Development of spatio-temporal pattern of Ca2+ on the chemotactic behaviour ofPhysarum plasmodium

Summary The development of a spatio-temporal pattern of Ca²⁺ concentration by a plasmodium ofPhysarum polycephalum during chemotaxis was studied using fura-2. Whenever the cell displayed coordinated migration in one direction as a whole body, a spatiotemporal pattern was established with a characteristic feature along the longitudinal axis. Calcium concentration oscillated with a period of a few minutes within the cell; the mean concentration at the front was higher than that at the rear. When the cell was given an attractant only at the rear end, the mean concentration rose at the site of application with an immediate increase in the frequency of oscillation. First, the change of the frequency is propagated toward the other end and then the mean level of the Ca²⁺ concentration at the non-stimulated site decreases. As a result, the Ca²⁺ gradient is reversed along the cell, which then begins to migrate in a coordinated manner in the reverse direction. This study showed that the spatiotemporal pattern of Ca²⁺ concentration is closely related to information processing for coordinated migration in chemotaxis. The role of the pattern in that process is discussed.     

Distribution of Acid and Alkaline Zones on the Cell Surface of Chara corallina under Stationary and Local Illumination

A scanning microprobe technique was used to study pH distribution near the cell surface of Chara corallina Klein ex Willd. under variations of light intensity, dark–light transitions, and local illumination of various cell parts. In darkness, the H⁺-transporting activity of plasmalemma was distributed homogeneously over the cell surface. However, after exposing the cell to weak light (irradiance 0.2–0.5 W/m²), individual alkaline peaks with pH of 1–2 units were observed in the longitudinal pH profile. The peaks in the longitudinal pH profile became more numerous with the increase in light intensity. The plot of pH as a function of light intensity included a steep transition from zero to its maximum amplitude. In strong light (100 W/m²), the pH bands alternated along the cell length with a periodic length of about 7 mm. It is shown that the light-induced formation of ring-shaped bands with H⁺-exporting and H⁺ sink activities is preceded by the appearance of irregularly located spots (patches). When small cell parts were illuminated and the light spot was suddenly shifted along the cell to another position, the alkaline bands reorganized in two ways. In some cases, this treatment was followed by a gradual shift in the band position (without attenuation in the peak height) toward the illuminated area. In other cases, the initial band disappeared after such treatment, and a new alkaline band emerged in the vicinity of the illuminated area. Despite the apparent similarity of regular bands in the longitudinal pH profiles, the properties of individual bands (variable sensitivity to light intensity changes, the ability of bands to move along the cell under external treatment) differ substantially. It is supposed that the light-induced formation of the pH profile is based on primary fluctuations of photosynthetic and transport activities in the chloroplast layer and the plasmalemma, as well as on further rearrangements of membrane domains that stabilize band locations.     

Oscillations of electric spatial patterns emerging from the homogeneous state in characean cells

Electric spatial patterns of bands formed along the cell wall of the characean internode were studied using a multi-electrode measuring system. The electric potential near the surface of the cell was measured by arranging about 25 electrodes along the cell at approximately 1.6 mm intervals. Since the time required for one scan over the cell length is only 1 s, the temporal change in the spatial pattern of surface electric potential can be readily observed. Oscillations were sometimes found as the electric pattern started to appear after the cell was illuminated. Fourier analysis shows that a single spatial mode arises gradually and then becomes stabilized in an oscillatory manner. A simple electric circuit model comprising three variables, i.e., a membrane potential, an electric current across the membrane and an electromotive force, can simulate well the oscillatory rise of bands. These results imply that the electric spatial pattern observed in characean internodes is a self-organized structure emerging far from equilibrium, known as a dissipative structure. Biophysical mechanisms of band formation are discussed.     

Fine structure of the band 3 protein in human red cell membranes: Freeze-fracture studies

The major red cell membrane protein, band 3, is a glycoprotein which extends across the membrane from the extracellular space into the cytoplasmic compartment. It is widely held that band 3 is a component of the intramembrane particles (IMP) which can be demonstrated by freeze-fracture electron microscopy. In this study, we find that the outer surface poles of the IMP can be seen by freeze-etching after they are unmasked by proteolysis under conditions which excise the surrounding sialopeptides from the membrane. The poles appear as distinctive projections, 30–50 Å in diameter, the “ES particles.” The ES particles remain associated with the outer surface of the membrane following cleavage of the band 3 polypeptide by chymotrypsin or pronase. This is consistent with previous biochemical studies which have shown that the 38,000-dalton outer surface segment of band 3 is intercalated in the lipid bilayer. A granulofibrillar component at the inner surface of the membrane is provisonally identified as the 40,000-dalton inner-surface domain of band 3.     

Microtubule arrays in regeneratingMougeotia protoplasts may be oriented by electric fields

Summary Initially non-polar protoplasts of the green algaMougeotia will regenerate to re-establish their original cylindrical cell shape. The orientation of the growth axis of regenerating protoplasts held in agarose was independent of both the direction of incident white light and gravity. Protoplasts elongated parallel to applied DC electric fields of approx. 0.2 Vcm^–1 (1 mV/protoplast) and greater, with an increasing percentage oriented with increasing field strength. At the maximum field strength used (10 mV/cell), 53% of protoplasts were oriented within +- 10° of the 0/180° axis of the field. In untreated controls, the orientation of elongation was random. Protoplast survival was unaffected by field treatment. Some protoplasts (up to 37% in 10 mV/cell fields) formed outgrowths towards the cathode and occasionally towards the anode. Regenerating protoplasts in fields displayed the normal sequence of microtubule reorganization. This means that the positioning of the ordered symmetrical array of microtubules centred on two foci that appears within 3 to 4 h, and the subsequent organization of microtubules by 8 to 12 h into a band that intersects both foci and which is transverse to the axis of elongation (Galway and Hardham 1986), may be controlled by externally applied electric fields. In the region of this microtubule band, the applied field causes the plasma membrane to be stretched parallel to the field (Bryant and Wolfe 1987). We suggest that microtubules may become oriented perpendicular to the direction of field-induced membrane stretching, and that membrane stretching may be one of the orienting mechanisms for membrane-linked microtubules in elongating plant cells.Abbreviations PBS phosphate buffered saline - PMM protoplast maintenance medium - DMM dilute maintenance medium - MES 2(N-morpholino)ethanesulfonic acid - TRIS tris(hydroxymethyl)aminomethane - ANOVA analysis of variance     

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

Summary The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na⁺ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal²²Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na⁺ flux was a linear function of mucosal Na⁺ concentration. Ouabain-sensitive Na⁺ flux and ouabain-sensitive, Na⁺-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na⁺ with the basolateral transport process is assumed to involve the interaction of some number of Na⁺ ions,n, with a maximal flux,M _max, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value forM _max of 287.8pm cm^–2 sec^–1 with an intracellular Na concentration of 2.0mm Na⁺ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na⁺-induced current, we calculate a Na⁺ to K⁺ coupling ratio of 1.40±0.07 for the transport process.     

Lipid-impregnated filters as a tool for studying the electric current-generating proteins.

Porous filters and collodion film impregnated with decane solution of phospholipids, were used for measurements of electric potential differences generated by bacteriorhodopsin, chromatophore redox chain, H⁺-ATPase, pyrophosphatase, and mitochondrial respiratory chain. It was shown that reconstituted proteoliposomes, containing e.g., bacteriorhodopsin or natural coupling membrane vesicles, such as Rhodospirillum rubrum chromatophores, can be attached to a filter surface by means of Ca²⁺ or Mg²⁺ ions. Addition of the respective energy source was found to result in electric potential difference being generated across the filter. This effect was measured directly by $\text{Ag}\text{AgCl}$ electrodes immersed into electrolyte solutions on both sides of the filter. Using a phospholipid-impregnated collodion film one can measure electric responses as fast as 300 nsec. The phospholipid-impregnated filters turned out to be sensitive and reliable electrodes for measuring the concentration of synthetic penetrating ions, such as phenyldicarbaundecaborane, tetraphenylborate, tetrapentylammonium, and tetraphenylphosphonium. By measuring changes in the concentration of these ions in the suspension of proteoliposomes, chromatophores, mitochondria, or bacterial cells, one can follow the formation and dissipation of transmembrane potential differences in these systems. It is shown that the phospholipid-impregnated filters are much more reliable and handy than planar phospholipid membranes previously used for studying electrogenic activity of electric current-producing membrane proteins.     

Mathematical model of the spatio-temporal dynamics of second messengers in visual transduction

A model describing the role of transversal and longitudinal diffusion of cGMP and Ca(2+) in signaling in the rod outer segment of vertebrates is developed. Utilizing a novel notion of surface-volume reaction and the mathematical theories of homogenization and concentrated capacity, the diffusion of cGMP and Ca(2+) in the inter-disc spaces is shown to be reducible to a one-parameter family of diffusion processes taking place on a single rod cross section; whereas the diffusion in the outer shell is shown to be reducible to a diffusion on a cylindrical surface. Moreover, the exterior flux of the former serves as a source term for the latter, alleviating the assumption of a well-stirred cytosol. A previous model of visual transduction that assumes a well-stirred rod outer segment cytosol (and thus contains no spatial information) can be recovered from this model by imposing a "bulk" assumption. The model shows that upon activation of a single rhodopsin, cGMP changes are local, and exhibit both a longitudinal and a transversal component. Consequently, membrane current is also highly localized. The spatial spread of the single photon response along the longitudinal axis of the outer segment is predicted to be 3-5 microm, consistent with experimental data. This approach represents a tool to analyze point-wise signaling dynamics without requiring averaging over the entire cell by global Michaelis-Menten kinetics.     

Transcellular ion currents and extension ofNeurospora crassa hyphae

Summary Hyphae ofNeurospora crassa, like many other tipgrowing organisms, drive endogenous electric currents through themselves such that positive charges flow into the apical region and exit from the trunk. In order to identify the ions that carry the current, the complete growth medium was replaced by media lacking various constituents. Omission of K⁺ or of phosphate diminished the zone of inward current, effectively shifting the current pattern towards the apex. Omission of glucose markedly reduced both inward and outward currents; addition of sodium azide virtually abolished the flow of electric current. Growing hyphae also generate a longitudinal pH gradient: the medium surrounding the apex is slightly more alkaline than the bulk phase, while medium adjacent to the trunk turns acid. The results suggest thatNeurospora hyphae generate a proton current; protons are expelled distally by the H⁺-ATPase and return into the apical region by a number of pathways, including the symport of protons with phosphate and potassium ions. Calcium influx may also contribute to the electric current that enters the apical region. There seems to be no simple obligatory linkage between the intensity of the transcellular electric current and the rate of hyphal extension. Calcium ions, however, are required in micromolar concentrations for extensions and morphogenesis of hyphal tips.     

Characteristics of a Pulse-Periodic Corona Discharge in Atmospheric Air

Pulse-periodic corona discharge in atmospheric air excited by applying a voltage pulse with a subnanosecond or microsecond rise time to a point electrode is studied experimentally. It is shown that, at a voltage rise rate of dU/dt ~10¹⁴ V/s, positive and negative ball-shaped streamers with a front velocity of ≥2 mm/ns form near the point electrode. As dU/dt is reduced to 10¹⁰?10¹¹ V/s, the streamer shape changes and becomes close to cylindrical. The propagation velocity of cylindrical streamers is found to be ~0.1 mm/ns at dU/dt ~ 2 × 10¹⁰ V/s. It is shown that the propagation direction of a cylindrical streamer can be changed by tilting the point electrode, on the axis of which the electric field strength reaches its maximum value. It is established that, for the negative polarity of the point electrode and a microsecond rise time of the voltage pulse, a higher voltage is required to form a cylindrical streamer than for the positive polarity of the point electrode.     

Electric birefringence study of the purple membrane of Halobacterium halobium

An electric birefringence study was carried out on aqueous suspensions of the purple membrane of Halobacterium halobium. In addition to the characterization of both native and modified membrane samples, the dependence of electric birefringence on pH and ionic strength was also investigated. The results indicate that purple membrane shows electric birefringence at a field strength as low as 200 V/cm. The permanent dipole moment and polarizability ranged from 20,500 debyes and 1.01 × 10^?14 cm³ for a purple membrane concentration of 0.40 mg/mL to 41,000 debyes and 2.05 × 10^?14 cm³ for a concentration of 0.80 mg/mL. It was also found that removal of the retinyl group of bacteriorhodopsin substantially decreases but does not eliminate the electric birefringence of the membrane. The solubilization of the membrane by Triton X-100, however, completely abolishes the electric birefringence. These experiments indicate that there is an interaction between adjacent bacteriorhodopsin molecules within the purple membrane via the retinyl chromophore moiety that builds up the permanent dipole moment. They also suggest that there are two types of response when purple membrane suspensions are placed in an electric field. One is an alignment of the disk-shaped particles with the field. The other is a stacking of the particles following their alignment by the electric field, which is promoted by the induced dipole moment.     

Effect of ion concentration,solution and membrane permittivity on electric energy storage and capacitance

Bio-membranes as capacitors store electric energy, but their permittivity is low whereas the permittivity of surrounding solution is high. To evaluate the effective capacitance of the membrane/solution system and determine the electric energy stored within the membrane and in the solution, we estimated their electric variables using Poisson-Nernst-Planck simulations. We calculated membrane and solution capacitances from stored electric energy. The effective capacitance was calculated by fitting a six-capacitance model to charges (fixed and ion) and associated potentials, because it cannot be considered as a result of membrane and solution capacitance in series. The electric energy stored within the membrane (typically much smaller than that in the solution), depends on the membrane permittivity, but also on the external electric field, surface charge density, water permittivity and ion concentration. The effect on capacitances is more specific. Solution capacitance rises with greater solution permittivity or ion concentration, but the membrane capacitance (much smaller than solution capacitance) is only influenced by its permittivity. Interestingly, the effective capacitance is independent of membrane or solution permittivity, but rises as the ion concentration increases and surface charge becomes positive. Experimental estimates of membrane capacitance are thus not necessarily a reliable index of its surface area.     

Electrical properties of the vertically growing root tip of Lepidium sativum L.

Electrical transmembrane potential differences and resistances in different tissues of intact root tips of Lepidium sativum L. were investigated in a humid atmosphere by conventional glass-microelectrode techniques with the reference electrode at the surface (apoplast) of the root. The resting potential (inside negative) in cells of the root cap rose from-80 mV in external cell layers (secretion cells) to approx.-140 mV in central cells (statocytes). Measurements of the electric input resistance within the apoplast of the root tip (calyptra, meristem and elongation zone) yielded a preference for longitudinal contact (resistance per length of tissue approx. 3.4 GOhm m^-1) compared with transversal contact (approx. 14 GOhm m^-1). Similarly, the symplastic coupling expressed as the characteristic length (L) where a signal is reduced to 1/c compared with the origin yielded L _y =390 m in the longitudinal (y) direction and L _x =140 m in the transversal (x) direction. Cable analytical treatment of the symplastic input resistances (approx. 10 MOhm) resulted in low membrane resistances in the y-direction at the ends of cells compared with the membrane resistances in the x-direction (approx. 0.2 Ohm m²) of the lateral membranes in the approximately cylindrical cells. This anisotropy is discussed in terms of model calculations. The resistivity of the symplast was calculated to be about 2.5 Ohm m. The input current-voltage relationship displayed a slight curvature with increasing slope for the more negative membrane potential typical of membranes with electrogenic pumps. Even after massive electrical stimulation in the range from-50 to-150mV carried out to trace current-voltage curves, electrical excitations (action potentials) were not detected in the cells investigated.Abbreviations el voltage recording electrodes - R resistance - V _r resting potential     

Flux of organic carbon in a riverine mangrove wetland in the Florida Coastal Everglades

Short-term (daily) and seasonal variations in concentration and flux of dissolved organic carbon (DOC) were examined over 15 tidal cycles in a riverine mangrove wetland along Shark River, Florida in 2003. Due to the influence of seasonal rainfall and wind patterns on Shark River’s hydrology, samplings were made to include wet, dry and transitional (Norte) seasons. We used a flume extending from a tidal creek to a basin forest to measure vertical (vegetated soil/water column) and horizontal (mangrove forest/tidal creek) flux of DOC. We found significant (p < 0.05) variations in surface water temperature, salinity, conductivity, pH and mean concentration of DOC with season. Water temperature and salinity followed seasonal patterns of air temperature and rainfall, while mean DOC concentration was highest during the dry season (May), followed by the wet (October) and ‘Norte’ (December) seasons. This pattern of DOC concentration may be due to a combination of litter production and inundation pattern of the wetland. In contrast to daily (between tides) variation in DOC flux between the mangrove forest and tidal creek, daily variations of mean water quality were not significant. However, within-tide variation of DOC flux, dissolved oxygen content and salinity was observed. This indicated that the length of inundation and water source (freshwater vs. saltwater) variation across tidal cycles influenced water quality and DOC flux in the water column. Net DOC export was measured in October and December, suggesting the mangrove forest was a source of DOC to the adjacent tidal creek during these periods. Net annual export of DOC from the fringe mangrove to both the tidal creek and basin mangrove forest was 56 g C m⁻² year⁻¹. The seasonal pattern in our flux results indicates that DOC flux from this mangrove forest may be governed by both freshwater discharge and tidal range.     

Studies with Composite Membranes: II. Measurement of Asymmetric Properties

Electrical potentials arising across composite membranes when they separate the same concentration of a (1:1) electrolyte or electrolytes have been measured. These potentials have been shown to arise from differences in the transport number of counterions contacting the two faces of the membrane which contained in its body a high concentration of electrolyte and polyelectrolyte. When the concentration of this trapped electrolyte or polyelectrolyte is low, the asymmetry potentials are small. Although measurements of current-voltage relations provided evidence for the existence of asymmetry between the two faces of the membrane, osmotic flow of water in either direction across the membrane and the salt flow in the two directions were symmetrical. These solvent and solute flux measurements lasted more than 30 hr. Short-term (about 4 hr) flux measurements, however, using tritiated water (THO), gave flows which were different in the two directions. Similarly, the salt flows measured using ²²Na isotope were different in the two directions. The usefulness of the present system as a model to use for studies concerned with carrier transport problems in biology has been pointed out.     

Inversion of extracellular current and axial voltage profile inChara andNitella

Summary Reducing the pH of the bathing solution from 8.2 to pH 6 can induce an inversion of the extracellular current pattern that develops at the surface ofChara corallina internodal cells. A similar result can be obtained on some cells by changing the medium to a pH value of 10. In noninvertingChara cells the currents were strongly reduced when the pH value of the medium was changed between 3 and 11. Simultaneous measurements of theChara transmembrane potential in the acid and alkaline regions revealed that a light-induced electrical potential gradient of approximately 24 mV was present in the axial (or longitudinal) direction. Correlated to the external current pattern inversion was an inversion of this internal longitudinal voltage gradient. Reillumination ofNitella cells, after a period of darkness, often resulted in a complete inversion of the extracellular current pattern. These results are discussed in terms of spatial and temporal control of membrane transport processes, and in particular the control of current loops that pass through these cells.     

Propagated disturbances of transverse potential gradient in intracellular fibrils as the source of motive forces for longitudinal transport in cells

Summary It is proposed as a working hypothesis that conformational changes propagated like waves along intracellular fibrils (tubules, microtubules, microfilaments) have an electric component,i.e., there are waves of disturbance of electric potential in the fibrils. The paper considers the unavoidable consequences of the wave. The latter is accompanied by local electric field in the boundary layer of cytoplasmic fluid. Both positively and negatively charged particles may be attracted to the fibril in certain regions of the field and, being attracted, the particle may be under the action of longitudinal component of electric force. When the force is strong enough to move the particle with wave velocity, the particle will travel smoothly along the fibril, otherwise the movement will be saltatory or of agitation type. Net electroosmotic flow in one direction in the boundary layer of fluid may be expected when the waves are propagated in series. Turbulent motion of the fluid caused by the waves may provide the basis for activated diffusion. Asymmetry of the wave may account for polar transport of this sort. The electric field transmitted along the fibril across a sieve pore in phloem may facilitate electroosmotically the flow through the pore. Quantitative requirements of the hypothesis that electric field generated by the waves may account for different aspects of longitudinal transport in cells are apparently met.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.