Imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy.
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Bacillus↗
Biomedical subjects
Max J Lab
Publications and source records attributed to Max J Lab.
Mechanosensitive-mediated interaction, integration, and cardiac control.
This review covers aspects of the cardiac mechanotransduction field at different levels, and advocates the possibility that mechanoelectro-chemical transduction forms part of a network of mechanically linked integration in heart-mechanically mediated integration (MMI). It assembles evidence and observations in the literature to promote this hypothesis. Mechanical components can provide the bond between interactions at molecular, cellular, and macro levels to enable the integration. Stretch-activated channels (SACs) exist in the heart, but stresses and strains can affect other membrane channels or receptors. A cellular mechanical change can thus promote several ionic or downstream changes. Cell signal cascades have been implicated and can affect membrane electrophysiology. MMI could shape intracellular and downstream signals using the cytoskeleton and intracellular Ca(2+). MMI also spans other regulatory systems and processes such as the autonomic nervous system (ANS) and operates throughout the whole heart as an integrative system. Finally, supporting the hypothesis, if elements of the normal integration become deranged it contributes to cardiovascular disease and, potentially, lethal arrhythmia.
Animals↗
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.
Animals↗
Stretch-induced regional mechanoelectric dispersion and arrhythmia in the right ventricle of anesthetized lambs.
Regional mechanical and electrophysiological changes accompany most ventricular arrhythmias and, it has been suggested, by mechanoelectric feedback. We hypothesized that an intervention producing regional mechanical dispersion was associated with regional, proarrhythmic electrical dispersion and studied the regional mechanoelectric feedback in the right ventricle (RV) of anesthetized lambs. Ten lambs were deeply anesthetized, and their hearts were exposed. Three tripodal devices, each incorporating three monophasic action potential electrodes and an integrated strain-gauge system, were placed on the RV apex outflow and inflow regions. Measurements were made before, during, and after 10-s pulmonary arterial occlusion. Pulmonary arterial occlusion increased RV pressure and overall regional segment length. Length excursion became out of phase with RV pressure beats immediately after occlusion, and the strain patterns were different in the three regions at the peak of occlusion. The occlusion resulted in different alterations in regional monophasic action potential morphology, including reduction in monophasic action potential amplitude and duration by different amounts and early afterdepolarizations that were unevenly distributed in the monophasic action potential recordings. This was associated with dispersion of repolarization and recovery time. The combination of electromechanical events precipitated a variety of arrhythmias. Acute RV distension is proarrhythmic, possibly through a causal relationship among mechanically induced afterdepolarizations, dispersion (heterogeneity) of mechanical strain, and dispersion of electrical recovery. The relationship among the different wall motions, the dispersion of repolarization, and arrhythmia underscored mechanoelectric feedback as an important part of arrhythmogenesis in pulmonary embolism and commotio cordis.
Action Potentials↗
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.
Adrenergic beta-Antagonists↗
Right ventricular myocardial responses to chronic pulmonary regurgitation in lambs: disturbances of activation and conduction.
Patients after repair of tetralogy of Fallot are at increased risk of arrhythmic death. Clinical data suggest that pulmonary regurgitation predisposes to these arrhythmias, although the cellular electrophysiologic effects of pulmonary regurgitation are unknown. We induced pulmonary regurgitation in lambs, and 3 mo later, having quantified the pulmonary regurgitant (PR) fraction, studied right ventricular mechanical and electrophysiologic properties in vivo and in vitro. The PR fraction was greater in PR (75 +/- 10%) than in sham-operated animals (8 +/- 4%; p < 0.01). In vivo, monophasic action potential duration and activation time, at rest and during acute right ventricular stretch, were similar in both groups. However, the dispersion of activation time was greater in PR animals at rest (13 +/- 1.1 versus 8 +/- 1.1 ms; p < 0.05). Furthermore, the dispersion of activation increased during right ventricular stretch in PR, but not in sham-operated animals. In vitro, myocardial force-frequency responses were similar in both groups, indicating preserved systolic performance, but mechanical restitution studies showed a prolonged refractory period (447 +/- 22 versus 370 +/- 26 ms; p < 0.05) and a decreased recovery time constant (184 +/- 19 versus 265 +/- 20 ms; p < 0.001) in PR animals, indicating altered calcium cycling. Furthermore, the myocardial conduction velocity was reduced in PR animals (31 +/- 3.58 versus 47.9 +/- 5.1 cm/s; p < 0.01), resulting from a 2-fold increase in intracellular resistance (437.25 +/- 125.93 versus 194 +/- 43.27 Omega. cm; p = 0.025). Chronic PR leads to inhomogeneity of right ventricular activation, alters myocardial calcium cycling, reduces conduction velocity, and increases intracellular resistivity. These may contribute to the development of arrhythmias associated with PR, including those in patients after tetralogy repair.
Animals↗
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.
Animals↗
Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: application to single virus-like particle entry into a cell.
We have developed a method for simultaneous recording of high-resolution topography and cell surface fluorescence in a single scan which we call scanning surface confocal microscopy. The resolution of the system allows imaging of individual fluorescent particles in the nanometer range on fixed or live cells. We used this technique to record the interaction of single virus-like particles with the cell surface and demonstrated that single particles sink into the membrane in invaginations reminiscent of caveolae or pinocytic vesicles. This method provides a technique for elucidating the interaction of individual viruses and other nanoparticles, such as gene therapy vectors, with target cells. Furthermore, this technique should find widespread application for studying the relationship of fluorescently tagged molecules with components of the cell plasma membrane.
Animals↗
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.
Animals↗
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.
Animals↗