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PubMed1991

Action potentials in Acetabularia: measurement and simulation of voltage-gated fluxes.

Amounts and temporal changes of the release of the tracer ions K+ (86Rb+), 22Na+, and 36Cl- as well as of H+ in the course of action potentials in Acetabularia have been recorded. New results and model calculations confirm in quantitative terms the involvement of three major ion transport systems X in the plasmalemma: Cl- pumps, K+ channels, and Cl- channels (which are marked in the following by the prefixes, P, K and C) with their equilibrium voltages XVe and voltage/time-dependent conductances, which can be described by the following, first approximation. Let the maximum (ohmic) conductance of each of the three populations of transporter species be about the same (pL, KL, CL = 1) but voltage gating be different: the pump (pVe about -200 mV) being inactivated (open, o----closed, c) at positive going transmembrane voltages, Vm; the K+ channels (KVe about -100 mV) are inactivated at negative going Vm; and the Cl- channels (CVe: around 0 mV), which are normally closed (c) at a resting Vm (near pVe) go through an intermediate open (o) state at more positive Vm before they enter a third "shut" state (s) in series. Model calculations, in which voltage sensitivities are expressed by the factor f = exp(VmF/(2RT], simulate the action potential fairly well with the following parameters (pkco: 10/fks-1, pkoc: 1000.f ks-1, Kkco: 200.f ks-1, Kkoc: 2/f ks-1, ckco: 500.f ks-1, fkoc: 5/f ks-1, Cks0: 0.1/f ks-1,Ckos: 20.f ks-1). It is also shown that the charge balance for the huge transient Cl- efflux, which frequently occurs during an action potential, can be accounted for by the observation of a corresponding release of Na+.

Acetabularia↗
PubMed1975

Germination of cysts in acetabularia mediterranea.

Techniques for the growth of uniformly reacting populations of cysts of Acetabularia mediterranea and for quantitative measurement of cyst germination have been developed. Cysts of A. mediterranea can be induced to germinated by exposure to the atmosphere. Germination rates are very low in young cysts. They increased during exposure to total darkness. This "maturation of cysts" is found to be completed after a period of 12-15 weeks. Germination rates of cysts that have passed the maturation period exceed 90 percent in continuous white light and 80 percent in darkness. Cysts germinate in less than two days in darkness and less than four days in light. The influence of temperature at a range of 15 degrees C to 25 degrees C on germination kinetics is studied in light and darkness. Germination is accelerated with increasing temperature up to 21 degrees C. At higher temperature germination is delayed in light but the time of germination remains constant in darknesss. Rates of germination are not altered by the influence of temperature in light while in darkness there is a dramatic decrease at temperatures higher than 21 degrees C. From these findings it is concluded that cyst germinationA. mediteranea does not need any light but is influenced by light dependent systems. The influence of light is strongest at elevated temperatures.

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PubMed1983

On-line data acquisition and evaluation of long-term measurements of circadian rhythms in individual cells of Acetabularia.

A multitasking time-sharing computer system was implemented for studies of different circadian rhythms in individual cells of the unicellular green alga, Acetabularia. This fully automatized system allows simultaneous data acquisition and analysis. Graphical presentation of untreated and mathematically treated data is permanently available on three graphic displays and on a digital plotter. The sampling rate for the data acquisition in each of the 60 channels connected to the system is 720/24 h. Provisions have been made to guarantee uninterrupted data uptake for these long-term measurements by including an auto-restart module and by providing extremely reliable software for the experimenter using menu techniques.

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PubMed1999

The roles of light and the nucleus in the regulation of reproductive onset in Acetabularia acetabulum.

At reproductive onset the marine green alga Acetabularia acetabulum (L.) P.C. Silva redirects growth from vertical elongation of the axis of the plant to lateral expansion of a disk-shaped reproductive structure, the "cap." We used amputation to synchronize cap initiation and to facilitate investigation of the light requirements during amputation-induced cap initiation. Following amputation of a nascent cap, most plants initiate one whorl of vegetative hairs and then a cap. Both hair and cap initiation required photosynthesis, as indicated by studies with 3-(3',4'-dichlorophenyl)-1, 1-dimethylurea, but did not require the nucleus. Amputation-induced hair initiation occurred in red light, but 10 min of blue light given in a background of red light significantly increased hair initiation, supporting previous studies that hair initiation is a blue-light-triggered photomorphogenic event. Amputation-induced cap initiation also occurred in red light, but daily 10-min flashes of blue light given in a background of red light did not significantly enhance cap initiation. We also examined the light requirements of intact plants at each phase of development. In the absence of blue light, juveniles and adults with </=13.7 +/- 4.3 whorls of hairs arrested in development and failed to initiate caps. In contrast, very late adults with >/=13.7 +/- 4.3 whorls of hairs initiated caps in the absence of blue light, suggesting that there is a point in late adult development beyond which cap initiation does not require blue light. Several plausible interpretations of the role of light and the nucleus in the regulation of reproductive onset are discussed to try to reconcile these data with those in the literature.

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PubMed2001

Relationships between growth, morphology and wall stress in the stalk of Acetabularia acetabulum.

With carbon particles we analyzed patterns of growth in Acetabularia acetabulum (Lam.) P.C. Silva, a giant unicell famous for classic development studies. We focused on the stalk apex, which generates the stalk, whorls of hairs, and whorls of gametophores. To gain visual and physical accessibility, we amputated the youngest whorls of hair and the original apex and performed experiments on the apex that regenerated. Video analysis indicated that most growth occurred near the tip of the new apex. Less growth occured throughout the cut-interwhorl. We also analyzed cell wall thickness along stalks cleared of cytoplasm. Correlating growth data to wall morphology suggests growth near the apex may be proportional to stress on the cell wall. We propose that turgor-pressure wall stress modulates local apical cell wall growth rates. A supplementary model, relating cell wall curvature and growth rate in the cut-interwhorl, characterizes how the stalk's final dimensions and nearly cylindrical shap may arise. See http://faculty.washington.edu/mandoli/vondassow for supplementary data, analysis, and mathematical appendices. We believe this is the first quantiative description of apex morphogenesis of A. acetabulum.

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PubMed2003

Growth inhibition and changes in morphology and actin distribution in Acetabularia acetabulum by phalloidin and phalloidin derivatives.

Effects on morphology and microfilament structure caused by phalloidin, phallacidin, and some semisynthetic phalloidin derivatives were studied in vegetative cells of the green alga Acetabularia acetabulum (L.) Silva. All phalloidin derivatives (except for phalloidin itself) caused growth stop of the alga after 1 day and (except for the fluorescein-labeled phalloidin) death of the cells after 4-7 days. Hair whorl tip growth and morphology as screened by light microscopy, as well as microfilament structure in tips, suggested that growth stop is correlated with a disorganization of actin filaments similar to that recently described for jasplakinolide (H. Sawitzky, S. Liebe, J. Willingale-Theune, D. Menzel, European Journal of Cell Biology 78: 424-433, 1999). Using rabbit muscle actin as a model target protein, we found that the toxic effects in vivo did not correlate with actin affinity values, suggesting that permeation through membranes must play a role. Indeed, the most lipophilic phalloidin derivatives benzoylphalloidin and dithiolanophalloidin were the most active in causing growth stop at ca. 100 microM. In comparison to the concentration of jasplakinolide required to cause similar effects (<3 microM), the two most active phalloidin derivatives exhibited an activity ca. 30 times lower. Nonetheless, lipophilic phalloidin derivatives can be used in algae, and probably also other cells, to modulate actin dynamics in vivo. In addition, we found that the fluorescent fluorescein isothiocyanate-phalloidin is able to enter living algal cells and stains actin structures brightly. Since it does not suppress actin dynamics, we suggest fluorescein isothiocyanate-phalloidin as a tool for studying rearrangements of actin structures in live cells, e.g., by confocal laser scanning microscopy.

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PubMed2005

Poly(A)+ RNA and cytoskeleton during cyst formation in the cap ray of Acetabularia peniculus.

The configuration and distribution of polyadenylated RNA (poly(A)+ RNA) during cyst formation in the cap rays of Acetabularia peniculus were demonstrated by fluorescence in situ hybridization using oligo(dT) as a probe, and the spatial and functional relationships between poly(A)+ RNA and microtubules or actin filaments were examined by immunofluorescence microscopy and cytoskeletal inhibitor treatment. Poly(A)+ RNA striations were present in the cytoplasm of early cap rays and associated with longitudinal actin bundles. Cytochalasin D destroyed the actin filaments and caused a dispersal of the striations. Poly(A)+ RNA striations occurred in the cytoplasm of the cap rays up to the stage when secondary nuclei migrated into the cap rays, but they disappeared after the secondary nuclei were settled in their positions. At that time, a mass of poly(A)+ RNA was present around each of the secondary nuclei and accumulated rRNA. This mass colocalized with microtubules radiating from the surface of each secondary nucleus and disappeared when the microtubules were depolymerized by butamifos, which did not affect the configuration of actin filaments. These masses of poly(A)+ RNA continued to exist even after the cap ray cytoplasm divided into cyst domains. Thus two distinct forms of poly(A)+ RNA population, striations and masses, appear in turn at consecutive stages of cyst formation and are associated with distinct cytoskeletal elements, actin filaments and microtubules, respectively.

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PubMed1975

Chloroplast membranes of the green alga Acetabularia mediterranea. I. Isolation of the photosystem II.

1. In the presence of Triton X-100, chloroplast membranes of the green alga Acetabularia mediterranea were disrupted into two subchloroplast fragments which differed in buoyant density. Each of these fractions had distinct and unique complements of polypeptides, indicating an almost complete separation of the two fragments. 2. One of the two subchloroplast fractions was enriched in chlorophyll b. It exhibited Photosystem II activity, was highly fluorescent and was composed of particles of approx. 50 A diameter. 3. The light-harvesting chlorophyll-protein complex of the Photosystem II-active fraction had a molecular weight of 67 000 and contained two different subunits of 23 000 and 21 500. The molecular ratio of these two subunits was 2:1.

Acetabularia↗
PubMed1977

The light-harvesting chlorophylla a/b.protein complex of the green alga Acetabularia mediterranea. Isolation and characterization of two subunits.

In the green alga Acetabularia mediterranea a light-harvesting chlorophyll a/b.protein complex of 67 000 daltons has been found which contains two polypeptide chains of 21 500 and 23 000 daltons. These two polypeptides were isolated on a preparative scale and were further characterized by several different methods. Both polypeptides proved to be very similar. While their amino acid and sugar compositions as well as their immunochemical properties were almost identical the tryptic peptides and the cyanogen bromide fragments of the two polypeptides revealed minor but significant differences. The 67 000-dalton chlorophyll a/b.protein complex and its two polypeptide components were compared to the light-harvesting chlorophyll a/b.protein of higher plants.

Acetabularia↗
PubMed1991

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 B1. A 66 kDa polypeptide present in the preparation cross-reacted with the anti-IgG fractions against the alpha and beta 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-beta 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.

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PubMed1992

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 ratio 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-substrate 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 decreased 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).

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PubMed1976

Covalently closed minicircular DNA associated with Acetabularia chloroplasts.

When Acetabularia cliftonii chloroplast DNA (p = 1.706 g/cm3) is centrifuged in an ethidium bromide-CsCl gradient, the lower band is enriched for DNA with a buoyant density of 1.712 g/cm3 containing small covalently closed circular molecules. The minicircles measure 4.15 +/- 0.30 mum in the closed conformation and 4.35 +/- 0.20 mum in the open conformation. They are not of nuclear or bacterial origin, and appear to exist as independent entities within the chloroplast, although a mitochondrial origin cannot be completely ruled out. No 40-45 mum circles, as found in other chloroplasts, were found in either ethidium bromide-CsCl fraction. None were found in total chloroplast DNA by any of a number of methods tried. Linear molecules up to 200 mum were measured in chloroplast lysates. The main chloroplast genome may consist of very large circular molecules which are broken by even small shear forces.

Acetabularia↗
PubMed1980

Protein synthesis by isolated Acetabularia chloroplasts. In vitro synthesis of the apoprotein of the P-700-chlorophyll alpha-protein complex (CP i).

Acetabularia chloroplasts can incorporate radioactive amino acids for up to several hours in vitro. The incorporation is sensitive to chloramphenicol and lincomycin, insensitive to cycloheximide, and completely light-dependent. At least 35 discrete labelled bands can be separated by SDS-polyacrylamide gel electrophoresis: 20--24 in the soluble fraction and 13--15 in the membrane fraction. Most of the label (80--85%) is in the membrane fraction, and 90% of that is in a polypeptide of 32 000 daltons. Chlorophyll-protein complexes were purified from in vitro labelled chloroplasts by SDS-polyacrylamide gel electrophoresis. CP I (P-700-chlorophyll alpha-protein complex) and its apoprotein were both labelled. This shows that the apoprotein is synthesized on chloroplast ribosomes, and can be integrated correctly into the thylakoid membrane in the absence of any cytoplasmic contribution. In contrast, no label was incorporated into the two polypeptides of CP II, the light-harvesting chlorophyll a/b complex.

Acetabularia↗
PubMed1975

Model for the positional differentiation of the cap in Acetabularia.

A late stage during the biological cycle of the unicellular alga Acetabularia is the differentiation of a cap at the apical end of the stalk. A minimal model of the spatio-temporal regulation of this event is proposed on the basis of biological data available and current hypotheses. This involves the interaction between a diffusing inhibitor specific to the translation of cap mRNAs and a graded distribution of these messengers. The model accounts for delayed protein synthesis which occurs preferably at the apex and is likely to initiate the formation of the cap. The biological and theoretical implications are discussed.

Acetabularia↗
PubMed1997

Exchangeability of the b subunit of the Cl(-)-translocating ATPase of Acetabularia acetabulum with the beta subunit of E. coli F1-ATPase: construction of the chimeric beta subunits and complementation studies.

The gene encoding the b subunit of the Cl(-)-translocating ATPase (aclB) was isolated from total RNA and poly(A)+ RNA of Acetabularia acetabulum and sequenced (total nucleotides of 3038 bp and an open reading frame with 478 amino acids). The deduced amino acid sequence showed high similarity to the beta subunit of the F type ATPases, but was different in the N-terminal 120 amino acids. The role of the N-terminal region was investigated using an F -ATPase beta-less mutant of E. coli, JP17. The JP17 strain expressing the aclB could not grow under conditions permitting oxidative phosphorylation, although ACLB was detected in the membrane fraction. The beta subunit was divided into three portions: amino acid position from 1 to 95 (portion A), 96 to 161 (portion B) and 162 to the C-terminus (portion C). The corresponding regions of ACLB were designated as portions A' (from 1 to 106), B' (from 107 to 172) and C' (from 173 to 478). Chimeric proteins with combinations of A-B'-C', A-B-C' and A'-B-C restored the function as the beta subunit in E. coli F0F1-complex, but those with combinations of A'-B'-C and A-B'-C had no function as the beta subunit. These findings suggested that portion B plays an important role in the assembly and function of the beta subunit in the F0F1-complex, while portion B' of ACLB exhibited inhibitory effects on assembly and function. In addition, portion A was also important for interaction of the beta subunit with the alpha subunit in E. coli F0F1-complex. These findings also suggested that the b subunit of the Cl(-)-translocating ATPase of A. acetabulum has a different function in the Cl(-)-translocating ATPase complex, although the primary structure resembled to the beta subunit of the F1-ATPase.

Acetabularia↗
PubMed1990

An application of membrane theory to tip morphogenesis in Acetabularia.

Focusing our attention on the cell wall and the plasmalemma (i.e. the cell membrane), we seek to show that the initiation of the regenerative, growing tip in the unicellular marine alga Acetabularia mediterranea, can be predicted using the techniques of thin-shell and elasticity theory. We build upon and extend the work of Goodwin & Trainor (1985, J. theor. Biol. 117, 79-106), Trainor & Goodwin (1986, Physica D. 21D, 137-145) and Brière & Goodwin (1988, J. theor. Biol. 131, 461-475) where the attention was focused on the calcium-regulated strain and stress fields of the cortical cytoplasm. Finally, we attempt to model the subsequent tip growth using a moving-boundary formulation, with cytosolic free calcium concentration and turgor pressure being the two variables responsible for the growth.

Acetabularia↗
PubMed2000

Analogous features of delayed luminescence from Acetabularia acetabulum and some solid state systems.

This paper compares some features of the delayed luminescence in the seconds range emitted from an unicellular alga Acetabularia acetabulum and from some solid state systems. Results lead us to think that such delayed luminescence is connected to the dimension of the ordered structures present in both biological and solid state systems. In particular, for the biological system examined, these structures are thought to be those which contribute to the dynamic formation of the cytoskeleton.

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