when coupling and aggregation events usually occur, this analogy can introduce severe errors when attempting to probe the dielectric characteristics of the suspended fraction. In the framework of this study, a theoretical examination of the effect of aggregation on the dielectric characteristics of spherical cellular suspension is presented. Here, small clusters of coupled and fused (gap connected) shelled spheres were used to imitate the presence of aggregates when suspended in a homogenous suspension of spherical cells. The permittivity spectra of the aggregate-cell mixtures were numerically calculated by applying computational solution of complex potential problem using 3D Boundary Element Method. The dispersion characteristics of the mixtures have been determined as function of both aggregates shape and concentration. Those reveal significant deviations in comparison to the characteristics of homogenous cellular suspension. Quantitative analyses of the induced fields and transmembrane potential gradients of the interacted cells suggest that those deviations are mainly induced due to changes occur on the polarization state of the membranes.

Dielectric dispersion of suspended cells using 3D reconstructed morphology model.

In the framework of this study, novel method for dispersion analysis of cellular suspensions is presented. The method is fundamentally based on the ability to reconstruct the exact 3D morphology of a given cell with resolution accuracy of few nanometers using AFM imaging. By applying a reverse engineering approach, the morphology of the cell is constructed based on a set of measured spatial points that describes its geometry. The permittivity spectrum of the reconstructed cell is then directly derived based on computational solution of complex potential problem using 3D Boundary Element Method. The applicability of the method is demonstrated both theoretically and experimentally with tight comparison to the well known shell models. This comparison reveals significant deviations between the models, and hence, emphasises the vast effect of morphology in dispersion analysis of cellular suspensions.

The effect of irregularity on the dielectric dispersion characteristics of spherical cellular suspension.

The dielectric dispersion characteristics of cellular suspensions are fundamentally determined based on the analogy to composite dielectric materials when periodically and discrete arrangement of cells is assumed. However, under native physiological conditions, when flocculation and clamping events usually occur, those assumptions are usually not valid. In the framework of this study, an examination of irregularity effect on the dispersion characteristics of spherical cellular suspensions is presented. Here, the permittivity spectra of the suspensions have been determined by both measurements of living K562 cell suspensions and finite numerical simulations. Based on the measured and simulated spectra, the dispersion characteristics of the suspensions, for several destinies and arrangements of cells, have been quantitatively analyzed using the Havriliak-Negami empirical formula. Generally, a strong correlation between the low dispersion characteristics was observed as the concentration and density of the cells was increased. In addition, all characteristics found to be significantly deviated in comparison to the characteristics of a periodically arrayed suspension. However, when low-dense arrangement was assumed, the correlation found to be much lower when all characteristics found to be less perturbated. Based on a simple model of interacting cells, it is suggested that those deviations are related to intercellular interactions between adjacent cells.

Modeling and measurement of a whole-cell bioluminescent biosensor based on a single photon avalanche diode.

Whole-cell biosensors are potential candidates for on-line and in situ environmental monitoring. In this work we present a new design of a whole-cell bioluminescence biosensor for water toxicity detection, based on genetically engineered Escherichia coli bacteria, carrying a recA::luxCDABE promoter-reporter fusion. Sensitive optical detection is achieved using a single photon avalanche photodiode (SPAD) working in the Geiger mode. The present work describes a simple mathematical model for the kinetic process of the bioluminescence based SOS toxin response of E. coli bacteria. We find that initially the bioluminescence signal depends on the time square and we show that the spectral intensity of the bioluminescence signal is inverse proportional to the frequency. We get excellent agreement between the theoretical model and the measured light signal. Furthermore, we present experimental results of the bioluminescent signal measurement using a SPAD and a photomultiplier, and demonstrate improvement of the measurement by applying a matched digital filter. Low intensity bioluminescence signals were measured after the whole-cell sensors were exposed to various toxicant concentrations (5, 15 and 20ppm).