Light-triggered pH banding profile in Chara cells revealed with a scanning pH microprobe and its relation to self-organization phenomena

When exposed to light, Characean cells develop a pattern of alternating alkaline and acid bands along the cell length. The bands were identified with a tip-sensitive antimony pH microelectrode positioned near one end of Chara internode at a distance of 50-100 microm from the cell wall. The stage with Chara cell was moved along its longitudinal axis at a computer-controlled speed (100 or 200 microm s(-1)) relative to the pH probe over a distance of 50 mm. Under sufficient uniform illumination of the cell (from 100 to 2.5 Wm(-2)), the homogeneous pH distribution becomes unstable and a banding pattern is formed, the spatial scale of which decreases with the light intensity. If the cell is locally illuminated, bands are formed only in the region of illumination. It is shown that the inhibition of cyclosis by cytochalasin B leads to the disappearance of the banding pattern. The addition of ammonium (weak base) inhibited the banding pattern, whereas acetate (weak acid) alleviated the inhibitory effect of ammonium and restored the pH banding. A model explaining the observed phenomena is formulated in terms of proton concentration outside and bicarbonate concentration inside the cell. It contains two diffusion equations for the corresponding ions with nonlinear boundary conditions determined by ion transport processes across the cell membrane. The model qualitatively explains most of the experimental observations. It describes the dependence of the pattern characteristics on the light intensity and reveals the role of cyclosis in this phenomenon.     

Transitions from alkaline spots to regular bands during pH pattern formation at the plasmalemma of<Emphasis Type="Italic"> Chara</Emphasis> cells

A scanning pH-microprobe was used to study pH patterns near the surface of Chara corallina cells at various light intensities and during light-induced transitions from homogeneous pH distribution to alternating pH bands. In the irradiance (PAR) range 4-400 micromol quanta m(-2) s(-1), the sustained pH profiles consisted of alternating acid and alkaline bands with a characteristic length of 7-10 mm and pH shifts as large as 2-3 units. At lower irradiance, the number of alkaline bands decreased while the amplitude of remaining peaks stayed high. On cyclic changes in light intensity, a hysteresis of pH banding was observed: the pH bands tolerated low irradiance in weakening light, but higher irradiance was required for their emergence after dark adaptation of the cell. The pH profiles measured for different paths of electrode scanning suggest that the pH pattern at low light level represents patches coexisting with bands. The exposure of the cell to high-intensity light led to formation of radially symmetrical bands. Transformations of the pH pattern induced by lowering the light intensity were similar to those induced by transcellular electric current (1.5-3 microA). The data suggest that band formation at the plasmalemma of Chara cells proceeds through the initial appearance of multiple patches with a localized H(+)-transporting activity and subsequent spot rearrangements (fusion, deletions, widening), leading to establishment of alternating bands.     

Ion flux interaction with cytoplasmic streaming in branchlets of Chara australis

Both parts of the actin-myosin complex involved in cytoplasmic streaming could be regulated by mineral ions. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell wall and plasma membrane. The transport of K(+) and Ca(2+) along pH bands in Chara branchlet internodal cells was characterized by using the MIFE system for non-invasive microelectrode measurement of ion fluxes. Branchlets formed acidic and alkaline bands with the pH ranging from 5 to 8. Different pH patterns were observed for different sides of the branchlets. Sides with cyclosis streaming acropetally generally showed greater variation in the profiles of pH and H(+) fluxes. Although a high correlation was not found between pH bands and Ca(2+) or K(+) fluxes, there was a positive correlation between Ca(2+) and K(+) fluxes themselves for both sides of the branchlets. Application of cytochalasin D, an inhibitor of cyclosis, had no immediate effect on pH and ion fluxes, however, the time of cyclosis cessation corresponded with a dramatic change in Ca(2+) and K(+) fluxes; pH profiles and H(+) fluxes were affected within 2 h. The evidence suggests that, in Chara branchlets, pH band formation and Gd(3+)-insensitive Ca(2+) transport systems are linked to the cyclosis machinery: (i) the pH band amplitude for the acropetally streaming side was larger than that for the basipetally streaming side; (ii) cessation of cytoplasmic streaming after cytochalasin D application resulted in changed pH banding profiles and H(+), Ca(2+) and K(+) fluxes; and (iii) the application of GdCl(3) or incubation in GdCl(3) solutions did not lead to the cessation of cytoplasmic streaming, although external Ca(2+) fluxes changed.     

Influence of culture medium pH on charasome development and chloride transport inChara corallina

Summary Internodal cells ofChara, grown in culture either at pH 5.7, 6.5 or 7.5, were studied to determine their chloride influx capability, the quantitative aspects of charasome morphology and the degree to which these two parameters could be correlated. In cells grown at pH 5.7 the charasomes were relatively small, were widely spaced on the plasma membrane, and contributed only a 0.6% increase to the surface area of the plasma membrane in the acid region of the cell. In contrast, the charasome membrane surface area of cells grown at pH 7.5 had increased × 19, the density of charasomes on the cell surface increased × 42, thus producing a × 3.57 increase in the acid region plasma membrane surface area. Chloride influx in cells grown at pH 7.5 was × 8.7–12.7 greater than in cells grown at pH 5.7. Cells that had been starved of chloride exhibited a × 2.4 average increase in the rate of chloride influx. Our observations establish the existence of a positive correlation between the rate of chloride influx and the increase in membrane surface area due to charasomes, although other factors, such as the effect of pH on transport-related enzymes, and the effect of charasome structure on chemical equilibria, may also be of importance.     

Microtubule organization differs between acid and alkaline bands in internodal cells ofChara but bands can develop in the absence of microtubules

We have studied the relationship between pH banding and the organization of cortical microtubules in the alga Chara corallina Klein ex Willd. Microtubules were visualized by immunofluorescence and also by imunogold-silver enhancement to allow immediate comparison of microtubule arrangement with visible structural cell features. In cells that are nearing growth completion, microtubule number and alignment change between acidic and alkaline bands over a distance of a few micrometres. Thus, it appears that the still unknown mechanisms for microtubule organization respond to the localized differences in membrane properties. Band formation was not prevented when microtubules were depolymerized with the herbicide oryzalin, demonstrating that microtubules are not necessary for pH bands to develop in these cells.Abbreviations DMSO dimethylsulfoxide - MT microtubule We thank Frank Gubler for helpful advice on immunogold-silver enhancement procedures, Brian Gunning for tuition in confocal microscopy, Ann Cork for assistance with photography and Dean Price for helpful discussions. G.O.W. gratefully acknowledges the receipt of a National Research Fellowship and a Queen Elizabeth II Fellowship from the Australian Research Council.     

The plasma membrane of young Chara internodal cells revealed by rapid freezing

Young elongating internodal cells of Chara globularis var. capillacea (Thuill.) Zanev. were rapidly frozen and freze-fractured in order to observed transient events occurring within the plasma membrane. Several structures have been observed. Relatively small depressions, varying in depth, are prolific and scattered at random over the plasma membrane. Charasomes and clusters of particle rosettes are common. Arrays of intramembrane particle lines are a characteristic feature of the internodal cell plasma membrane. The charasomes and the arrays of particle lines occupy a considerable proportion of the plasma membrane. In these young cells, substantial movement must take place across this membrane and its basic structure must fluctuate accordingly. The innumerable small depressions may represent pinocytotic and secretory processes. The array of intramembrane particle lines may represent stages in fusion between the membranes of vesicles within the cytoplasm and the plasma membrane. The technique of ultra-rapid freezing allows these events and their intermediate stages to be visualised; some features of the membrane may only be seen by this method.     

Structure and possible function(s) of charasomes; complex plasmalemma-cell wall elaborations present in some characean species

Summary Charasomes, complex membrane structures, were found along the longitudinal walls of internodal and lateral branch cells ofChara corallina andC. braunii, but not along their transverse walls or in other cell types. Charasome-complexes were larger and more numerous in the lateral branch cells than in internodal cells. InC. corallina, a dioecious species, especially large elaboration of charasome material occurs in the lateral branch cells of the female plant, sometimes reaching a cross-sectional width which is as great as that of the adjacent cell wall. Chara internodes transport hydroxyl (OH^–) out of the cell and bicarbonate (HCO₃ ^–) into the cell. Spatial distribution of charasomes along the cell was examined with respect to these transport phenomena, which occur at specific identifiable regions along the cell. Charasome-complexes were always found in regions in which HCO₃ ^– transport occurs but were often fewer, reduced in size or absent in areas of OH^– efflux.Nitella flexilis exhibited similar patterns of OH^– and HCO₃ ^– transport along the cell; however, there was a complete absence of charasomes. Ultrastructural examinations onNitella translucens indicated that charasomes were also absent in this species. The observation that charasomes are present in both transport regions ofChara but are totally lacking in the twoNitella spp. indicates that the charasome-complex is not involved in transport of either substance. Other possible functions for the charasomes, including a role in osmoregulation, are discussed.Charasome substructure is the same in bothChara species, consisting of a mass of short (50 nm average length) anastomosing tubules (30 nm average diameter) derived from the plasmalemma. The interior of the tubules is open to the cytoplasm while the area surrounding the tubules is ultimately open to the wall and thus can be considered to be wall space. Charasomes are quite variable in size and shape, but are roughly globular, with the bulk of the structure projecting into the cell cytoplasm. Tubular components of the charasome were sometimes seen to extend into the microfibrillar wall matrix. A three dimensional model of the charasome-complex presented details the great complexity of this membrane system.     

FM dyes label sterol-rich plasma membrane domains and are internalized independently of the cytoskeleton in characean internodal cells

We applied the endocytic markers FM1-43, FM4-64 and filipin to internodal cells of the green alga Chara corallina. Both FM dyes stained stable, long-living plasma membrane patches with a diameter of up to 1 microm. After 5 min, FM dyes labeled cortical, trembling structures up to 500 nm in size. After 15 min, FM dyes localized to endoplasmic organelles up to 1 microm in diameter, which migrated actively along actin bundles or participated in cytoplasmic mass streaming. After 30-60 min, FM fluorescence appeared in the membrane of small, endoplasmic vacuoles but not in that of the central vacuole. Some of the FM-labeled organelles were also stained by neutral red and lysotracker yellow, indicative of acidic compartments. Filipin, a sterol-specific marker, likewise labeled plasma membrane domains which co-localized with the FM patches. However, internalization of filipin could not be observed. KCN, cytochalasin D, latrunculin B and oryzalin had no effect on size, shape and distribution of FM- and filipin-labeled plasma membrane domains. Internalization of FM dyes was inhibited by KCN but not by drugs which interfere with the actin or microtubule cytoskeleton. Our data indicate that the plasma membrane of characean internodal cells contains discrete domains which are enriched in sterols and probably correspond to clusters of lipid rafts. The inhibitor experiments suggest that FM uptake is active but independent of actin filaments, actin polymerization and microtubules. The possible function of the sterol-rich, FM labeled plasma membrane areas and the significance of actin-independent FM internalization (via endocytosis or energy-dependent flippases) are discussed.     

Spatial coordination of chloroplast and plasma membrane activities in <Emphasis Type="Italic">chara</Emphasis> cells and its disruption through inactivation of 14-3-3 proteins

In Chara corallina cells exposed to continuous light, external pH (pH(o)) and photosystem II (PSII) photochemical yield show correlated banding patterns. Photosynthetic activity is low in cell regions producing alkaline zones and high in the acid regions. We addressed the question whether (and how) photosynthetic activity and plasma membrane (PM) H+-pumping and H+-conductance are coupled in the different bands. First, PM H+-pump activity was stimulated with fusicoccin. This resulted in a more acidic pH in the acid bands without disturbing the correlation of photosynthetic electron transport and H+ fluxes across the PM. Next, H+-pump activity was reduced through microinjection of a phosphorylated peptide matching the canonical 14-3-3 binding motif RSTpSTP in the acid cell region. Microinjection induced a rapid (~5 min) rise in pH(o) by ca. 1.0 unit near the injection site, whereas the injection of the non-phosphorylated peptide had no effect. This pH rise confirms the supposed inhibition of the H+-pump upon the detachment of 14-3-3 proteins from the H+-ATPase. However, the PSII yield in the cell regions corresponding to the new alkaline peak remained high, which violated the normal inverse relations between the pH(o) and PSII photochemical yield. We conclude that the injection of the competitive inhibitor of the H+-ATPase disrupts the balanced operation of PM H+-transport and photosynthetic electron flow and promotes electron flow through alternative pathways.     

Ca2+ and phosphate releases from calcified Chara cell walls in concentrated KCl solution

Ca2+ and P(i) (inorganic phosphate) releases from isolated calcified and uncalcified Chara cell walls were measured with a Ca(2+)-selective electrode and colorimetry, and their ionic relations were analysed on the basis of the electroneutrality rule. The results showed that (1) not only Ca2+ but also P(i) can be released from isolated calcified Chara cell walls into pure deionized water and 100 mM KCl solution, and (2) the positive charge due to the Ca2+ released cannot be neutralized only by the negative charge from the simultaneously released P(i). These findings suggest that calcium bands of calcified Chara cell walls are composed of mainly CaCO(3) and CaHPO(4) and some anions other than P(i) should be released simultaneously with the Ca2+ and P(i). More Ca2+ and P(i) can be solubilized from isolated Chara cell walls in 100 mM KCl solution than in pure deionized water. The pH value of 100 mM KCl solution in which isolated uncalcified young Chara cell walls have been immersed is a little lower than that of pure deionized water in which the same isolated uncalcified young Chara cell walls have been immersed, suggesting that some acidic substances are solubilized by 100 mM KCl. To explain this from the viewpoint of solution chemistry, the solubilities of pure CaCO(3) and pure CaHPO(4) in water and 100 mM KCl solution were measured with a Ca2+ -selective electrode and their pH values with a glass pH electrode. The conclusion reached was that the Ca2+ release from isolated Chara cell walls is accompanied by the release of P(i), CO(2-)(3) and acidic substances. This suggests that the so-called calcium bands and/or ionic relations, including ion exchange, in Chara cell walls are chemically or physicochemically more complex than they are currently considered to be.     

Effect of a Single Excitation Stimulus on Photosynthetic Activity and Light-dependent pH Banding in <Emphasis Type="Italic">Chara</Emphasis> Cells

Using pH microelectrodes and a Micro-scopy PAM (pulse-amplitude modulated) chlorophyll fluorometer, it is shown that a propagation of an action potential in Chara corallina leads to transient suppression of spatially periodic pH profiles along the illuminated cell. The suppression was manifested as a large pH decrease in the alkaline zones and a slight pH increase in the acid zones. The propagating action potential diminished the maximum yield of chlorophyll fluorescence (F_m′) in the alkaline cell regions, as well as the quantum yield of photosystem II photochemistry, without affecting F_m′ in the acid cell regions. The results indicate an interference of membrane excitation in the mechanisms responsible for pH banding patterns in Characean algae. Apparently, the electrical excitation of the plasma membrane in the alkaline cell regions initiates a pathway that can modulate membrane events at the thylakoid membrane.     

Characean charasome-complex and plasmalemma vesicle development

Summary We report on an unusual phenomenon which occurs in some characean algae as a normal plasma membrane activity and also in association with charasome formation. The phenomenon of formation of coated invaginations of the plasma membrane was observed in twoChara and 6Nitella species. These invaginations are coated on their cytoplasmic surface, are 50–60 nm in diameter and rarely exceed 60 nm in length. They are abundant in the young cells ofChara andNitella and also occur in mature cells, but at a lower frequency.N. translucent is an exception in that coated invaginations were few in the young cells and absent in mature cells. Coated vesicles (50–60 nm diameter) were closely associated with these invaginations. Our observations suggest the vesicles may be derived from the invaginations by endocytosis.A close relationship was noted between the development of charasomes (plasmalemma modifications) and coated invaginations. Numerous coated invaginations are seen along the membranes of young charasomes; these invaginations appear to be associated with growth of the charasomes. Coated vesicles were not associated with the coated invaginations of the charasome membrane. The tubular network of cytoplasm and wall space seen in the mature charasome may be formed by fusion of coated invaginations of the developing charasomes, leaving cytoplasmic strands between the fused portions. Coated invaginations were not present along charasomes of the mature cells.     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy

The discovery that the lipids constituting the plasma membrane are not randomly distributed, but instead are able to form laterally segregated lipid domains with different properties has given hints how the formation of such lipid domains influences and regulates many processes occurring at the plasma membrane. While in model systems these lipid domains can be easily accessed and their properties studied, it is still challenging to determine the properties of cholesterol rich lipid domains, the so called “Rafts”, in the plasma membrane of living cells due to their small size and transient nature. One promising technique to address such issues is fluorescence lifetime imaging (FLIM) microscopy, as spatially resolved images make the visualization of the lateral lipid distribution possible, while at the same time the fluorescence lifetime of a membrane probe yields information about the bilayer structure and organization of the lipids in lipid domains and various properties like preferential protein-protein interactions or the enrichment of membrane probes. This review aims to give an overview of the techniques underlying FLIM probes which can be applied to investigate the formation of lipid domains and their respective properties in model membrane and biological systems. Also a short technical introduction into the techniques of a FLIM microscope is given.     

Plasma membrane coat and a coated vesicle-associated reticulum of membranes: their structure and possible interrelationship in Chara corallina

Primary fixation with buffered glutaraldehyde plus 2.0 mM CaCl2 and 0.1% tannic acid results in the preservation of certain portions of the plasma membrane coat of Chara when seen with the electron microscope. Such a coat is not observable after fixation with glutaraldehyde alone. The coat appears to be present on all the above ground, vegetative cells of the male plant. Within complex invaginations of the plasma membrane, which are known as charasomes, the coat has two structural components, a central core that is either tubular or solid and a fibrous or granular peripheral region that surrounds the core. The coat material appears to be at least partially derived, via exocytosis, from the contents of single membrane-bound organelles known as glycosomes. Glycosomes seem to originate from within an assemblage of membranes and coated vesicles that can be described, in purely structural terms, as a partially coated reticulum. Such a reticulum is distinguishable from Golgi stacks because the reticulum (a) is not composed of stacked membranes, (b) is extensively involved with large, clearly detailed coated vesicles and coated invaginations, (c) is closely associated with glycosomes, and (d) is only slightly stained by the zinc-iodide- osmium tetraoxide reagent.     

Modeling of hysteresis in pH pattern formation along the cell membrane of algae Chara corallina

It is known that illumination of the algae Chara corallina results in the formation along the membrane of regions with inhomogeneous distribution of pH. It was shown that, in a particular range of illumination intensities, two states with different pH distribution are realized at one and the same value of light intensity: an entirely homogeneous state and completely formed structures (pattern). The transition from the homogeneous state to the pattern formation takes place at one value of light intensity, and the back transition, at another light intensity, i.e., the hysteresis is observed. This phenomenon was studied by mathematical modeling. The mechanism of hysteresis is discussed.     

Artificial control of cytoplasmic pH and its bearing on cytoplasmic streaming,electrogenesis and excitability of characeae cells

Effects of changing the cytoplasmic pH on the cytoplasmic streaming, membrane potential and membrane excitability were studied in tonoplast-free cells ofChara australis andNitellopsis obtusa. The cytoplasmic pH was varied by internal perfusion of pH-buffered media.Nitellopsis cells were perfused only once, whileChara cells were perfused twice to control the pH more accurately. In both materials the rate of cytoplasmic streaming was maximum at about pH 7, low at pH 8.5–9 and almost zero at pH 5–5.5. The membrane potential was most negative at about pH 7. InChara the membrane potential supported by Mg·ATP was strongly inhibited at pH 5.5, and almost zero at pH 9, supporting the results obtained by Fujiiet al. (1979) on cells ofChara australis which were perfused once. The action potential could be induced by electrical stimulation inChara at pH 6.0–9.0 and inNitellopsis at pH 6.6–7.9. The membrane resistance ofNitellopsis was high at acidic and neutral pH values and low at alkaline pH, while that ofChara was low at both acidic and alkaline pH values.     

Modelling of pattern formation and oscillations in pH and transmembrane potential near the cell membrane of Chara corallina

A mathematical model of potencial-dependent proton transfer across the membrane of Chara corallina cells is considered. To construct the model, partial differential equations describing the system dynamics in time and in space were used. The variables of the model are the proton concentration and membrane potential. The model describes the experimentally observed inhomogeneous distribution of transmembrane potential and pH along the membrane and oscillations of the potential and pH in time. A mechanism of the distribution of pH and membrane potential along the Chara corallina cell is suggested.     

Pil1 Controls Eisosome Biogenesis

The molecular composition of plasma membranes is constantly remodeled by endocytosis and exocytosis. Eisosomes are large cytoplasmic protein assemblies that localize to specialized domains on the yeast plasma membrane. They are of uniform size and immobile, and their disruption leads to large aberrant plasma membrane invaginations and endocytic defects. It is unknown how eisosomes are formed or inherited and what governs their size, distribution, and location. Here we show that eisosomes are formed de novo in the bud of dividing cells. They colonize newly formed membrane at a fixed density in a polarized wave proceeding from the bud neck to the bud tip and become anchored at the site of their formation. Pil1, one of the two main eisosome subunits, emerges as the central regulator of eisosome biogenesis that determines both size and location of eisosomes. Lowering Pil1 expression leads to normal-sized eisosomes at a reduced density, suggesting that eisosomes must be of a minimal size. Conversely, raising Pil1 expression leads to larger eisosomes at a fixed density, suggesting that under these conditions eisosome nucleation sites are limiting. Pil1 expression is regulated by the cell cycle, which synchronizes eisosome formation with plasma membrane growth. Our results establish a first framework of the molecular principles that define eisosome assembly and distribution.     

Microfilaments and microtubules control the shape, motility, and subcellular distribution of cortical mitochondria in characean internodal cells

Summary. The shape, motility, and subcellular distribution of mitochondria in characean internodal cells were studied by visualizing fluorescent dyes with confocal laser scanning microscopy and conducting drug-inhibitor experiments. Shape, size, number, and distribution of mitochondria varied according to the growth status and the metabolic activity within the cell. Vermiform (sausage-shaped), disc-, or amoeba-like mitochondria were present in elongating internodes, whereas very young cells and older cells that had completed growth contained short, rodlike organelles only. Mitochondria were evenly distributed and passively transported in the streaming endoplasm. In the cortex, mitochondria were sandwiched between the plasma membrane and the stationary chloroplast files and distributed in relation to the pattern of pH banding. Highest mitochondrial densities were found at the acid, photosynthetically more active regions, whereas the alkaline sites contained fewer and smaller mitochondria. In the cortex of elongating cells, small mitochondria moved slowly along microtubules or actin filaments. The shape and motility of giant mitochondria depended on the simultaneous interaction with both cytoskeletal systems. There was no microtubule-dependent motility in the cortex of nonelongating mature cells and mitochondria only occasionally travelled along actin filaments. These observations suggest that mitochondria of characean internodes possess motor proteins for microtubules and actin filaments, both of which can be used either as tracks for migration or for immobilization. The cortical cytoskeleton probably controls the spatiotemporal distribution of mitochondria within the cell and promotes their association with chloroplasts, which is necessary for exchange of metabolites during photosynthesis and detoxification.Correspondence and reprints: Arbeitsgruppe Pflanzenphysiologie, Fachbereich Zellbiologie, Universität Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.     

Interaction of preS domains of hepatitis B virus with phospholipid vesicles

The role of preS domains of the hepatitis B virus (HBV) envelope proteins in the first steps of viral infection has been restricted to their implication in virus attachment to a putative hepatocyte receptor. In order to explore a fusion activity in these regions, we used recombinant preS domains to characterize their interaction with liposomes. Binding experiments carried out with NBD-labeled proteins indicated that preS were able to interact in a monomeric way with acidic phospholipid vesicles, being the partition coefficient similar to that described for peptides which can insert deeply into bilayers. Fluorescence depolarization of DPH-labeled vesicles confirmed the specificity for negative charged phospholipids. Upon interaction the proteins induced aggregation, lipid mixing and release of internal contents of acidic vesicles at both acid and neutral pH in a concentration-dependent manner. Taken together, all these data indicate that preS domains are able to insert into the hydrophobic core of the bilayer. Moreover, the insertion resulted in a protein conformational change which increased the helical content. Therefore all these results suggest that, besides their participation in the recognition of a cellular receptor, the preS domains could be involved in the fusion mechanism of HBV with the plasma membrane of target cells.