Alga Acetabularia Research Articles

Calcium localization duringAcetabulariawhorl formation: evidence supporting a two-stage hierarchical mechanism

ABSTRACTThe giant single-celled marine alga Acetabularia mediterranea (or A. acetabulum) repeatedly generates whorls of hairs at its growing tip. Theoretical considerations, and physicochemical analysis of the effect of extracellular calcium concentration on hair spacing, have suggested a two-stage mechanism, in which whorl pattern is preceded by a simple annular pattern. This analysis requires that, at the time of whorl determination, morphogenetically related calcium should still be distributed in the first-stage annular pattern. This paper reports studies of calcium distribution at closely spaced developmental stages from unpatterned growing tip to whorl expression. The sequence observed is that expected from the theoretical analysis. The fluorescence chelate used, chloro tetracycline, is believed to be specific for membrane-bound calcium. The results therefore suggest, but do not prove, that morphogenetically related calcium is attached to membrane-bound structures rather than Intracellular ones.

Differential labelling of the vegetative primary nucleus of the marine green alga Acetabularia by lectins, anti-immunoglobulin-antibodies and antinuclear autoimmunosera.

Differential labelling of the vegetative primary nucleus of the marine green alga Acetabularia by lectins, anti-immunoglobulin-antibodies and antinuclear autoimmunosera.

Spontaneous Action Potential Initiation and Propagation in Regenerating Cell Segments ofAcetabularia mediterranea

Spontaneous Action Potential Initiation and Propagation in Regenerating Cell Segments of<i>Acetabularia mediterranea</i>

Antibodies specific for vertebrate actin, myosin, actinin, or vinculin recognize epitopes in the giant nucleus of the marine green alga Acetabularia

AbstractThe vegetative giant nucleus of the unicellular and uninucleate marine green alga Acetabularia is a depot of conservative epitopes, homologous to antigenic determinants of vertebrate actin, myosin, actinin, and vinculin. The epitopes appear at the nucleolar surface (actin, actinin, vinculin determinants), in the caryoplasm (actin, myosin determinants), along “caryoplasmic” filaments (actin determinants), and in “nuclear envelope plus perinuclear bodies” (actin, myosin, actinin, vinculin determinants). The structure homologies of the nuclear antigenic determinants to those of the vertebrate muscular and/or microfilamental proteins were deduced from in situ cross‐reaction of anti‐chicken actin (cross‐reaction also with rabbit actin), anti‐chicken alpha actinin, anti‐chicken vinculin, and anti‐bovine myosin (cross‐reaction also with chicken myosin), respectively, by indirect immunofluorescence microscopy. Artifacts which arise from the binding of contaminating unspecific markers or from unspecific adherence of specific ones to the algal nucleus have been overcome by the use of both polyclonal and/or monoclonal immunoglobulins as primary markers and different types of second markers each conjugated with fluorescein isothiocyanate (FITC). Fluorescein staining of primary markers was performed either with fluorescent anti‐immunoglobulin antibodies in a one‐step (AIA‐FITC) or highly sensitive two‐step procedure (AIA/AIA‐FITC), covalently labeled F(ab′)2 specific for either Fc or F(ab′)2 (the latter anti‐fragment antibody excluded both possible interactions between nuclear “lectins” and glycosidic residues of Fc and staining of glycosidic nuclear antigens by AIAs or anti‐Fc specific for the glycosidic part of the immunoglobulin antigen) or fluorescent complex “protein A‐biotin‐avidin” (PABA‐FITC, a highly sensitive nonimmunoglobulin second reagent). Three of four different AIA‐FITC preparations tested alone and also “F(ab′)2 anti‐Fc” showed reactivities with the nucleoli and the nuclear envelope. This indicates the presence of glycosidic determinants at the sites of reaction. Each of the other fluorescent markers, including AIA/AIA‐FITC, reacted with the primary marker only, although they were different in sensitivity.The staining patterns of nuclear actin epitopes differed in certain details if primary marker (monoclonal against polyclonal), second marker (AIA‐FITC against PABA‐FITC), or nuclear preparation (degree of nuclear flattening by the cover slide and salt condition) were changed. It suggests that type and number of actin epitopes, valence, affinity, and number of anti‐actin clones, but also subclass or class specificity of the second marker and accessibility of the nuclear actin determinant(s), were involved. The nuclear actin and myosin epitopes stained most intensively in a “high salt” environment (100 mM PBS, 50 mM/1 KC1; pH 7.2) if compared to “low salt staining.” This indicates local concentrations and/or accessibility of antigenic determinants which were hidden in “low salt” (1.5 mN/1 Na2HPO4, 8mM/1 KH2PO4, 2.7 mM/1KCl, 137 mM/1 KCl, 137 mM/1 NaCl; pH 7.4) conditions.

Evidence for a circaseptan and a circasemiseptan growth response to light/dark cycle shifts in nucleated and enucleated Acetabularia cells, respectively.

Nucleated as well as enucleated Acetabularia mediterranea cells were subjected to 14 different patterns of shifts in a regimen of 12 hr of light alternating with 12 hr of darkness in four 30-day long experiments. With one exception, which might be due to a circannual modulation, these experiments showed that nucleated cells had maximal growth rates when a shift was performed every 7th or 15th day. In enucleated cells, maxima were observed on shift schedules that were about 3-4 days rather than about 7 days apart. The results indicate that in the unicellular green alga Acetabularia a rhythm of about 7 days (circaseptan) exists and that removal of the nucleus results in a circaseptan frequency multiplication.

Determination of transmembrane currents from external potential measurements by the method of regularization.

A novel non-invasive technique for measuring the transmembrane currents during action potential depolarizations of stalk segments of the giant unicellular alga Acetabularia mediterranea is described. This involves measurement of spatial samples of the time-dependent potential in the external medium (sea water) and an inverse transformation to give the current distribution at the cell surface. A detailed description of the method of regularization needed to handle this ill-conditioned inversion problem is presented in this paper, along with the results of test experiments to demonstrate its validity. An example of its application to the data recorded from an actual cell is also given.

Quantitative control of Acetabularia morphogenesis by extracellular calcium: A test of kinetic theory

The unicellular marine alga Acetabularia repeatedly produces whorls of hairs at its growing tip. The spacing between hair initials, measured when they first become visible, depends linearly on the reciprocal of calcium concentration in the culture medium. Some hypothetical mechanisms of reaction-diffusion theory predict that pattern spacings will depend on inverse powers of the concentrations of precursors to the morphogenetic substances. Accordingly, we plot spacing as ordinates vs 1/[Ca 2+ ], and examine the consequences of treating this as a plot of 1/(bound Ca) vs 1/(free Ca), i.e. as being analogous to a Lineweaver-Burk plot. Thus we calculate values of binding constant, Δ H 0 and Δ S 0 resembling those for endothermic, entropy-driven binding of calcium to anion paris in small ligands and (binding constant known, not Δ H 0 and Δ S 0 to phospholipid bilayers and some instances of protein side-chains. The probable regions of localization of calcium during morphogenesis are discussed in relation to a two-stage model for whorl formation published earlier from this laboratory. The first stage forms an annular distribution of a precursor which feeds into the whorl-forming second stage. Preliminary data on calcium-chlorotetracycline fluorescence give tentative support to the identification of this precursor with the bound-calcium species.

Band-Type Dissipative Structure in Ion Transport Systems with Cylindrical Shape

A theory is presented for an appearance of band-type pattern of electric potential accompanied with electric current surrounding a unit or many-cell system of a cylindrical shape. A flux continuity at the membrane is reduced to a nonlinear equation composed of passive and active fluxes through a diffusion equation for protons. Another equation concerns the concentration of a diffusive substance weakening the pump activity. It is shown that, when an external parameter is varied from the homogeneous state, a symmetry breaking along a longitudinal axis usually appears before the one along a circumferential direction. A TDGL equation for spatial modes, which can describe both normally and invertedly bifurcating cases, is derived. An observation of hysteretic bands of chloroplasts in the green alga Acetabularia is discussed within the present theoretical scheme.

Cllmodulin in tip-growing plant cells, visualized by fluorescing calmodulin-binding phenothiazines

Calmodulin (CaM) was visualized light-microscopically by the fluorescent CaM inhibitors fluphenazine and chlorpromazine, both phenothiazines, during polar tip growth of pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya spec. and in the alga Acetabularia mediterranea, as well as during multipolar tip growth in Micrasterias denticulata. Young pollen tubes and root hairs showed tip fluorescence; at later stages and in the growing parts of the other subjects the fluorescence was almost uniform. After treatment with cytochalasin B, punctuate fluorescence occurred in the clear zone adjacent to the tip of pollen tubes. The observations indicate that there is CaM in all our tested systems detectable with this method. It may play a key role in starting polar growth. As in pollen tubes, CaM might be in part associated with the microfilament network at the tip, and thus regulate vesicle transport and cytoplasmic streaming.

Enhancer-controlled expression of the simian virus 40 T-antigen in the green alga Acetabularia.

Nuclei from Acetabularia mediterranea were isolated, microinjected with simian virus 40 (SV40) DNA and fused with cytoplasts from the same species. Various times after fusion of the injected nuclei the fusion products were screened for expression of the T-antigen by indirect immunofluorescence. One and two days after injection a bright fluorescence could be observed in the nuclei of Acetabularia. On the basis of this immunofluorescence we conclude that in Acetabularia cells the T-antigen is expressed and accumulated in the nucleus. Moreover, evidence is presented that the Acetabularia cell recognizes the SV40 enhancer sequence. The expression product of the SV40 DNA appears significantly earlier than the expression products of other foreign genes in Acetabularia. The results suggest that the well characterized SV40 can be used as a vector system for the introduction and expression of foreign genes in Acetabularia.

A synthetic growth medium for the green algaAcetabularia mediterranea

A simple, synthetic growth medium for the green alga Acetabularia mediterranea Lamouroux is developed. The medium is based on a commercial sea salt mixture. Except for sodium nitrate no other additives are necessary for optimal growth. The cells display a normal morphogenesis while passing through the developmental cycle. The growth response, chlorophyll and protein synthesis are similar to those of cells grown in the classic Erd-Schreiber medium based on natural sea-water. The synthetic sea-water supplemented with nitrate only has proved to be suitable for the long term maintenance of the cells into the following generation. This medium might be adaptable for the culture of other species of green algae.

Temporal morphology and cap formation in Acetabularia--I. Light-dark cycle perturbations.

Cap formation, a major developmental process in the alga Acetabularia, is influenced by a single perturbation of the entraining light-dark schedule and thus, presumably, of the circadian rhythms. This perturbation is brought about several weeks before cap formation, the most conspicuous expression of morphogenesis in Acetabularia. The effect is more pronounced on cap formation than on growth. It varies in importance with the circadian time at which the perturbation was brought about. The effect is dependent on the developmental state of the alga: transfer carried out during the logarithmic phase of growth produces a delay whose importance decreases with time. When carried out during the phase of slow terminal growth, the transfer induces a transitory acceleration of cap formation. When the algae approach their final length, no effect is elicited. Photoperiodism seems to be involved.

Turing's model and pattern adjustment after temperature shock, with application to Acetabularia whorls

The giant single-celled alga Acetabularia repeatedly forms whorls of hairs during vegetative growth. The spacing between hair initials, measured when they first become visible, is temperature-sensitive. If the temperature is changed rapidly from 18°C to 29°C, the corresponding change in spacing takes place over several hours, roughly linearly. This functional form suggests that, if the pattern is readjusting from, typically, nine hairs to twelve, the intermediate values ten and eleven are favoured at intermediate times. Turing's reaction—diffusion model describes pattern determination in terms of the competitive growth and decay of patterns with different spacings at spacing-dependent rates. After the competitive rates have been changed by temperature shock, appearance of intermediate patterns is an expected feature of the Turing mechanism. Lacalli & Harrison 1979 indicated the ranges of relative values of rate constants in the Turing model which are appropriate to various kinds of morphogenetic behaviour. For any change which alters the ratio (system size)/(pattern spacing) = number of parts in pattern, the adjustment of pattern will be rapid only for a rather restricted range of values of the rate constants. A set of values proposed in this connection in the 1979 paper for pattern changes in Drosophila wing discs gives a good account of the changes in A. mediterranea whorls after temperature shock. The values required for diffusivities suggest that the morphogens are membrane-bound.

Chronobiology at the cellular and molecular levels: models and mechanisms for circadian timekeeping.

This review considers cellular chronobiology and examines, at least in a superficial way, several classes of models and mechanisms that have been proposed for circadian rhythmicity and some of the experimental approaches that have appeared to be most productive. After a brief discussion of temporal organization and the metabolic, epigenetic, and circadian time domains, the general properties of circadian rhythms are enumerated. A survey of independent oscillations in isolated organs, tissues, and cells is followed by a review of selected circadian rhythms in eukaryotic microorganisms, with particular emphasis placed on the rhythm of cell division in the algal flagellate Euglena as a model system illustrating temporal differentiation. In the ensuing section, experimental approaches to circadian clock mechanisms are considered. The dissection of the clock by the use of chemical inhibitors is illustrated for the rhythm of bioluminescence in the marine dinoflagellate Gonyaulax and for the rhythm of photosynthetic capacity in the unicellular green alga Acetabularia. Alternatively, genetic analysis of circadian oscillators is considered in the green alga Chlamydomonas and in the bread mold Neurospora, both of which have yielded clock mutants and mutants having biochemical lesions that exhibit altered clock properties. On the basis of the evidence generated by these experimental approaches, several classes of biochemical and molecular models for circadian clocks have been proposed. These include strictly molecular models, feedback loop (network) models, transcriptional (tape-reading) models, and membrane models; some of their key elements and predictions are discussed. Finally, a number of general unsolved problems at the cellular level are briefly mentioned: cell cycle interfaces, the evolution of circadian rhythmicity, the possibility of multiple cellular oscillators, chronopharmacology and chronotherapy, and cell-cycle clocks in development and aging.

Electrogenic Cl- pump in Acetabularia.

Measurements of this transmembrane potential difference (V) under various conditions have demonstrated the operation of an electrogenic Cl- pump in the outer plasma membrane (plasmalemma) of the unicellular marine alga Acetabularia. In preparations of partly purified membranes (containing plasmalemma), there is Cl- stimulated, N,N'-dicyclohexylcarbodiimide-insensitive, vanadate-sensitive ATPase activity with a pH optimum around pH 6.5. These properties are consistent with the assumption that the electrogenic Cl- pump is an ATPase. In order to investigate electrical details of the "Mitchellian" type of charge-translocating enzyme, steady-state current-voltage curves of the electrogenic pump (Ip(V)) were measured in vivo under dark and light conditions and analysed by two-state reaction kinetic model. This model with the resulting parameters predicts V-sensitive, undirectional Cl- effluxes through the pump. The predictions of this model agree with the experimental results. Green light causes a fast decrease of V, which is explained as a disturbance of the pump cycle. Relaxation studies on this effect and reaction kinetic analysis of Ip(V) under different external Cl- concentrations are used to develop a consistent three-state model of the pump that includes the order of and absolute rate constants of individual reactions, states of charge, stoichiometry, voltage-sensitivity and density of the pump molecules in the membrane.

The nature of the light-harvesting complex as defined by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Reelectrophoresis of the oligomer form (CP II∗) of the chlorophyll a b light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II 1) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl a b proteins. Neither of these bands can be attributed to the Chl a b complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II 1 and CP II 2 of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II 1 and CP II 2 are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels.

The chlorophyll-protein complexes of Acetabularia. A novel chlorophyll [formula omitted] complex which forms oligomers

(1) Five minor chlorophyll-protein complexes were isolated from thylakoid membranes of the green alga Acetabularia by SDS-polyacrylamide gel electrophoresis, after SDS or octylglucoside solubilization. None of them were related to CP I (Photosystem I reaction center core) or CP II (chlorophyll a b light-harvesting complex). (2) Two complexes (CPa-1 and CPa-2) contained only chlorophyll (Chl) a, with absorption maxima of 673 and 671 nm, and fluorescence emission maxima of 683 nm compared to 676 nm for CP II. The complexes had apparent molecular masses of 43–47 and 38–40 kDa, and contained a single polypeptide of 41 and 37 kDa, respectively. They each account for about 3% of the total chlorophyll. (3) Three complexes had identical spectra, with Chl a b ratios of 3–4 compared to 2 for thylakoid membranes, and a pronounced shoulder around 485 nm indicating enrichment in carotenoids. One of them was the complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) and the other two were slightly different oligomeric forms of CP 29. They could be formed from CP 29 during reelectrophoresis; but about half the complex was isolated originally in an oligomeric form. Together they account for at least 7% of the total chlorophyll. Their function is unknown.

Chloroplast DNA of Acetabularia mediterranea: Cell cycle related changes in distribution

Chloroplast DNA (cpDNA) distribution in the giant unicellular, uninucleate alga Acetabularia mediterranea was analyzed with the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) at various stages of the cell cycle. The number of chloroplasts exhibiting DNA/DAPI fluorescence changes during the cell's developmental cycle: (1) all chloroplasts in germlings contain DNA; (2) the number of plastids with DNA declines during polar growth of the vegetative cell; (3) it increases again prior to the transition from the vegetative to the generative phase; (4) several nucleoids of low fluorescence intensity are present in the chloroplasts of the gametes. The temporal distribution of the number of chloroplasts with DNA appears to be linked to the different mode of chloroplast division and growth during the various stages of development. The chloroplast cycle in relation to the cell cycle is discussed.

Widespread Distribution of Some Minor Chlorophyll-Protein Complexes in Some Plants and Algae

The use of the non-ionic detergent octyl beta-d-glucoside has allowed the demonstration in spinach of a chlorophyll a+b-protein complex of apparent molecular weight 29 kilodaltons (Camm and Green, 1980, Plant Physiol 66: 428-432). Complexes analogous to this one also can be demonstrated in three grasses, in dicots of the Chenopodiaceae, Fabaceae, and Asteraceae, and in the siphonaceous green alga Acetabularia mediterranea. These complexes are clearly distinguishable from the light harvesting complex on the basis of the chlorophyll a/b ratio, apparent molecular weight, and polypeptide composition. In addition, most plants surveyed contained two, not one, minor chlorophyll a complexes as well as the chlorophyll a complex of Photosystem I.

Heterogeneity of chloroplasts in Acetabularia mediterranea. Heterogeneous distribution and morphology of chloroplast DNA

A high percentage of chloroplasts in the siphonaceous green alga Acetabularia mediterranea lacks DNA: staining with the sensitive DNA-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) revealed that DNA was present only in 47–51% of the total plastid population. The distribution of DNA-containing chloroplasts appeared heterogeneous, exhibiting an apicobasal gradient. DNA could be detected in 64, 46, 35, and 36% of the plastids from the apical, subapical, middle, and basal part of the cell, respectively. DNA morphology also exhibited heterogeneity. Three types of nucleoid were distinguished: (1) round compact nucleoids; (2) long thin nucleoids characteristic of chloroplasts of the cell apex and the whorls; and (3) elaborate nucleoids appearing to consist of several subunits, which were more typical of the middle and basal part of the cell. On the basis of the nucleoid morphology and the decrease in DNA-containing plastids from the apex towards the basis of the cell, we propose a model for the development of plastids lacking DNA in relation to chloroplast replication.