Possible involvement of the membrane potential in the gravitactic orientation of Euglena gracilis

Euglena gracilis, a unicellular photosynthetic flagellate, uses light and gravity as environmental hints to reach and stay in regions optimal for growth and reproduction. The current model of gravitaxis (the orientation with respect to the earth's gravitational field) is based on the specific density difference between cell body and medium. The resulting sedimentation of the cell body applies a force to the lower membrane. This force activates mechano-sensitive ion channels. The resulting ion flux changes the membrane potential, which in turn triggers reorientational movements of the trailing flagellum. One possibility for recording the predicted membrane potential changes during reorientation is the use of potential-sensitive dyes, such as Oxonol VI. The absorption changes of the dye indicating potential changes were recorded with a custom-made photometer, which allows a high precision measurement with a high temporal resolution. After a gravitactic stimulation, a short period of hyperpolarization was detected, followed by a massive depolarization of the cell. The membrane potential returned to initial values after a period of approximately 200 s. Parallel measurements of the precision of orientation and the membrane potential showed a close relationship between both phenomena. The obtained results support the current model of gravitaxis of Euglena gracilis     

A simple colorimetric method for detecting cell viability in cultures of eukaryotic microorganisms

The dye methylene blue can be taken up by dead or severely damaged cells, but not by living cells. Based on this fact, a method was devised which permits quantitative determinations of injured cells in populations of microorganisms such asSaccharomyces cerevisiae, Rhodotorula glutinis, andEuglena gracilis. The percentage of damaged cells was determined by measuring, at 664 nm, the optical density of cell suspensions pretreated with 0.15 mM methylene blue for 6 min, a condition that does not affect cell integrity as determined by oxygen consumption and release of potassium ions. This technique is faster and simpler than the classical dye-exclusion and plate-counting methods.     

A quantitative resolution of the spectra of a membrane potential indicator,diS-C3-(5), bound to cell components and to red blood cells

Summary Cationic cyanine dyes have been widely used to measure electrical potentials of red blood cells and other membrane preparations. A quantitative analysis of the binding of the most extensively studied of these dyes, diS-C₃-(5), to red blood cells and their constituents is presented here. Absorption spectra were recorded for the dye in suspensions of isolated red cell membranes and in solutions of cell lysate. The dependence of the spectra on the concentrations of dye and cell constituents shows that the dye binds to these membranes as monomers with an absorbance maximum at 670 nm instead of 650 nm as for free aqueous dye and that the dye binds to oxyhaemoglobin partly as monomer but primarily as dimer, with absorbance maxima ca. 670 and 595 nm, respectively. Quantitative estimates are derived for all binding constants and extinction coefficients. These estimates are applied to suspensions of whole cells to predict the dye binding, absorbance spectra, and calibration curves of binding and fluorescencevs. membrane voltage. Satisfactory agreement is found with binding and absorbance data for whole cells at zero membrane potential and with the binding and fluorescence data reported by Hladky and Rink (J. Physiol. (London) 263:287, 1976) for cells driven to positive and negative potentials using valinomycin. The marked tendency of oxyhaemoglobin to bind dye as dimer is not shared by some other proteins tested, including deocyhaemoglobin and oxymyoglobin.     

Photodynamic action of merocyanine 540 on carcinoma of cervix cells

Results of the studies carried out on localization and photodynamic action of merocyanine 540 (MC540) on carcinoma of cervix (HeLa) cells are presented. Fluorescence microscopic study showed that when HeLa cells were incubated with MC540 in dark, the dye localized in plasma membrane of cells. Photoirradiation of cells in presence of MC540 led to enhancement of dye uptake, intracellular localization of dye and a dose dependent decrease in cell survival. Clonogenic assay showed 96% cell killing at a light dose of 42 kJ/m2. Photosensitization of cells resulted in loss of membrane integrity, decrease in plasma membrane fluidity and reduction in mitochondrial dehydrogenase activity as measured by tetrazolium reduction (MTT) assay. At a given light dose, the relative change in plasma membrane properties was higher than the reduction in activity of mitochondrial enzyme. These results suggest plasma membrane is a primary target of photosensitization of HeLa cells by MC540.     

Possible involvement of the membrane potential in the gravitactic orientation of Euglena gracilis

Euglena gracilis, a unicellular photosynthetic flagellate, uses light and gravity as environmental hints to reach and stay in regions optimal for growth and reproduction. The current model of gravitaxis (the orientation with respect to the earth's gravitational field) is based on the specific density difference between cell body and medium. The resulting sedimentation of the cell body applies a force to the lower membrane. This force activates mechano-sensitive ion channels. The resulting ion flux changes the membrane potential, which in turn triggers reorientational movements of the trailing flagellum. One possibility for recording the predicted membrane potential changes during reorientation is the use of potential-sensitive dyes, such as Oxonol VI. The absorption changes of the dye indicating potential changes were recorded with a custom-made photometer, which allows a high precision measurement with a high temporal resolution. After a gravitactic stimulation, a short period of hyperpolarization was detected, followed by a massive depolarization of the cell. The membrane potential returned to initial values after a period of approximately 200 s. Parallel measurements of the precision of orientation and the membrane potential showed a close relationship between both phenomena. The obtained results support the current model of gravitaxis of Euglena gracilis.     

Changes of the plastid system of Euglena gracilis induced with streptomycin and dihydrostreptomycin

Summary Streptomycin-like antibiotics cause hereditary and irreversible aplastidity of Euglena gracilis by inhibiting the replication of plastids, while normal cell division is maintained.Therefore, a gradual dilution of plastids takes place in a multiplying culture. Streptomycin was found to be more effective as bleaching agent than dihydrostreptomycin. The cells of Euglena gracilis are totally deprived of plastids by streptomycin treatment after 4.5 cell divisions, while 9 cell divisions are required with dihydrostreptomycin. In addition to the inhibition of plastid replication both antibiotics bring about formation of pathological plastids, both in growing and in stationary cultures. In this latter case pathological plastids are released from cells only after further cell division has taken place.     

Fluorescence marking of neuropile glial cells in the central nervous system of the leech Hirudo medicinalis

Summary Neuropile glial (NG) cells in the central nervous system of the medicinal leech, Hirudo medicinalis L., were studied by histological and intracellular electrophysiological methods. Potential profiles of single leech ganglia were mapped by advancing an electrolyte-filled microelectrode into the ganglion as far as the NG cell. A small negative potential usually appeared during or immediately after penetration of the ganglion sheath. Most of the ganglia in the chain (ganglia 1–4 and 7–21) have Retzius-cell-bodies of normal size; in these, the potential associated with the ganglion sheath was followed by a jump to a more negative potential. Superimposed action potentials were associated with entry of the electrode into a Retzius cell. When the electrode tip passed out of the cell into the center of the ganglion, another potential change was observed, namely that to the membrane potential of the anterior NG cell. This membrane potential averaged -60.2 mV and ranged from -50 to -73 mV. In ganglia 5 and 6 the Retzius-cell-bodies are particularly small, and no changes of potential associated with these cells were observed; the first potential to appear after the electrode passed through the sheath of the ganglion was the membrane potential of the NG cell. Potential profiles like those of ganglia 5 and 6 are recorded in the posterior parts of all ganglia.Potential profiles of single leech ganglia were also recorded with microelectrodes filled with the fluorescent dye Procion Yellow M4-RAN. When the presumed membrane potential of an NG cell appeared, the dye was injected into the ganglion. Subsequent histological examination with the fluorescence microscope revealed that all of the dye was contained in NG cells.Supported by a Fellowship (Heisenberg-Stipendium, Schl 169/5) and grants (Schl 169/2, 4) to W.R.S. from the Deutsche ForschungsgemeinschaftThe authors thank Gisela Geiger for excellent assistance during this work     

Morphological change of cell-membrane-integrated crystalline structure induced by cell shape change inEuglena gracilis

Summary Fourier transform and an image filtering technique were used for structural analysis of the pellicular strip inEuglena gracilis. Freeze-fracture images of a two-dimensional crystalline structure in the plasma membrane were taken from elongated and rounded cells. Their lattice constants were precisely determined from Fourier transform patterns, and masked filter images were generated. Although differences in their lattice constants could not be detected, the Fourier transform patterns showed a significant difference between these two stages of cell shape. In elongated cells, the pseudo-crystal lattice was generated in the half periodicity of the minor striation. In contrast, it disappeared when the cells became rounded. The filtered images confirmed the crystallographic disagreement between these two stages in cell shape, probably reflecting morphological changes of integrated plasma membrane particles. Four particles detected in a unit structure showed quite similar shapes across the pseudo-lattice in elongated cells, though a unit structure in rounded cells consisted of four heterogeneous particles with opaque boundaries. A 39 kDa integral plasma membrane protein (IP39) is known as a major component in the plasma membrane ofE. gracilis. The compositions of monomeric and oligomeric forms of IP39 were determined in both elongated and rounded cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed the possibility that an oligomerization of IP39 proceeds during rounding-up movement of the cell. These morphological and stoichiometrical changes of the integrated plasma membrane proteins may suggest an active involvement in cell shape change.     

Effects of Pro --> peptoid residue substitution on cell selectivity and mechanism of antibacterial action of tritrpticin-amide antimicrobial peptide

To investigate the effect of Pro --> peptoid residue substitution on cell selectivity and the mechanism of antibacterial action of Pro-containing beta-turn antimicrobial peptides, we synthesized tritrpticin-amide (TP, VRRFPWWWPFLRR-NH(2)) and its peptoid residue-substituted peptides in which two Pro residues at positions 5 and 9 are replaced with Nleu (Leu peptoid residue), Nphe (Phe peptoid residue), or Nlys (Lys peptoid residue). Peptides with Pro --> Nphe (TPf) or Pro --> Nleu substitution (TPl) retained antibacterial activity but had significantly higher toxicity to mammalian cells. In contrast, Pro --> Nlys substitution (TPk) increased the antibacterial activity but decreased the toxicity to mammalian cells. Tryptophan fluorescence studies indicated that the bacterial cell selectivity of TPk is closely correlated with a preferential interaction with negatively charged phospholipids. Interestingly, TPk was much less effective at depolarizing of the membrane potential of Staphylococus aureus and Escherichia coli spheroplasts and causing the leakage of a fluorescent dye entrapped within negatively charged vesicles. Furthermore, confocal laser-scanning microscopy showed that TPk effectively penetrated the membrane of both E. coli and S. aureus and accumulated in the cytoplasm, whereas TP and TPf did not penetrate the cell membrane but remained outside or on the cell membrane. These results suggest that the bactericidal action of TPk is due to inhibition of the intracellular components after penetration of the bacterial cell membrane. In addition, TPK with Lys substitution effectively depolarized the membrane potential of S. aureus and E. coli spheroplasts. TPK induced rapid and effective dye leakage from bacterial membrane-mimicking liposomes and did not penetrate the bacterial cell membranes. These results suggested that the ability of TPk to penetrate the bacterial cell membranes appears to involve the dual effects that are related to the increase in the positive charge and the peptide's backbone change by peptoid residue substitution. Collectively, our results showed that Pro --> Nlys substitution in Pro-containing beta-turn antimicrobial peptides is a promising strategy for the design of new short bacterial cell-selective antimicrobial peptides with intracellular mechanisms of action.     

Fluorescence staining of living cells with fluorescamine

Summary The interaction of fluorescamine with living plant and animal cells was investigated to determine which subcellular structures and molecular species might react with the dye and to assess its effects on cell viability and function.Plasma and nuclear membranes ofXenopus erythrocytes, mitochondria of sea urchin sperm, growing apices of Timothy root hairs, and various organelles ofNitella andEuglena were labelled as judged by fluorescence microscopy. Cytoplasmic fluorescence was particulate inNitella and easily displaced by moderate centrifugal fields in sea urchin eggs. Chloroplasts and nuclei isolated from cells labelledin vivo exhibited fluorescamine dependent fluorescence.Reaction seemed to have little or no effect on cell viability (Euglena) photoautotrophic growth (Euglena), cell motility (sperm), fertilizability (sperm or egg), embryonic development (sea urchin), or cytoplasmic streaming (Nitella, Timothy).Quantitative fluorometric analysis of thein vivo reactants in sperm indicated a reaction preference for phospholipid over protein compared to control cells dissociated in SDS prior to labelling. The bulk of labelled lipid was phosphatidylethanolamine.These results suggest that fluorescamine is a true vital dye which can label the cell surface as well as penetrate deeply within cells to label a variety of organelles. The distribution of fluorescence and results of chemical analysis suggest thatin vivo the dye may preferentially react with membrane.     

ELECTROPHYSIOLOGICAL INVESTIGATIONS OF THE RED ALGA GRIFFITHSIA PAGIFIG AKYL. 1

The membrane potentials of the plasmalemma and tonoplast in Griffithsia pacifica Kyl were measured and shown to be significantly different. No consistent cell wall potential was discovered. Adjacent cells in the filament were demonstrated to be electrolonically coupled with a mean coupling coefficient of 0.13. Cobaltions did not move between cells. No dye coupling was seen using the fluorescent dye Lucifer yellow CH. These observations provide new information on the electrophysiology of red algae and indicate that there is intercellular continuity between the cells of a filament of Griffithsia pacifica.     

9-Aminoacridine, a fluorescent probe of the thiamine carrier in yeast cells

The uptake of 9-aminoacridine is studied in the yeast Saccharomyces cerevisiae by fluorescence and absorbance measurements of the dye. Uptake of the dye proceeds via two pathways. One pathway consists of a diffusion of the non-protonated form. At high pH (7.5) this pathway is the predominant one, and the dye distributes between the cell inner and the medium according to the ratio of the proton concentrations in the two compartments. In other words, at high pH 9-aminoacridine behaves as a probe of the H⁺ gradient across the yeast cell membrane. At low external pH (4.5) a second pathway is involved. Much greater accumulation ratios for the dye are observed than can be accounted for by the H⁺ gradient across the membrane. The transport system predominantly responsible for the great accumulation of the dye appears to be inducible, to require metabolic energy and to be saturable. This transport system is competitively inhibited by thiamine, and also by dibenzyldimethylammonium and thiaminedisulfide, two specific inhibitors of the thiamine carrier in the yeast. On the other hand, the thiamine uptake by the yeast cells is competitively inhibited by 9-aminoacridine. In addition, uptake of 9-aminoacridine is greatly reduced in the thiamine transport-negative mutant of S. cerevisiae, PT-R2. It is concluded that at low pH 9-aminoacridine is taken up by yeast via the thiamine carrier of the cell and that, consequently, the dye may be applied as a probe of this transport system.     

Suppression of the plasma membrane H<Superscript>+</Superscript>-conductance on the background of high H<Superscript>+</Superscript>-pump activity in dithiothreitol-treated <Emphasis Type="Italic">Chara</Emphasis> cells

Photosynthesizing cells of characean algae exposed to light are able to produce pH bands corresponding to alternate areas with dominant H⁺-pump activity and high H⁺-conductance of the cell membrane. The action potential generation temporally arrests the counter-directed H⁺ fluxes, which gives rise to opposite pH shifts in different cell regions and represents a suitable indicator for activities of the plasma membrane H⁺-transporting systems. Measurements of pH near the cell surface by means of microelectrodes and microspectrophotometry in the presence of pH-indicating dye thymol blue have shown that the treatment of cells with dithiothreitol (SH-group reducing agent) suppresses pH changes induced by the action potential generation in the alkaline cell areas and considerably increases the concurrent pH changes in the acid regions. Measurements of plasma membrane resistance in the alkaline zones revealed that dithiothreitol inhibits the light-dependent conductance of the resting cell and diminishes the conductance inactivation caused by the action potential generation. The data suggest that the reduction of accessible disulfide bonds results in the decrease of H⁺-conductance, whereas the activity of plasma membrane H⁺-pump remains unimpaired or is even enhanced.     

EFFECTS OF DARKNESS AND OF STREPTOMYCIN ON THE FINE STRUCTURE OF EUGLENA GRACILIS

Siegesmund , Kenneth A., Walter G. Rosen , and Stanley R. Gawlik . (Marquette (J., Milwaukee, Wis.) Effects of darkness and of streptomycin on the fine structure of Euglena gracilis. Amer. Jour. Bot. 49 (2) : 137–145. Illus. 1962.—Dark-grown Euglena gracilis cells, transferred from streptomycin (SM)-containing medium to SM-free medium 5 days before transfer to light, turn green normally, indicating that proplastids are unaffected by SM. SM-bleached cells, grown in light, contain numerous bodies composed of concentric lamellae (CL bodies). These differ from chloroplasts in that their lamellae lack electron-dense dots, are not coalesced, and they lack a 3-layered limiting membrane and pyrenoids. CL bodies are absent from dark-grown normal and dark-grown SM-bleached cells, as well as from light-grown normal cells. It is suggested that CL bodies result from a derangement of chloroplast synthesis caused by SM blockage of chlorophyll synthesis.     

The effect of the fluorescent probe, 3,3′-dipropylthiodicarbocyanine iodide,on the membrane potential of Ehrlich ascites tumor cells

The effects of the potential-sensitive fluorescent dye, 3,3′dipropylthiodicarbocyanine iodide, on factors establishing the membrane potential of Ehrlich ascites tumor cells have been tested. The dye itself induces membrane hyperpolarization as monitored by electrophysiological methods. In addition, the dye inhibits active (Na⁺+K⁺-transport and increases cell membrane permeability to K⁺ by about 65% in these cells.     

Evaluation of cell damage caused by cold sampling and quenching for metabolome analysis

Cell damage during sampling and quenching for metabolome analysis have been investigated at whole sample level using an OD-based method and ATP loss investigation, and at single cell level by means of flow cytometry. Escherichia coli was cultivated in shake flasks and sampled into several cold quenching solutions during exponential growth phase varying quenching solution composition and sampling temperature. For single cell analysis, the samples were incubated with selective propidium iodide dye and analysed via flow cytometry to differentiate between intact and damaged cells. It was found that every combination of quenching solution, temperature, or cooling rate tested influenced the E. coli cell membrane integrity indicating rupture which will not only let the dye in, but also intracellular ATP out of the cells, which is not desired in in vivo metabolome analysis.     

Antibiotics and apochlorosis

Hydroxylamine, an inhibitor of deoxyribonucleic acids (DNA), ribonucleic acids (RNA) and proteosynthesis interferes with the bleaching effect of streptomycin on growing cells ofEuglena gracilis. The addition of hydroxylamine to a green autotrophic culture ofEuglena gracilis inhibits, depigmentation of the culture by streptomycin. Otherwise, streptomycin alone, without, hydroxylamine, is a powerful bleaching agent and when added to a growing culture ofEuglena gracilis, transforms the green, autotrophic cells to permanently colourless, heterotrophic cells. Phenethyl alcohol, an inhibitor of RNA synthesis, and chloramphenicol, an inhibitor of proteosynthesis, do not block the bleaching effect of streptomycin. It can be concluded from these results that the bleaching effect of streptomycin is related to its interference in the plastid DNA.     

Uptake of the fluorescent indicator atebrin into acidic vacuoles in the halotolerant alga Dunaliella satina

The fluorescent indicator atebrin (3-chloro-9-(4-diethylamino-1-methylbutyl)-7-methyoxy-acridine) is taken up by Dunaliella salina cells at alkaline external pH and accumulates in acidic vacuoles. The uptake is unaffected by light, by photosynthetic inhibitors, by protonophores or by ionophores; however, the dye can be released by amines, indicating that it is specifically accumulating in acidic vacuoles. Amines induce a biphasic enhancement of atebrin fluorescence — a fast phase, accompanied by redistribution within the cell, consistent with release of the dye from the vacuoles to the cytoplasm, and a slow phase, correlated with release of atebrin from the cells. These results are interpreted to indicate a slow equilibration of atebrin across the plasma membrane and a fast equilibration across the vacuolar membrane. Part of the dye cannot be released by the amines, and appears to be internally bound. Atebrin uptake is inhibited by cholesteryl hemisuccinate and is stimulated by lysophosphatidylcholine, indicating that modification of the lipid composition of the plasma membrane affects the permeability to atebrin. Analysis of the pH dependence of atebrin uptake indicates that the dye enters the cells by fluid-phase permeation. Different stresses enhance the rate of atebrin uptake and release, indicating that they modify plasma-membrane structure or composition. Atebrin may serve as a specific marker for acidic vacuoles, as an indicator for amine uptake, and as a probe for subtle changes in the permeability of the plasma membrane.Abbreviations Atebrin 3-chloro-9-(4-diethylamino-1-methylbutyl)-7-methoxy-acridine - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea - SF-6847 3,5-ditertbutyl-4-hydroxybenzylidenemalonitrile     

Antimicrobial action of N‐(n‐dodecyl)diethanolamine on Escherichia coli: effects on enzymes and growing cultures

Aims: This study investigates the effects of N‐(n‐dodecyl)diethanolamine (DDA) on enzymes and growing cells of Escherichia coli NCIMB 8277. Methods and Results: Enzyme activities in the presence of DDA were determined by measuring substrate‐dependent oxygen consumption by whole cells, or of NADH formation or oxidation by cell extracts. Lysis of growing cells was followed by measuring changes in turbidity and cell count. DDA promptly arrested oxygen uptake on pyruvate and acetate, due to cofactor loss rather than to enzyme denaturation, since cell‐free glyceraldehyde‐3‐phosphate and NADH dehydrogenases remained active. Formate and succinate oxidation by membrane‐bound enzyme systems independent of cofactors was likewise unaffected. DDA lysed growing cells at rates related to drug concentration, pH, and the previous growth rate. Conclusions: Loss of cellular enzyme activity following addition of DDA is due to cofactor leakage and not to enzyme denaturation. Whereas nongrowing cells remain intact in the presence of DDA, actively‐growing organisms undergo lysis, consistent with autolysin action. Significance and Impact of the Study: Cell lysis, not normally observed with membrane‐active antimicrobials, also occurs with cetrimide, and may be dependent on the alkyl chain length in these compounds. The action on growing cells parallels that of penicillin and daptomycin, which bears a decanoyl residue that penetrates the cell membrane, causing leakage and membrane depolarization.