Acetabularia Acetabulum Research Articles

Improved visualization of F-actin in the green alga Acetabularia by microwave-accelerated fixation and simultaneous FITC-Phalloidin staining.

By employing a new procedure we have been able to visualize a highly intense actin cytoskeleton in the unicellular green alga Acetabularia acetabulum Silva. The protocol described in this study involves microwave-accelerated simultaneous permeabilization with 10% dimethyl sulphoxide, fixation with 1% glutaraldehyde and incubation with 0.5 microM fluorescein-isothiocyanate-conjugated Phalloidin. Comparison of the images of the actin cytoskeleton of the stalk, as visualized by methods used previously, with those obtained in our own experiments shows that the actin filaments were preserved completely in an excellent condition. The required time for each procedure could be reduced from 12 h for the most commonly used immunofluorescence technique to 35 min. Moreover, it has been possible to observe the actin filament system of hair whorls, rhizoid and tip. Previously, the actin cytoskeleton of these parts of the cell could not be visualized by conventional techniques. It is shown that each region of the cell-stalk, tip, rhizoid and side branches-displays characteristic degrees of actin bundling and regularity of actin alignment.

Coordination of cellular events that precede reproductive onset in Acetabularia acetabulum: evidence for a 'loop' in development.

Amputated apices from vegetative wildtype cells of the uninucleate green alga Acetabularia acetabulum can differentiate a reproductive structure of 'cap' in the absence of the nucleus (Hämmerling, J. (1932) Biologisches Zentralblatt 52, 42-61). To define the limits of the ability of wildtype cells to control reproductive differentiation, we determined when during development apices from wildtype cells first acquired the ability to make a cap in the absence of the nucleus and, conversely, when cells with a nucleus lost the ability to recover from the loss of their apices. To see when the apex acquired the ability to make a cap without the nucleus, we removed apices from cells varying either the developmental age of the cells or the cellular volume left with the apex. Cells must have attained the adult phase of development before the enucleate apex could survive amputation and make a cap. Apices removed from cells early in adult growth required more cell volume to make a cap without the nucleus than did apices removed from cells late in adult growth. To define the limits of the cell to recapitulate development when reproduction falters, we analyzed development in cells whose caps either had been amputated or had spontaneously aborted. After loss of the first cap, cells repeated part of vegetative growth and then made a second cap. The ability to make a second cap after amputation of the first one was lost 15-20 days after cap initiation. Our data suggest that internal cues, cell age and size, are used to regulate reproductive onset in Acetabularia acetabulum and add to our understanding of how reproduction is coordinated in this giant cell.

Kurkku, a Phenotype of Acetabularia acetabulum That Is Arrested in Vegetative Growth, Can Be Rescued with Wild-Type Cytoplasm.

We isolated several spontaneous phenotypes in the giant unicell Acetabularia acetabulum that have vegetative terminal morphologies. Because they arrest in vegetative development, these cell lines are effectively immortalized. However, they had to be rescued before they could be studied via classical genetics because no heterozygotes from the original self-crosses were found, that is, the wild-type siblings yielded only wild-type progeny. We attempted to rescue these phenotypes in three ways: by amputating the cell apex, by "piggybacking" the mutant nucleus through development in a binucleate heterokaryon, and by replacing the abnormal apex with a wild-type apex. We used one of our immortal cell lines, kurkku, which has a terminal phenotype consistent with arrest early in the juvenile phase of vegetative development, as a prototype for these rescue methods. The kurkku phenotype segregated 1:3 in the original self-cross in which it arose as if it were a single, recessive Mendelian trait. Although amputation failed to rescue kurkku, we succeeded in compensating for the defect both in binucleate heterokaryons and in apical grafts to wild-type cells. kurkku was always recovered in the progeny of the self-crosses of these grafts. These unique ways of analyzing vegetative mutants, combined with the ability to then perform classical genetics, may make A. acetabulum a powerful unicellular model system for the study of vegetative phase change in plants.

High-resolution measurement of circadian periodicities in Acetabularia.

Well-expressed endogenous circadian rhythms in Acetabularia acetabulum were spectrally analyzed and recorded in time-period distributions. The stability of the circadian periods under constant conditions and their changes could be monitored continually in step sizes close to the circadian period length. The resolution of period estimates of the circadian component was increased by a factor of approximately 4-10 by adapting analyzed interval lengths to full period sizes of the corresponding main component. Methodological aspects of the applied algorithms are discussed by means of examples that measure the temperature dependency of the circadian period.

Chloroplast ATPase in Acetabularia acetabulum: purification and characterization of chloroplast F1-ATPase.

ATPases were isolated from chloroplasts of the unicellular marine alga Acetabularia acetabulum. Two preparations of ATPase, a chloroplast-enriched fraction and an alpha beta gamma-complex were compared. The alpha beta gamma-complex was released into an EDTA solution and purified by anion-exchange chromatography, hydrophobic chromatography, and gel permeation chromatography. The subunit composition of this enzyme appeared to be 52-53 (alpha), 51 (beta), and 40 (gamma) kDa from SDS-PAGE. ATPase activity was enriched about 260-fold to a specific activity of approximate 4.1 U.mg protein-1. The catalytic properties of the alpha beta gamma-complex were as follows: pH optimum at 7.5; substrate specificity, ATP > ITP, GTP > UTP = CTP (Km for ATP 0.2 mM); divalent cation requirement, Mg2+ = Mn2+ = Co2+ > Zn2+ > Ni2+ > Ca2+; ATPase activity was inhibited by monovalent anions (NO3-, SCN-), while monovalent cations had neither inhibitory nor stimulatory effect. Orthovanadate had no inhibitory effect on the enzyme activity of alpha beta gamma-complex. Azide was the most effective inhibitor of the alpha beta gamma-complex. N-Terminal amino acid sequences of the alpha and beta subunits were not obtained and appeared to be blocked. The gamma subunit gave a sequence of AGLKEMKD-XIGSVXNTKKI, which showed 60% similarity to the gamma subunits of spinach and Chlamydomonas reinhardtii CF1-ATPase and EF1-ATPase.

Roles of anions in cells: studies on a Cl(-)-translocating ATPase and sulfate uptake system in Acetabularia acetabulum

Biochemical and molecular biological approaches to two anion translocators, a Cl(-)-translocating ATPase and sulfate permease were described for Acetabularia acetabulum, a unicellular marine alga. The primary structures of an almost complete cDNA clone of the 50 kDa subunit and a partial cDNA clone of the 54 kDa subunit of the Cl(-)-ATPase were highly similar to the beta and alpha subunits of the F type ATPase, respectively. A partial cDNA clone encoding the alpha subunit of chloroplast ATPase, and partial cDNA clones coding for the beta subunits of chloroplast and mitochondrial ATPases were also obtained from A. acetabulum. The presence of a small multigene family for the F type ATPases with different ion specificities was strongly suggested for the organism. Sulfate uptake system in this organism was also studied and partial cDNA clones encoding CysA and sulfate binding proteins were obtained. ca. 1.7 kb RNA for cysA gene and 1.55 kb for sbp gene were detected by Northern analysis, respectively. A putative malK gene was also partially cloned.

Characterization of ion channels from Acetabularia plasma membrane in planar lipid bilayers.

Plasma membrane from Acetabularia acetabulum was prepared by aqueous-polymer two-phase partitioning and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers by stirring in the presence of a (cis:trans) 325:100 mM KCl gradient. Under these conditions five distinct K(+)-selective channels were observed which had unitary chord-conductances (determined between 30 mV either side of the reversal potential) and frequencies of incorporation (in parentheses) of 1,600 pS (26%), 485 pS (21%), 259 pS (53%), 140 pS (37%) and 27 pS (37%). Two Cl(-)-selective channels were also observed, which had unitary chord-conductances of 8 and 48 pS and were present in 21 and 16% of bilayers, respectively. The voltage dependencies of channel open probability (Po), open-state time constant (tau o) and closed-state time constant (tau c) were determined for the 259, 140 and 27 pS K+ channels. The Po of all three channels increased with increasingly positive membrane potentials. Thus, since these channels were oriented with their extracellular face adjacent to the cis chamber, which was grounded, all would exhibit outward rectification in vivo. Changes in Po were effected by modulation of tau c in all channels, which shortened as membrane potentials became more positive, and also of tau o in the 140 and 27 pS channels, which increased as membrane potentials became more positive. Extracellular (cis) KCl concentration (and/or the KCl gradient across the bilayer) affected the Po of all three K+ channels, shifting the Po/membrane potential relationship in the direction of the change in the potassium reversal potential. In all channels this was achieved largely by changes in tau c.

Vanadate-sensitive ATPase in the plasmalemma of Acetabularia: biochemical and kinetic characterization

The electrogenic Cl(-)-pump in the plasmalemma of the marine alga Acetabularia acetabulum (L.) Silva has been suggested to be an unusual type of ATPase (Gradmann, 1989, Methods Enzymol. 174, 490). For a biochemical treatment of this issue, a plasmalemma-rich membrane fraction from Acetabularia has been prepared by phase-partitioning. About 80% of the ATPase activity in this material is inhibited by vanadate (K I50=1-2 μM). The phosphohydrolytic properties of the corresponding enzyme were further investigated. Its primary substrate MgATP(2-) (K m about 270 μM). Compared with other plasmalemma ATPases, it has an extremely alkaline pH optimum (pH 8-8.5), a weak sensitivity to diethyl-stilbestrol and to N,N'-dicyclohexylcarbodiimide, a strong selectivity for Mg(2+) over alternative divalent cations, and a weak selectivity for ATP over other phosphohydrolytic substrates. It is insensitive to KCl at concentrations up to 200 mM. Both ATP(4-) and Mg2ATP inhibit the ATPase, satisfying a relationship for competitive inhibition by 2ATP(4-) (K IATP=1.56 mM) and noncompetitive inhibition by Mg2ATP (K IMg2ATP=1.35 mM). Since no transport experiments are reported in this study, the ion species (H(+) or Cl(-)) that is transferred by this ATPase is not identified.

Improvement of reconstitution of the Cl −-translocating ATPase isolated from Acetabularia acetabulum into liposomes and several anion pump characteristics

The improved reconstitution of the Mono Q-III fraction, a Cl −-translocating ATPase, isolated from Acetabularia acetabulum (Ikeda et al. (1990) Biochemistry 29, 2057–2065) into liposomes rendered transport properties of this enzyme clear. The liposomes were prepared by the reversed-phase method using egg lecithin and cholesterol in a molar ration of 2:1 and the purified ATPase was incorporated into the liposomes by a dialysis for 3 h. About 80% of the ATPase was incorporated into the liposomes. The weight ratio of the enzyme to lipid was 1:400–600. A sigmoid curve was obtained when the Cl −-transport activity of the enzyme was plotted against Cl − concentration. Hill's plot afforded a half-subsrate concentration [S] 0.5 of 45 mM and a Hill's coefficient n of 2.33. Effects of Br − and F − on the Cl −-transport were also examined in the reconstituted system, both halide ions decrease the 36Cl − efflux significantly. These kinetic data are in good agreement with the electrophysiological data presented by Tittor et al. (1983) J. Membr. Biol. 75, 129–139).

Dynamics of a peculiar plant-herbivore relationship: the photosynthetic ascoglossan Elysia timida and the chlorophycean Acetabularia acetabulum

The ascoglossan mollusc Elysia timida Risso, 1818 retains functional chloroplasts from its algal food, the chlorophycean Acetabularia acetabulum (L.). Photosynthates from the plastids are an important source of organic nutrients for the mollusc. Chloroplast exploitation has an ecological function, allowing the ascoglossan to live entirely on an algal diet which is of limited, seasonal availability to other herbivores. Between October 1987 and July 1988, the annual evolution of the molluscan and algal populations was studied in a cove of Mazarron Bay, southeast Spain. The population density of the mollusc is highly dependent on its food supply, being controlled by the seasonal life cycle of the algal population. During its life cycle, the degree of grazing by the mollusc decreases with increasing algal calcification, the cell walls of the alga progressively calcify, and the eventually highly calcificied stalks are completely resistant to ascoglossan grazing. In contrast, the exploitation of the algal chloroplasts retained by the molluscs increases during the seasonal cycle. The progressively increasing scarcity of food during the seasonal cycle may have led to the retention of symbiotic chloroplasts by E. timida. The developmental strategy of the ascoglossan also changes during the year: when food is abundant (in November, December, January, February and March) it is direct, with no planktonic larval phase, when food is scarce (in October, April, May and June) it is lecithotrophic, with a short planktonic larval phase. Chloroplast retention acts as a buffer, alleviating the effects of annual changes in density, structure and abundance of the alga on the nutritional state of the molluse.

Cytoplasmic Free Ca2+in the Marine Alga Acetabularia: Measurement with Ca2+–selective Microelectrodes and Kinetic Analysis

Neutral carrier-based Ca 2+ -selective microelectrodes have been examined for application in concentrated multi-ion solutions, Calculations with data from the literature and our calibration series with Ca 2+ -EGTA buffers (a convenient algorithm for their calculation is given) provide the physico-chemical conditions for determination of submicromolar concentrations of free Ca 2+ in the cytoplasm (with about 400 mM K + and 70 mM Na + ) of the marine alga Acetabularia acetabulum. The experimental results give a cytoplasmic concentration of 560 nM free Ca + corresponding to 140 nM activity

A vacuolar ATPase and pyrophosphatase in Acetabularia acetabulum

Vacuole-rich fractions were isolated from Acetabularia acetabulum by Ficoll step gradient centrifugation. The tonoplast-rich vesicles showed ATP-dependent and pyrophosphate-dependent H +-transport activities. ATP-dependent H +-transport and ATPase activity were both inhibited by the addition of a specific inhibitor of vacuolar ATPase, bafilomycin B 1. A 66 kDa polypeptide present in the preparation cross-reacted with the anti-IgG fractions against the α and β subunits of Halobacterium halobium ATPase and with the antibody against the A subunit (68 kDa subunit) of mung bean vacuolar ATPase. A 56 kDa polypeptide present in the vacuole preparation showed cross-reactivity with the antibody against the B subunit (57 kDa) of mung bean vacuolar ATPase but not with the anti-β subunit of H. halobium ATPase. A 73 kDa polypeptide cross-reacted with the antibody against inorganic pyrophosphatase of mung bean vacuoles. These results suggest that vacuolar membrane of A. acetabulum equipped energy transducing systems similar to those found in other plant vacuoles.

A vibrating electrode analysis of extracellular ion currents in Acetabularia acetabulum

ABSTRACT An investigation of extracellular ionic currents in Acetabularia acetabulum is presented. Utilising the vibrating electrode technique it is shown that there are large (up to 380μAcm-2) light-dependent and small (up to lOμAcm-2) light-independent extracellular ionic currents around Acetabularia. The current density was greatest at the rhizoid and diminished towards the developing apex where there was no measurable current. Ion-replacement and ion-transport inhibitor studies indicated that Cl-was the main constituent of the light-dependent and light-independent currents. Calcium ions were found to contribute about 5–15% of the light-independent current and <1% of the light-dependent current, as deduced from cobalt inhibition studies. Possible mechanisms responsible for these phenomena are discussed briefly.

A chloride-translocating adenosine triphosphatase in Acetabularia acetabulum. 2. Reconstitution of the enzyme into liposomes and effect of net charges of liposomes on chloride permeability and reconstitution

The Mono Q-III fraction, a Mg2(+)-ATPase, isolated from Acetabularia acetabulum was reconstituted into liposomes of various net charges prepared by the reversed-phase method and tested for a Cl(-)-translocating activity. The liposomes from a mixture of egg lecithin, dicetyl phosphate, and cholesterol (63:18:9 mole ratio, negative liposomes) and from a mixture of egg lecithin and cholesterol (63:9 mole ratio, neutral liposomes) were less leaky than positive liposomes from asolectin, and from a mixture of egg lecithin, stearylamine, and cholesterol (63:18:9 mole ratio). A significant increase in 36Cl- efflux from the negative and neutral liposomes was observed by addition of ATP in the presence of valinomycin after incorporation of the enzyme by short-term dialysis. The ATP-driven 36Cl- efflux was inhibited by addition of azide, an inhibitor of the ATPase. The preincubation of the enzyme with phenylglyoxal, an arginine-modifying reagent, inactivated ATP-mediated 36Cl- efflux, but the ATPase activity of the preparation was not affected. When chloride was replaced by 35SO4(2)-, no ATP-dependent 35SO4(2)- efflux was detectable from the proteoliposomes. Proton-translocating activity of the enzyme was also tested, and no fluorescent quenching of 9-ACMA was observed.

Response of the unicellular giant algaAcetabularia Acetabulumto cadmium toxicity and accumulation∗

Accumulation of cadmium by the unicellular marine alga Acetabularia acetabulum (=A. mediterranea) was studied by using the radioactive isotope 109Cd. Column chromatography (Sephacryl S‐200) of labelled extracts showed that after a short incubation (1–2 h) 109Cd was eluted at the position characteristic for ionic cadmium and for low molecular weight compounds. However, after a longer incubation (one week or more), 109Cd was present also in high molecular weight molecules. Cadmium is very toxic to Acetabularia: cell growth and cap formation were reduced at concentration as low as 1 μg L‐1. For concentrations higher than 0.1 mg L‐1, various morphological anomalies appeared, such as formation of short and thick stalks and condensation of cytoplasm in apical or basal parts, eventually followed by cytolysis.

Graft incompatibility between Acetabularia and Batophora (Dasycladales, Rhodophyta)

Abstract Graft experiments between Acetabularia acetabulum L. (Silva) and Batophora oerstedii J. Agardh have shown that incompatibility exists between these two Dasycladaceae. The grafted nucleated basal fragments of Acetabularia die or eventually regenerate a new cell. The enucleated halves of Batophora survive for more than 3 months and develop numerous sporangia which remain sterile.

The effects of blue and red light on the transcellular electric potential, cytoplasmic streaming and rRNA transport in Acetabularia acetabulum

The effects of blue and red light on the transcellular electric potential, cytoplasmic streaming and rRNA transport in Acetabularia acetabulum