Scanning ion conductance microscopy reveals how a functional renal epithelial monolayer maintains its integrity.

BACKGROUND: To function as a transport barrier a renal tubule epithelial monolayer needs to maintain its integrity when, sudden hypertonic stress causes cell shrinkage, new cells are added, or cells in the monolayer die. However, the mechanism used to achieve this is largely unknown. Scanning ion conductance microscopy (SICM) has been shown to be suitable for imaging the surface of live renal cells with high topographic resolution, and can be used to elucidate how a functional renal epithelial monolayer maintains its integrity. METHODS: SICM was used for high spatial resolution topographic imaging of Xenopuslaevis renal epithelial A6 cells cultured on membrane filter inserts. RESULTS: The SICM images of A6 cells showed that the epithelial monolayer maintains its integrity under hypertonic stress, and during cell division and death. Sequential SICM topographic images revealed detailed structural changes and their time course for these protective processes, which involve highly cooperative cell movement. Some "balloon-like" structures were observed at susceptible tight junction regions, which were proposed to help cell maintaining the monolayer permeability integrity. CONCLUSION: SICM is a powerful tool for research on living renal epithelial cells, and has been used to elucidate how a functional epithelial monolayer maintains its integrity. Using this technique we have observed that during hypertonic stress and regeneration, an organized sequence of events protect the loss of integrity of monolayer so that tight junctions and cell-cell contact are maintained and disruption to the function of whole monolayer is prevented.

The use of scanning ion conductance microscopy to image A6 cells.

BACKGROUND: Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. METHOD: Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. RESULTS: This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 microm(-2) for extended cells to 2.5 microm(2) for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. CONCLUSIONS: Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells.

Esmolol is antiarrhythmic in doxorubicin-induced arrhythmia in cultured cardiomyocytes - determination by novel rapid cardiomyocyte assay.

Cardiac toxicity is an uncommon but potentially serious complication of cancer therapy, especially with anthracyclines. One of the most effective anticancer drugs is doxorubicin, but its value is limited by the risk of developing cardiomyopathy and ventricular arrhythmia. When applied to a network of periodically contracting cardiomyocytes in culture, doxorubicin induces rhythm disturbances. Using a novel rapid assay based on non-invasive ion-conductance microscopy we show that the beta-antagonist esmolol can restore rhythm in doxorubicin-treated cultures of cardiomyocytes. Moreover, esmolol pre-treatment can protect the culture from doxorubicin-induced arrhythmia.

Dynamic assembly of surface structures in living cells.

Although the dynamics of cell membranes and associated structures is vital for cell function, little is known due to lack of suitable methods. We found, using scanning ion conductance microscopy, that microvilli, membrane projections supported by internal actin bundles, undergo a life cycle: fast height-dependent growth, relatively short steady state, and slow height-independent retraction. The microvilli can aggregate into relatively stable structures where the steady state is extended. We suggest that the intrinsic dynamics of microvilli, combined with their ability to make stable structures, allows them to act as elementary "building blocks" for the assembly of specialized structures on the cell surface.

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

High-resolution scanning patch-clamp: new insights into cell function.

Cell specialization is often governed by the spatial distribution of ion channels and receptors on the cell surface. So far, little is known about functional ion channel localization. This is due to a lack of satisfactory methods for investigating ion channels in an intact cell and simultaneously determining the channels' positions accurately. We have developed a novel high-resolution scanning patch-clamp technique that enables the study of ion channels, not only in small cells, such as sperm, but in submicrometer cellular structures, such as epithelial microvilli, fine neuronal dendrites, and, particularly, T-tubule openings of cardiac myocytes. In cardiac myocytes, as in most excitable cells, action potential propagation depends essentially on the properties of ion channels that are functionally and spatially coupled. We found that the L-type calcium and chloride channels are distributed and colocalized in the region of T-tubule openings, but not in other regions of the myocyte. In addition, chloride channels were found in narrowly defined regions of Z-grooves. This finding suggests a new synergism between these types of channels that may be relevant for action potential propagation along the T-tubule system and excitation-contraction coupling.

Stochastic resonance in thermally activated reactions: Application to biological ion channels.

At the molecular level many thermally activated reactions can be viewed as Poisson trains of events whose instantaneous rates are defined by the reaction activation barrier height and an effective collision frequency. When the barrier height depends on an external parameter, variation in this parameter induces variation in the event rate. Extending our previous work, we offer a detailed theoretical analysis of signal transduction properties of these reactions considering the external parameter as an input signal and the train of resulting events as an output signal. The addition of noise to the system input facilitates signal transduction in two ways. First, for a linear relationship between the barrier height and the external parameter the output signal power grows exponentially with the mean square fluctuation of the noise. Second, for noise of a sufficiently high bandwidth, its addition increases output signal quality measured as the signal-to-noise ratio (SNR). The output SNR reaches a maximum at optimal noise intensity defined by the reaction sensitivity to the external parameter, reaction initial rate, and the noise bandwidth. We apply this theory to ion channels of excitable biological membranes. Based on classical results of Hodgkin and Huxley we show that open/closed transitions of voltage-gated ion channels can be treated as thermally activated reactions whose activation barriers change linearly with applied transmembrane voltage. As an experimental example we discuss our recent results obtained with polypeptide alamethicin incorporated into planar lipid bilayers.(c) 1998 American Institute of Physics.