A cell-free system for light-dependent nuclear import of phytochrome.

Translocation from the cytosol to the nucleus is an essential step in phytochrome (phy) signal transduction. In the case of phytochrome A (phyA), this step occurs with the help of FHY1 (far-red-elongated hypocotyl 1), a specific transport protein. To investigate the components involved in phyA transport, we used a cell-free system that facilitates the controlled addition of transport factors. For this purpose, we isolated nuclei from the unicellular green algae Acetabularia acetabulum. These nuclei are up to 100 mum in diameter and allow easy detection of imported proteins. Experiments with isolated nuclei of Acetabularia showed that FHY1 is sufficient for phyA transport. The reconstituted system demonstrates all the characteristics of phytochrome transport in Arabidopsis thaliana. In addition, FHY1 was also actively exported from the nucleus, consistent with its role as a shuttle protein in plants. Therefore, we believe that isolated Acetabularia nuclei may be used as a general tool to study nuclear transport of plant proteins.

Development and organization of the central vacuole of Acetabularia acetabulum.

* Here we analyzed the shape of the central vacuole of Acetabularia acetabulum by visualizing its development during diplophase (from juvenility through reproduction) and haplophase (from meiosis through mating). * Light microscopy and whole-organism applications of a pH-sensitive dye, neutral red, were used to visualize the anatomy of the central vacuole. We studied connectivity within the thallus by locally applying dye to morphologically distinct regions (rhizoid, stalk, apex, hairs) and observing dye movements. * In vegetative thalli most of the rhizoid, stalk and young hairs stained with dye. In reproductive structures (caps, gametangia) dye also stained the majority of the interiors. When applied to small areas, dye moved at different rates through each region of the thallus (e.g. within the stalk). Dye moved from younger hairs, but not from older hairs, into the stalk. Errors in incorporation of central vacuole into gametangia occurred at <10(-5). * These data indicate that the central vacuole of A. acetabulum is a ramified polar organelle with, potentially, a gel-like sap that actively remodels its morphology during development.

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.

Dynamics and pharmacological perturbations of the endoplasmic reticulum in the unicellular green alga Acetabularia.

The giant unicellular green alga Acetabularia was labeled with the lipophilic fluorochrome DiOC6 (3,3'-dihexyloxacarbocyanine) and examined by confocal laser scanning microscopy to study the distribution of the endoplasmic reticulum (ER) and its dynamic changes after the application of inhibitors. In control cells, a two-dimensional polygonal network of ER sheets and tubulus is suspended between parallel, longitudinally oriented bands. These bands coincide with the main physical tracks of organelle transport. All treatments that inhibited organelle motility caused a transformation of the polygonal network into confluent large patches of lamellar ER sheets. The shape of the lamellar sheets and residual activities of the ER were dependent on the inhibitors used. The largest ER lamellae were obtained after cytochalasin D (CD) treatment which effectively stopped cytoplasmic streaming. CD also caused the formation of a network of fine tubules overlapping with the lamellar sheets. Okadaic acid, a specific inhibitor of serine/threonine-protein phosphatases, also caused inhibition of organelle movement and enlargement of lamellar areas. Tension in the cytoplasm appeared to be reduced, as judged from the convexly curved lamellar rims and wavy connecting ER tubules. In contrast, N-ethylmaleimide, a sulfhydryl group blocking reagent, rapidly stopped streaming and halted all activities of the ER in a rigor-like state. These effects are interpreted in the context of actin-based motility phenomena prevalent in Acetabularia, and regulatory principles are discussed that might underlie ER dynamics.

Na+ transport in Acetabularia bypasses conductance of plasmalemma.

Na(+)-selective microelectrodes with the sensor ETH 227 have been used to measure the cytoplasmic Na+ concentration, [Na+]c, in Acetabularia. In the steady-state, [Na+]c is about 60 mM (external 460 mM). Steps in external Na+ concentration, [Na+]o, cause biexponential relaxations of [Na+]c which have formally been described by a serial three-compartment model (outside<==>compartment 1<==>compartment 2). From the initial slopes (some mMsec-1) net uptake and release of about 3 mumolm-2sec-1 Na+ are determined. Surprisingly, but consistent with previous tracer flux measurements (Mummert, H., Gradmann, D. 1991. J. Membrane Biol, 124:255-263), these Na+ fluxes are not accompanied by corresponding changes of the transplasmalemma voltage. [Na+]c is neither affected by the membrane voltage, nor by electrochemical gradients of H+ or Cl- across the plasmalemma, nor by cytoplasmic ATP. The results suggest a powerful vesicular transport system for ions which bypasses the conductance of the plasmalemma. In addition, transient increases of [Na+]c have been observed to take place facultatively during action potentials. The exponential distribution of the amplitudes of these transients (many small and few large peaks) points to local events in the more ore less close vicinity of the Na+ recording electrode. These events are suggested to consist of disruption of endoplasmic vesicles due to a loss of pressure in the cytoplasm.

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.

Magnetic resonance imaging approaching microscopic scale: maturation stages of Acetabularia mediterranea reproductive caps.

The reproductive cap of the giant single-celled alga Acetabularia mediterranea (or A. acetabulum) has rays tapering from a width of about 400 microns at the circumference of the cap to about 30 microns at their junction with the stalk of the cell. This is ideal geometry for testing the current limits of spatial resolution of proton magnetic resonance imaging. In this work, resolution of features down to 40 microns is achieved. Maturation of the cap rays involves a major cytoplasmic reorganization, from continuous cytoplasm and a central vacuole in each ray to bundles of cysts surrounded by aqueous solution. This work shows that an intermediate stage in the change can be highlighted in images by relaxation time (T1) contrasting.

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.

Temporal morphology and cap formation in Acetabularia--II. Effects of morphactin and auxin.

In order to support the hypothesis that circadian rhythms are implicated in cap formation, experiments were undertaken on the possible time-dependency of the effects of (a) a competitive inhibitor of auxins, morphactin and (b) of auxin (IAA). It was found that: (i) the inhibitory effect of morphactin varies dramatically with the time at which the several weeks' treatment was first begun; (ii) the maximum inhibition varies with development and decreases with time; (iii) IAA accelerates cap formation when the algae are submitted to IAA during the exponential growth phase; the effect is time dependent and decreases with time; (iv) IAA first applied on smaller algae has a transient inhibitory effect which is time dependent; (v) anucleate fragments also respond differentially to an IAA treatment begun at several times in the 24-hr cycle, most clearly when newly formed mRNA have been accumulated and (vi) the effect of iAA is not cumulative with that of a LD shift; that of morphactin is not, or only slightly, improved by a LD shift.

Apicobasal gradient of chloroplast DNA synthesis and distribution in Acetabularia.

Autoradiographic and biochemical experiments have revealed the presence, in vegetative cells of Acetabularia, of an apicobasal gradient of penetration and incorporation of labelled DNA precursors into the chloroplasts. Staining of chloroplasts with the DNA-specific fluorochrome DAPI has shown that the number of chloroplasts without DNA increases from the apex towards the base of the cell. All together, our findings support the existence of an apicobasal gradient of chloroplast DNA synthesis and distribution in Acetabularia.

Use of the fluorochrome 4'6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA.

Use of the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. (a) Wild-type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. (b) In vegetative cells of the giant-celled marine algae Acetabularia and Batophora, only about a quarter of the chloroplasts have even one discernible chloroplast DNA particle, and such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. By contrast, 98% of chloroplasts in developing Acetabularia cysts contain chDNA, with as many as nine nucleoids per chloroplast. (c) DAPI-stained chloroplasts of chromophyte algae display the peripheral ring of DNA expected from electron microscope studies. However, these rings are not uniform in thickness, but are necklace-like, with the appearance of beads on a string. Since the multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast, a common structural plan may underlie chDNA morphology in both groups of algae.

Accumulation of peroxidase in the cap rays of Acetabularia during the development of gametangia.

Accumulation of peroxidase was demonstrated by light and electron microscopy to occur in Acetabularia in certain regions of the cap rays in relation to the development of the gametangia (cysts). Peroxidase was found to be incorporated into special, cell wall-like obstructions that separate the cap rays from the stalk when the secondary nuclei have settled in the cap rays. It is assumed that peroxidase acts as an anti-microbial protectant of the gametangia.