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In the green alga Scenedesmus acutus, Golgi bodies are located near the nucleus and supplied with transition vesicles that bud from the outer nuclear envelope membrane. Using this alga, we have shown previously that thiamine pyrophosphatase (TPPase), a marker enzyme of Golgi bodies, migrates in vesicles from the Golgi bodies to the ER via the nuclear envelope in the presence of BFA (Noguchi et al., Protoplasma 201, 202-212, 1998). In this study we demonstrate that both cytochalasin B and oryzalin (microtubule-disrupting agent) inhibit the BFA-induced migration of TPPase from Golgi bodies to the nuclear envelope. However, only actin filaments--not microtubules--can be detected between the nuclear envelope and the Golgi bodies in both BFA-treated and untreated cells. These observations suggest that actin filaments mediate the BFA-induced retrograde transport of vesicles. This mechanism differs from that found in mammalian cells, in which microtubules mediate BFA-induced retrograde transport by the elongation of membrane tubules from the Golgi cisternae. We also discuss the non-participation of the cytoskeleton in anterograde transport from the nuclear envelope to the Golgi bodies.

The arrangement of the microtubule cytoskeleton in tip-growing and gravisensing Chara rhizoids has been documented by immunofluorescence microscopy. Predominantly axially oriented undulating bundles of cortical microtubules were found in the basal zone of the rhizoids and colocalized with the microfilament bundles underlying the cytoplasmic streaming. Microtubules penetrate the subapical zone, forming a three-dimensional network that envelops the nucleus and organelles. Microtubules are present up to 5 to 10 microns basal from the apical cytoplasmic region containing the statoliths. No microtubules were found in the apical zone of the rhizoid which is the site of tip growth and gravitropism. Depolymerization of microtubules by application of oryzalin does not affect cytoplasmic streaming and gravitropic growth until the relatively stationary and polarly organized apical and subapical cytoplasm is converted into streaming cytoplasm. When the statoliths and the apical cytoplasm are included in the cytoplasmic streaming, tip growth and gravitropism are stopped. Oryzalin-induced disruption of the microtubule cytoskeleton also results in a rearrangement of the dense network of apical and subapical microfilaments into thicker bundles, whereas disruption of the microfilament cytoskeleton by cytochalasin D had no effect on the organization of the microtubule cytoskeleton. It is, therefore, concluded that the arrangement of microtubules is essential for the polar cytoplasmic zonation and the functionally polar organization of the actin cytoskeleton which is responsible for the motile processes in rhizoids. Microtubules are not involved in the primary events of gravitropism in Chara rhizoids.

Highly purified Golgi membranes were isolated from the scaly green flagellate Scherffelia dubia using osmotic shock for controlled cell rupture, differential centrifugations and a discontinuous sucrose density gradient centrifugation. Three Golgi membrane fractions (based on the distribution of IDPase activity in the gradient) at densities 1.14 g/ml, 1.17 g/ml and 1.20 g/ml were obtained. The specific IDPase activity in these fractions was enriched about 78-fold compared to the crude cell homogenate. The Golgi membrane fractions were further characterized by electron microscopy, SDS-PAGE and lectin blotting. The low density fraction (1.14 g/ml) contained two distinct vesicle populations and scale precursors associated with the outer surface of the larger-size vesicles. The medium density fraction (1.17 g/ml) contained in addition to the larger vesicles, multilamellate vesicles and semicircular cisternae. Finally, in the high density fraction (1.20 g/ml) in addition to small and large vesicles, a tubular membrane reticulum was observed. The three Golgi membrane fractions revealed the same complex overall polypeptide composition when analyzed by SDS-PAGE, but gradual quantitative differences in the polypeptide profile between fractions were observed. The lectins GNA, DSA, and AAA bound to several glycoproteins in all Golgi membrane fractions. Deglycosylation with N-glycosidase F showed that all carbohydrate structures recognized by GNA and DSA, and one recognized by AAA were of the N-glycosidic type indicating the presence of both "high mannose" and "processed" N-glycans in the Golgi apparatus of S. dubia.

Glycoproteins associated with one type of flagellar scale (p-scale) isolated from the flagellate green alga Tetraselmis striata (Prasinophyceae) were shown to bind the mannose-specific lectin GNA (Galanthus nivalis agglutinin). Enzymatic deglycosylation of the glycoproteins with N-glycosidase F led to an electrophoretic mobility shift to lower molecular masses in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and abolished GNA-binding strongly indicating that most of the scale-associated glycoproteins contain asparagine-linked oligosaccharide side chains presumably of the high mannose type. To evaluate the significance of N-linked glycoproteins for scale structure and integrity, p-scales were digested with various proteases or extracted with 8 M urea and their ultrastructure and protein composition determined. The results show that while scale-associated N-linked glycoproteins do not determine the overall structure of the scale subunits (which consist of complex polysaccharides), they are apparently involved in mediating linkages between scale subunits; we have tentatively identified one glycoprotein of Mr 280,000 which may link outer scale subunits to one another. In addition, some scale-associated N-linked glycoproteins may provide connections between the layer of p-scales and the underlying flagellar membrane.

We report on the isolation and purification of structurally intact eyespot apparatuses from the naked, biflagellate green alga Spermatozopsis similis. Two eyespot-enriched fractions, separated by sucrose gradient centrifugation, retained the typical reflective properties of eyespots in situ as demonstrated by reflection confocal laser scanning microscopy. Ultrastructurally, both fractions contained eyespot plates consisting of a single layer of lipid globules. Structurally intact eyespot apparatuses, including patches of plasma membrane and chloroplast envelope overlying the eyespot plate and a single thylakoid subtending the eyespot plate, were particularly enriched in one of the two fractions (fraction 2a). Measurement of several marker enzymes and chlorophyll content (less than 0.001% of total) established the absence of most other cell organelles from the eyespot fractions. The absorption spectra of the two fractions were dominated by carotenoids with an additional shoulder at 540 nm. Following extraction with organic solvents and sodium dodecyl sulfate polyacrylamide gel electrophoresis, several proteins were found to be considerably enriched in the two fractions. In addition to several proteins in the high Mr range, at least 4 polypeptides of 35, 29, 23, and 20 kDa are selectively enriched in fraction 2a with the 29 and 20 kDa proteins being the most prominent. The presence of glycoproteins in fraction 2a was demonstrated by binding of the mannose-specific lectin Galanthus nivalis agglutinin to several high molecular weight polypeptides. In addition, a hydrophobic component with abnormal electrophoretic mobility that reacts strongly with periodic acid-Schiff and thymol/sulfuric acid was prominent in both fractions. Mass isolation and purification of the intact phototactic apparatus of a flagellate green alga now greatly facilitates the biochemical and molecular characterization of the signal transduction chain involved in green algal phototaxis.

The reflection properties of different types of eyespots in three unicellular, flagellated green algae (Tetraselmis chui, Chlamydomonas eugametos, Hafniomonas reticulata) were investigated using confocal laser scanning microscopy in the epireflection mode. The eyespots differed with respect to the number of eyespot lipid globule layers and surface appearance (concave/convex). A strong reflection signal was observed in all eyespots, and a detailed quantitative analysis by optical xy (horizontal) and xz (vertical) sectioning was performed. By applying both sectioning capabilities, multi- and single/double-layered eyespots as well as concave and convex eyespot surfaces could be distinguished using living, immobilized cells. Focusing of the reflected light was only observed in eyespots with concave surfaces. In xz series of multi-layered eyespots at reduced laser intensities (0.01%), the intensity profiles of the reflection revealed a series of alternating maxima and minima with increasing reflection intensities toward the cell surface. At very low laser intensities (0.001%), multi-layered eyespots exhibited about twice the reflection intensity at the presumptive photoreceptor site compared to single/double-layered eyespots. Our results provide the first experimental evidence to support the proposal that multi-layered eyespots act as interference reflectors in photoaxis of green algae.

Triton-treated cells of the coenocytic green alga, Ernodesmis verticillata, manifest longitudinal contraction and centripetal closure of the cytoplasm in the presence of exogenous free calcium ions. Complete closure of the cytoplasm around the vacuolar remnants is obtained with 1 mM ATP present in the activation medium. The cytoplasmic contractions in these cell models closely resemble normal wound healing motility phenomena previously reported for this organism, thereby providing a system for studying the contractions associated with cellular wound healing. Dye-exclusion experiments with eosin B reveal that the tritonized models are freely permeable, whereas untreated cells do not take up the stain. Ultrastructurally, activated (contracting) models possess a network of microfilaments beneath the plasma membrane remnants in regions of active contraction. Microtubules are associated with the microfilaments, but they also occur throughout the periphery of the entire cells, even in unactivated models. This suggests that microfilaments may be involved in localized contractions leading to wound healing, whereas microtubules are probably providing a cytoskeletal framework upon which the microfilaments act. Colchicine (5 mM) has no effect on contraction in the models; extensive experiments with cytochalasins B and D (up to 100 micrograms/ml) have failed to show regular, reproducible inhibition of contraction also. Trifluoperazine (10 microM) or La3+ (1 mM) decreases the rate and magnitude of contraction to some extent, perhaps by preventing Ca2+ interaction with the contractile apparatus. The threshold concentration of free Ca2+ necessary to activate contraction in the models appears to be 10(-6) M, an order of magnitude higher than the preliminary value reported for non-permeabilized, wounded cells. Free Mg2+ also must be present for activation of models, with a minimum concentration of ca. 5 mM. The requirement of ATP for contraction can be demonstrated by the fact that either excessive washing of cell models prior to activation or inclusion of uncoupling agents in the solutions largely prevents activation of the models unless ATP is added to the Ca2+ -containing activation medium. These results provide further evidence that cellular wound healing in Ernodesmis is similar to the majority of other non-muscle cell motility phenomena in requiring ATP and micromolar amounts of free Ca2+ and that microfilaments are implicated in the contractile process. Moreover, the Ernodesmis cell models should be excellent systems for the further probing of cytoplasmic contractions associated with wound healing at the cellular level of organization.

Cells of Oocystis solitaria after treatment with inhibitors of a) microfibril orientation b) microfibril synthesis, and c) microfibril crystallization have been investigated with the freeze-fracturing technique. Changes in morphology of the E-face of the plasma membrane as a result of these treatments have been recorded. Terminal complexes are removed by cycloheximide, but not by colchicine or congo red treatments. Microfibril imprints are removed by congo red but not by colchicine or cycloheximide treatments. Congo red induces in addition the presence of increased numbers of paired, thickened, terminal complexes. Prolonged (4-6 h) treatment with congo red causes the insertion of a second set (oriented at 90 degrees to the first set) of parallel terminal complexes in the E-face. These results are discussed in relation to plasma membrane turnover.

As an extension of the observation and interpretation regarding the different microtubule systems of Micrasterias denticulata [12, 19], the existence of intertubular structures, such as microfilaments, which are strongly marked in osmotically treated cells, is especially interesting. The complex of microtubules and microfilaments occurs during post-telophase nuclear migration, probably engaged in the mechanism of movement. The arrangement of microtubules either parallel or perpendicular to the nuclear membrane is characteristic for the stage of nuclear migration. Another microtubule system, the microtubule band in the cortical protoplasm of the isthmus region [12], is described during morphogenesis of the new half cell. Osmotically treated cells in the stage of septum formation demonstrate the presence of cross-linked microtubules near the plasmalemma and microtubule bundles, situated in the protoplasm between the secondary wall and the chloroplast, probably representing the microtubule system in the cortical protoplasm of the old half cell described by Kiermayer [12, 16]. The frequent appearance of microtubules and intertubular structures in differentiating cells of Micrasterias denticulata after osmotic treatment is discussed along with implication for stabilization of microtubules, cross bridges, and microfilaments.

In the giant marine green alga Bryopsis the chloroplasts are attached with their flattened, ventral sides to the inner surface of the cortical cytoplasm. They move at speeds up to 60 microns/min in the direction of the long axis of the cell either in a coordinated fashion or independently of each other. Intracellular sedimentation of chloroplasts by centrifugation leaves an intact cell cortex in which the movement of mitochondria and nuclei--normally obscured by chloroplasts--can be observed. Mitochondria display a saltatory type of movement alongside an extensive, two-dimensional system of phase-translucent channels. Nuclei appear to be entangled in the channel system and move in an unusual, rolling fashion. With a new technique involving the microsurgical removal of the chemically fixed cytoplasm from the confinement of the cell wall, this unique cell type is made accessible to immunocytochemical procedures. Microtubules (MT) can be visualized using a variety of tubulin antibodies, while actin only reacts with one monoclonal antibody out of several antibodies tested. Microtubules form a dense, two-dimensional palisade of bundles extending longitudinally in the cortical cytoplasm. Parallel arrays of actin fibers closely, but not exclusively, colocalize with the MT bundles. Particularly strong actin staining is observed near converging MT bundles underneath the tip regions of the chloroplasts. Because of the extensive superposition of actin and MTs, both cytoskeletal elements could potentially cooperate in creating the diversity of organelle movements in this alga. The respective roles of MTs and actin in chloroplast movement are experimentally tested in the accompanying paper.

The cortical cytoplasm of the giant cells of Bryopsis contains hundreds of interconnected microtubule (MT) bundles aligned along the cell's long axis. Actin fibers show an extensive but not exclusive superposition with these MT bundles. Chloroplasts move parallel to the bundles. Colchicine (0.5 mM), vinblastine (0.1 mM), and the herbicide ami-prophosmethyl (APM, 1-5 microM) strongly inhibit chloroplast movement and severely disrupt both the MT and the actin network. Additionally, APM leads to the appearance of large actin bundles up to 5 microns in diameter and several tens of micron in length. Erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA, 1 mM) does not block chloroplast movement, but affects chloroplast behavior by causing transient aggregations. The MT network is not significantly changed by EHNA, but actin fibers converge in large, radially symmetric complexes in regions of chloroplast aggregates. Cytochalasin D (CD, 1-10 micrograms/ml) leads to a significant but transient reduction of chloroplast speed within the first 60 min, as the actin network breaks down into small foci. Within the next 1 to 3 h of treatment, these foci segregate into massive clusters where chloroplasts remain immobilized. At the same time, chloroplast movement recovers in other areas of the cell. This recovery coincides with the reappearance of actin filament bundles in these cell regions. The MT cytoskeleton is not significantly affected by CD. These data are inconsistent with a mechanism of chloroplast movement in Bryopsis based solely on either MTs or actin, but instead they suggest an intimate interaction of both cytoskeletal networks in maintaining the spatial organization of the cytoplasm and in supporting chloroplast movement.

Perfused Chara cells capable of resuming ATP-dependent cytoplasmic streaming in low free Ca++ solutions have been examined by electron microscopy for myosin-like filaments. Filaments 44 nm in diameter and up to 3 micron in length have been found associated with the endoplasmic reticulum that along with mitochondria, microbodies and dictyosomes from the endoplasm becomes immobilised around the sub-cortical actin bundles when ATP is depleted. Such endoplasmic filaments have not been detected in association with mitochondria or microbodies and they have not been found in the stationary cortex. These filaments are extracted from the perfused cell by ATP unless motility-inhibiting levels of cytochalasin B are present. The filaments are not detectable in cells inactivated in solutions containing high (10(-4) M) Ca++ concentrations even when the Ca++ level is subsequently lowered. Consistent with their being required for motility, cytoplasmic streaming cannot be effeiciently reactivated by ATP in such filament-depleted cells. The possibility is discussed that the filaments contain myosin and that the endoplasmic reticulum with which they are associated has a major role in generating and transmitting the motive force for streaming.

The chloroplasts of the green alga, Ulva lactuca L., migrate rhythmically between the outer (periclinal) cell walls in the daytime ("face" position) and the anticlinal cell walls at night ("profile" position). Both NaN3 and colchicine inhibit chloroplast movement mainly in the direction from profile towards face position. Differential drug sensitivity is suggestive of different mechanisms for the two directions of chloroplast migration. UV light reverses the inhibition by colchicine, presumably through the formation of lumicolchicine, the non-tubulin-binding isomer of colchicine. This result is indicative of microtubule involvement. Interpretation of the effects of azide is complicated by changes in the biological clock (phase delay and lengthening of the period). Cytochalasin B has no effect on chloroplast movement when added alone, but when added with colchicine it prevents colchicine inhibition of movement. We hypothesize that chloroplast position is controlled by a balance between two opposing movement systems with differential drug sensitivity.