A tribute to Fredric Stewart Fay: June 5, 1943 - March 18, 1997.
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Biophysics↗
Biomedical subjects
L M Loew
Publications and source records attributed to L M Loew.
Intracellular fluorescent probe concentrations by confocal microscopy.
A general method is described that takes advantage of the optical sectioning properties of a confocal microscope to enable measurement of both absolute and relative concentrations of fluorescent molecules inside cells. For compartments within cells that are substantially larger than the point spread function, the fluorescence intensity is simply proportional to the concentration of the fluorophore. For small compartments, the fluorescence intensity is diluted by contributions from regions outside the compartment. Corrections for this dilution can be estimated via calibrations that are based on the intensity distribution found in a computationally synthesized model for a cell or organelle that has been blurred by convolution with the microscope point spread function. The method is illustrated with four test cases: estimation of intracellular concentration of a fluorescent calcium indicator; estimation of the relative distribution between the neurite and soma of a neuronal cell of the InsP3 receptor on the endoplasmic reticulum; estimation of the distribution of the bradykinin receptor along the surface of a neuronal cell; and relative distribution of a potentiometric dye between the mitochondria and cytosol as a means of assaying mitochondrial membrane potential.
3T3 Cells↗
Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons.
An experimental method has been established to measure the electric properties of a cell membrane by combination of patch clamp and dual-wavelength ratio imaging of a fluorescent potentiometric dye, 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl ]pyridinium betaine (di-8-ANEPPS). Pairs of fluorescence images from the dye-stained membrane of neuroblastoma N1E-115 cells excited at two wavelengths were initially obtained to calculate ratio images corresponding to the resting transmembrane potential. Subsequently, a whole-cell patch was established and the membrane potential clamped to levels varying from -100 to +60 mV; at each voltage, a pair of dual-wavelength images were acquired to develop a calibration of the fluorescence ratio. Using this method, the resting potentials could accurately be measured showing that the differentiated cells were 17 mV more polarized than undifferentiated cells. The combination of electrical and optical methods can also follow changes in other membrane electric properties, such as dipole potential, and thus permit a detailed analysis of the membrane electrical properties underlying the voltage regulation of ion channels.
Animals↗
A general computational framework for modeling cellular structure and function.
The "Virtual Cell" provides a general system for testing cell biological mechanisms and creates a framework for encapsulating the burgeoning knowledge base comprising the distribution and dynamics of intracellular biochemical processes. It approaches the problem by associating biochemical and electrophysiological data describing individual reactions with experimental microscopic image data describing their subcellular localizations. Individual processes are collected within a physical and computational infrastructure that accommodates any molecular mechanism expressible as rate equations or membrane fluxes. An illustration of the method is provided by a dynamic simulation of IP3-mediated Ca2+ release from endoplasmic reticulum in a neuronal cell. The results can be directly compared to experimental observations and provide insight into the role of experimentally inaccessible components of the overall mechanism.
Animals↗
Dye screening and signal-to-noise ratio for retrogradely transported voltage-sensitive dyes.
Using a novel method for retrogradely labeling specific neuronal populations, we tested different styryl dyes in attempt to find dyes whose staining would be specific, rapid, and lead to large activity dependent signals. The dyes were injected into the ventral roots of the isolated chick spinal cord from embryos at days E9-E12. The voltage-sensitive dye signals were recorded from synaptically activated motoneurons using a 464 element photodiode array. The best labeling and optical signals were obtained using the relatively hydrophobic dyes di-8-ANEPPQ and di-12-ANEPEQ. Over the 24 h period we examined, these dyes bound specifically to the cells with axons in the ventral roots. The dyes responded with an increase in fluorescence of 1-3% (delta F/F) in response to synaptic depolarization of the motoneurons. The signal-to-noise ratio obtained in a single trial from a detector that received light from a 14 x 14 microns2 area of the motoneuron population was about 10:1. Nonetheless, signals on neighboring diodes were similar, suggesting that we were not detecting the activity of individual neurons. Retrograde labeling and optical recording with voltage-sensitive dyes provides a means for monitoring the activity of identified neurons in situations where microelectrode recordings are not feasible.
Animals↗
Graded fibronectin receptor aggregation in migrating cells.
We have examined the distribution of the integrin fibronectin receptor in migrating NIH 3T3 fibroblasts to test the hypothesis that cell locomotion involves regional differences of adhesive receptor aggregation. A distinct asymmetry of fibronectin receptor aggregation was observed on the surface of migrating NIH 3T3 fibroblasts. Direct current electric fields were used to stimulate directional cell migration and thus allow the quantitative correlation of receptor asymmetries to the direction of cell locomotion. Digital particle analysis of fluorescent confocal micrographs demonstrates that the leading half of cells has a higher proportion of receptors contained in small clusters than does the trailing half. Conversely, larger receptor aggregates are more prevalent in the rear of the cell than in the front. We have also observed a gradient of extracellular fibronectin fibril assembly, similar to that described for the fibronectin receptor. Extracellular fibronectin appears in progressively larger fibrils across the ventral cell surface from front to rear, with dense meshworks often deposited as a trail behind the cell. Large fibronectin receptor clusters toward the rear of the cell generally do not correlate with focal contacts, and are thus most likely aggregated by cell surface-bound fibronectin fibrils and not by adhesion to the substratum. These results suggest that spatial variations in the degree of adhesive receptor aggregation are created in fibroblasts during the processes of migration and matrix synthesis.
3T3 Cells↗
Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.
Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells.
Animals↗
Physiological cytosolic Ca2+ transients evoke concurrent mitochondrial depolarizations.
Calcium, a ubiquitous second messenger, stimulates the activity of several mitochondrial dehydrogenases. This has led to the suggestion that the same messenger that signals cell activation could also activate mitochondrial electron/proton transport, thereby meeting demands for increased cellular energy. To test this in live cells, quantitative three-dimensional microscopy and ratio imaging were used to measure membrane potential of individual mitochondria and cytosolic calcium distribution. Mitochondria reversibly depolarized as cytosolic calcium rose and then fell following physiological stimulation. Thus, the dominant response of the mitochondrion to a rise in cytosolic [Ca2+] is to draw on the electrochemical potential, possibly to accelerate processes directly involved in ATP synthesis and calcium homeostasis.
Adenosine Triphosphate↗
Distinct electric potentials in soma and neurite membranes.
Structurally similar voltage-dependent ion channels may behave differently in different locations along the surface of a neuron. A possible reason could be that channels experience nonuniform electrical potentials along the plasmalemma. Here, we map the electrical potentials along the membrane of differentiated N1E-115 neuroblastoma cells with a potential-sensitive dye. We find that the intramembrane potential gradient is indeed more positive in the membranes of neurites than in the membranes of somata. This is not attributable to differences in ion conductances or surface charge densities between the membranes of neurites and somata; instead, it can be explained by differences in lipid composition. The spatial variation in intramembrane electrical potential may help account for electrophysiological and functional differences between neurites and somata.
Animals↗
Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.
The electrostatic potentials associated with cell membranes include the transmembrane potential (delta psi), the surface potential (psi s), and the dipole potential (psi D). psi D, which originates from oriented dipoles at the surface of the membrane, rises steeply just within the membrane to approximately 300 mV. Here we show that the potential-sensitive fluorescent dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6- naphthyl]vinyl]pyridinium betaine (di-8-ANEPPS) can be used to measure changes in the intramembrane dipole potential. Increasing the content of cholesterol and 6-ketocholestanol (KC), which are known to increase psi D in the bilayer, results in an increase in the ratio, R, of the dye fluorescence excited at 440 nm to that excited at 530 nm in a lipid vesicle suspension; increasing the content of phloretin, which lowers psi D, decreases R. Control experiments show that the ratio is insensitive to changes in the membrane's microviscosity. The lack of an isosbestic point in the fluorescence excitation and emission spectra of the dye at various concentrations of KC and phloretin argues against 1:1 chemical complexation between the dye and KC or phloretin. The macromolecular nonionic surfactant Pluronic F127 catalyzes the insertion of KC and phloretin into lipid vesicle and cell membranes, permitting convenient and controlled modulation of dipole potential. The sensitivity of R to psi D is 10-fold larger than to delta psi, whereas it is insensitive to changes in psi S. This can be understood in terms of the location of the dye chromophore with respect to the electric field profile associated with each of these potentials. These results suggest that the gradient in dipole potential occurs over a span s5 A, a short distance below the membrane-water interface. These approaches are easily adaptable to study the influence of dipole potentials on cell membrane physiology.
Animals↗
Electric field-directed fibroblast locomotion involves cell surface molecular reorganization and is calcium independent.
Directional cellular locomotion is thought to involve localized intracellular calcium changes and the lateral transport of cell surface molecules. We have examined the roles of both calcium and cell surface glycoprotein redistribution in the directional migration of two murine fibroblastic cell lines, NIH 3T3 and SV101. These cell types exhibit persistent, cathode directed motility when exposed to direct current electric fields. Using time lapse phase contrast microscopy and image analysis, we have determined that electric field-directed locomotion in each cell type is a calcium independent process. Both exhibit cathode directed motility in the absence of extracellular calcium, and electric fields cause no detectable elevations or gradients of cytosolic free calcium. We find evidence suggesting that galvanotaxis in these cells involves the lateral redistribution of plasma membrane glycoproteins. Electric fields cause the lateral migration of plasma membrane concanavalin A receptors toward the cathode in both NIH 3T3 and SV101 fibroblasts. Exposure of directionally migrating cells to Con A inhibits the normal change of cell direction following a reversal of electric field polarity. Additionally, when cells are plated on Con A-coated substrata so that Con A receptors mediate cell-substratum adhesion, cathode-directed locomotion and a cathodal accumulation of Con A receptors are observed. Immunofluorescent labeling of the fibronectin receptor in NIH 3T3 fibroblasts suggests the recruitment of integrins from large clusters to form a more diffuse distribution toward the cathode in field-treated cells. Our results indicate that the mechanism of electric field directed locomotion in NIH 3T3 and SV101 fibroblasts involves the lateral redistribution of plasma membrane glycoproteins involved in cell-substratum adhesion.
Animals↗
Probing membrane potential with nonlinear optics.
The nonlinear optical phenomenon of second harmonic generation is shown to have intrinsic sensitivity to the voltage across a biological membrane. Our results demonstrate that this second order nonlinear optical process can be used to monitor membrane voltage with excellent signal to noise and other crucial advantages. These advantages suggest extensive use of this novel approach as an important new tool in elucidating membrane potential changes in biological systems. For this first demonstration of the effect we use a chiral styryl dye which exhibits gigantic second harmonic signals. Possible mechanisms of the voltage dependence of the second harmonic signal are discussed.
Fluorescent Dyes↗
Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria.
Because of its importance in the chemiosmotic theory, mitochondrial membrane potential has been the object of many investigations. Significantly, however, quantitative data on how energy transduction might be regulated or perturbed by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have not been possible because of their small size, their intrinsic motility, and the absence of appropriate analytical reagents. In this article, we combine techniques for rapid, high resolution, quantitative three-dimensional imaging microscopy and mathematical modeling to determine accurate distributions of a potentiometric fluorescent probe between the cytosol and individual mitochondria inside a living cell. Analysis of this distribution via the Nernst equation permits assignment of potentials to each of the imaged mitochondrial membranes. The mitochondrial membrane potentials are distributed over a narrow range centered at -150 mV within the neurites of differentiated neuroblastoma cells. We find that the membrane potential of a single mitochondrion is generally remarkably stable over times of 40-80 s, but significant fluctuations can occasionally be seen. The motility of individual mitochondria is not directly correlated to membrane potential, but mitochondria do become immobile after prolonged treatment with respiratory inhibitors or uncouplers. Thus, three spatial dimensions, a key physiological parameter, and their changes over time are all quantitated for objects at the resolution limit of light microscopy. The methods described may be readily extended to permit investigations of how mitochondrial function is integrated with other processes in the intact cell.
Algorithms↗
Confocal microscopy of potentiometric fluorescent dyes.
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Animals↗
Voltage-sensitive dyes: measurement of membrane potentials induced by DC and AC electric fields.
Dye indicators of membrane potential have been available for the past 15 years and have been employed in numerous studies of cell physiology. Since the cell membrane is a likely primary site for the cascade of events resulting in a biological response to electromagnetic fields, methodologies for monitoring the membrane voltage will be critical. This laboratory has developed a series of dyes by using theoretical molecular orbital calculations to predict electrochromic structures. The spectra of electrochromic dyes are altered via a direct coupling of the molecular electronic states with an electric field. This mechanism has the advantage of providing both high temporal and high spatial resolution because the effect is instantaneous and is localized to the level of individual indicator molecules. It therefore can have significant advantages over traditional microelectrode techniques because fast changes in potential can be monitored simultaneously over many different regions of a biological preparation. We have used these dyes to monitor membrane potentials induced by both DC and AC electric fields. In a series of studies with a model membrane system, a spherical lipid bilayer, we showed that the potential develops on the membrane in good agreement with a time-dependent solution to Laplace's equation. Cell membranes can also be stained with voltage sensitive dyes. In experiments with dc fields, we are able to map the variation of the induced membrane potential along the surface of the cell by employing digital video fluorescence microscopy. We can also use the fluorescence microscope to detect membrane potential induced by ac fields by using a phase-sensitive detection scheme to extract the corresponding change in the light intensity from the fluorescent indicator. The technology can be extended to more complex biological preparations and can be used in conjunction with other optical techniques such as those which monitor intracellular ion concentrations. It may, therefore, prove highly valuable for the elucidation of biological responses to electromagnetic fields.
Animals↗
A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations.
The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.
Animals↗
Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth.
Our study explores the mechanisms behind neurite galvanotropism. Using phase, differential interference contrast and ratiometric fluorescence microscopy, we reveal four responses of N1E-115 mouse neuroblastoma cells to 0.1-1.0 mV/microns uniform DC electric fields: cathode-directed neurite initiation and elongation, cathode-biased growth cone filopodial protrusions, transient cathode-localized calcium increases, and persistent cathode-localized membrane depolarizations. These newly demonstrated events are temporally and spatially correlated, suggesting that they are causally related. The calcium increases are prevented by calcium channel blockers and by the removal of extracellular calcium. We therefore propose that the observed field-induced membrane depolarizations activate voltage-dependent calcium channels, resulting in cathode-localized calcium influx. This, in turn, may initiate the observed cathode-biased growth cone filopodial protrusions, followed by the cathode-directed neurite elongation.
Animals↗
Cytotoxic T lymphocyte (CTL)-mediated cytolysis proceeds in the absence of Na+/H+ antiport activity: regulation of cytosolic pH by the Na+/H+ antiport in a cloned CTL.
Cytotoxic T lymphocyte (CTL)-mediated cytolysis of specifically bound target cells (TC) is thought to be triggered by cross-linking the T-cell antigen receptor (TcR). Biochemical events associated with TcR cross-linking include increased intracellular calcium levels [Ca2+]i, hydrolysis of phosphatidylinositol (PI), and an increase in intracellular pH [pH]i. Whereas CTL-mediated cytolysis of some TC is calcium-dependent, and PI hydrolysis is speculated to trigger the CTL lethal hit via activation of PKC, little is known about changes in [pH]i relating to activation of the lethal hit stage. We report regulation of [pH]i in a cloned CTL by the electroneutral Na+/H+ antiport during activation with PMA and specific antigen-bearing TC. Furthermore, using 5-(N-methyl-N-isobutyl) amiloride (MIBA), a potent antiport inhibitor, we demonstrate that Na+/H+ exchange is not required for activation of CTL cytolytic activity.
Acid-Base Equilibrium↗