Transformation of the Golgi apparatus in the cell cycle,especially at the resting and earliest developmental stages of a green alga,Micrasterias americana

Summary Transformation of the Golgi apparatus inMicrasterias americana at various stages after full growth and at the earliest stage of cell growth was investigated using an electron microscope. Silver-hexamine staining and the acid phosphatase (ACPase) test were also carried out. In cells cultured for two days after full growth, dictyosomes began to produce hypertrophied vesicles (HVs) along their five peripheral reagions. The HVs contained fibrous material, which was stained by silver-hexamine, and small granules which reacted with ACPase. The HVs were pinched off the dictyosomes and fused with each other and with the vacuoles. In the earliest stage of cell growth, the cisternae of the dictyosomes were stretched in one direction, which modified the shape from circular to elliptical and the dictyosomes curved along the long axis of the ellipse. These curved dictyosomes which produced middle sized vesicles (MVs) from the distal networks, divided into two identical parts along the short axis of the ellipse.     

Electrophysiological characteristics of the unicellular green alga Micrasterias torreyi

The electrophysiological characteristics of the unicellular green alga Micrasterias torreyi Bail. are studied here for the first time using microelectrode techniques. The resting potential of the plasma membrane varied between –39.5 and –42.2 mV for different developmental stages of the dividing cell and was –41.7 mV ( se = 3.2, n = 9) in the interphase cells. The resting potential of the chloroplast envelope was lower, –53.9 mV ( se = 3.6, n = 15). Supraoptimal K⁺ (20 m M ) had no clear effects on the plasma membrane but caused a depolarization of 10 mV in the chloroplast. Additional external Ca²⁺ (10 m M ) depolarized the membrane potential quite strongly (by 23 mV). Low external pH did not affect the resting potential of the cell. There is a marked difference in the resting potential values between non-vacuolated cells (about –40 mV), to which Micrasterias belongs, and vacuolated plant cells (–100 to –250 mV). This indicates the participation of the tonoplast in the transport of ions and charged molecules in vacuolated cells. Na⁺ and Cl⁻, which play an important role in ion metabolism in most plant cells, are not needed by Micrasterias .     

Cortical microtubules and cortical microfilaments in the green alga,Micrasterias pinnatifida

Summary Cortical microtubules and cortical microfilaments were visualized in cells ofMicrasterias pinnatifida treated by freeze-substitution, and the pattern of their distribution was reconstructed from serial sections. Most cortical microtubules accompanied the long microfilaments that ran parallel to the microtubules. Cortical microfilaments not accompanied by the microtubules were also found. They were short and slightly curved. Both types of cortical microfilament were not grouped into bundles, and were 6–7 nm in diameter, a value that corresponds to the diameter of filaments of F-actin.     

PCD and autophagy in the unicellular green alga Micrasterias denticulata

Programmed cell death (PCD) plays a central role in normal plant development and is also induced by various biotic and abiotic stress factors. In the unicellular freshwater green alga Micrasterias denticulata morphological and biochemical hallmarks such as the appearance of autophagosomes, increased production of ROS and degradation of genomic DNA into small fragments (“DNA laddering”) indicate PCD. Our data not only demonstrate that Micrasterias is capable of performing PCD under salt stress, but also that it is triggered by the ionic and not osmotic component of salinity. Additionally, results from the present and previous studies suggest that different inducers may lead to different cell death pathways in one and the same organism.     

Chondramides, novel cyclodepsipeptides from myxobacteria, influence cell development and induce actin filament polymerization in the green alga Micrasterias

The effects of chondramides A-D, new actin targeting cyclodepsipeptides from the myxobacterium Chondromyces crocatus, are probed on the unicellular green alga Micrasterias denticulata, a model organism for studies on cytomorphogenesis. All four chondramides readily enter the cells and cause severe shape malformations when applied during growth. However, the four derivatives have different lowest effective concentrations. Chondramide A: 20 microM, chondramide B: 15 microM, chondramide C: 5 microM chondramide D: 10 microM. At the ultrastructural level, chondramide C, the most effective drug, causes the appearance of abnormal, dense F-actin bundles, and a substantial increase in ER, which covers large parts of the developing semicell. Also the secondary cell wall is malformed by the drug. When chondramide C effects are investigated by means of indirect immunofluorescence, alterations of the F-actin system are also visible. Instead of the cortical F-actin network of untreated controls, distinct parts of the cell are covered by abundant F-actin aggregations. Phalloidin staining of chondramide C treated cells results in a decreased fluorescence in a time-dependent manner due to binding competitions between these drugs. F-actin polymerizing and bundling capacities of chondramides A-D are presented in Micrasterias for the first time, and may in future make this substances a useful tool for cell biological research.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Cytoskeleton mediating transport between the ER system and the Golgi apparatus in the green alga Scenedesmus acutus

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.     

Scale-invariant pattern in the alga Micrasterias

Spatial structures arise in a variety of different physical, chemical and biological systems. A striking example is found during morphogenesis in the single-celled alga Micrasterias, where cell extensions called lobes branch repeatedly to produce a highly regular, apparently self-similar pattern. Lobe outgrowth in Micrasterias is thought to be controlled by the local accumulation of growth determinants at the lobe tips. These tip-growth sites undergo successive spatial bifurcations, leading to the recursively branched, final cell form. I have tested for scale invariance of this form, by measuring the distribution of tips as a function of position along the cell perimeter in mature Micrasterias cells of four different species. This tip distribution should reflect the steady-state distribution of growth determinants at the end of the spatial bifurcation process. For each cell measured, the distribution of tips resembled a Cantor set with three levels of constant, nested scaling. Significantly, roughly the same scale factor (3.0) was found at each scaling level in individual cells, and among different cells in each of the four species measured. These data suggest that scaling by this factor is intrinsic to the pattern formation process in Micrasterias.     

Nitric oxide suppresses growth and development in the unicellular green alga Micrasterias denticulata

Nitric oxide (NO), a key molecule in inter- and intracellular signalling, is implicated in developmental processes, host defense, and apoptosis in higher plants. We investigated the effect of NO on development in the unicellular green alga, Micrasterias denticulata, using two different NO donors, S-nitroso-N-acetyl-dl-penicillamine (SNAP) and sodium nitroprusside (SNP). Investigations at the light microsopic level revealed that both NO donors suppressed cell growth. Ultrastructural analyses were performed with SNAP- as well as SNP-treated cells and, additionally, with the control compound N-acetyl-d-penicillamine (NAP). Cells incubated with NO donors lacked a secondary wall and dictyosomal function was impaired, whereas NAP-treated cells showed no difference in development and organelle structure compared to control cells. Moreover, cisternae of the Golgi stacks were slightly involute and no vesicles were pinched off after SNAP and SNP incubation. The NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, potassium salt) abrogated the effect of SNP, thus confirming that inhibition of cell growth is due to nitric oxide. Addition of iodoacetic acid, an inhibitor of cysteine-containing enzymes, like glyceraldehyde-3-phosphate dehydrogenase (GAPDH), evoked similar effects on cell growth and secondary wall formation as obtained by treatment with NO donors. Therefore, we hypothesize that NO inhibits activity of enzymes involved in the secretory pathway, such as GAPDH, via S-nitrosylation of the cysteine residue and, consequently, modulates cell growth in M. denticulata.     

Growth kinetics of the Golgi apparatus during the cell cycle in onion root meristems

In onion root meristems, the number of dictyosomes per cell shows a kinetics of growth strongly related to the cell cycle. During the interphase of steady-state proliferative cells, the volume density and numerical density of the Golgi apparatus decrease to reach minimum values in late-interphase cells, characterized by their greatest length. This pattern is also found in the total volume occupied by Golgi apparatus. Once in mitosis, the above-mentioned parameters begin to increase reaching maximum mean values in telophase. After the experimental uncoupling of chromosome and growth cycles by presynchronization with hydroxyurea, we found a similar behaviour pattern in the Golgi apparatus: decreasing values during interphase and a triggering of Golgi-apparatus growth in prophase independently of the bigger cell sizes reached in mitosis as an effect of pretreatment with hydroxyurea. These results indicate a cyclic kinetics of this subcellular component in higher-plant meristems, coupled with early mitotic events.     

Kinesin-like proteins are involved in postmitotic nuclear migration of the unicellular green alga Micrasterias denticulata

The unicellular green alga Micrasterias denticulata performs a two-directional postmitotic nuclear migration during development, a passive migration into the growing semicell, and a microtubule mediated backward migration towards the cell centre. The present study provides first evidence for force generation by motor proteins of the kinesin family in this process. The new kinesin specific inhibitor adociasulfate-2 causes abnormal nuclear displacement at 18 microM. AMP-PNP, a non hydrolyseable ATP analogue or the general ATPase inhibitors calyculin A and sodium orthovanadate also disturb nuclear migration. In addition kinesin-like proteins are detected by means of immunoblotting using antibodies against brain kinesin, plant derived antibodies to kinesin-like proteins and a calmodulin binding kinesin-like protein. Immunoelectron microscopy suggests a correlation of conventional kinesin-like proteins, but not of the calmodulin binding kinesin-like protein to the microtubule apparatus associated with the migrating nucleus.     

Physiological and structural changes in the chloroplast of the green alga Micrasterias denticulata induced by UV-B simulation

Exposure of postmitotic growing and non-growing cells of the unicellular green alga Micrasterias denticulata to different UV-B cut-off wavelengths together with simulated sunlight in a sun simulator has revealed a marked resistence of the algae against strong irradiation. While down to a cut-off wavelength of 284 nm irradiated during the most sensitive stage of cell development chloroplast ultrastructure remains unaffected, severe changes in arrangement and structure of stroma and grana thylakoids occur only at the lowest cut-off wavelengths of 280 and 275 nm. The structural alterations end up in a more or less complete desintegration of grana and stroma thylakoids with the remaining membraneous structures appearing in negative staining thus indicating drastic changes in membrane composition. Photosynthetic activity determined by chlorophyll fluorescence (ratio of variable to maximal fluorescence) and oxygen evolution responded more sensitively to UV-B irradiation. With decreasing UV cut-off wavelengths and prolonged incubation a decrease of photochemistry of PS II occured reaching its lowest values after 60 min at 275 and 280 nm. Oxygen production was even maintained under strong UV irradiation with a cut-off wavelenght of 275 nm up to 15 min. With prolonged UV-B treatment any activity was lost. HPLC separations of pigments exhibited the appearance of break-down products (mainly derivatives of chl b and chl a) with decreasing cut-off wavelength and increasing exposure time. The xanthophyll cycle pigments seemed to be unaffected at least for an irradiation period of 60 to 90 min at low UV cut-offs. Possible mechanisms of UV stress avoidance or protection are discussed with regard to the varying altitudes of the natural habitats of the algae.     

Genome-wide analysis of core cell cycle genes in the unicellular green alga Ostreococcus tauri

The cell cycle has been extensively studied in various organisms, and the recent access to an overwhelming amount of genomic data has given birth to a new integrated approach called comparative genomics. Comparing the cell cycle across species shows that its regulation is evolutionarily conserved; the best-known example is the pivotal role of cyclin-dependent kinases in all the eukaryotic lineages hitherto investigated. Interestingly, the molecular network associated with the activity of the CDK-cyclin complexes is also evolutionarily conserved, thus, defining a core cell cycle set of genes together with lineage-specific adaptations. In this paper, we describe the core cell cycle genes of Ostreococcus tauri, the smallest free-living eukaryotic cell having a minimal cellular organization with a nucleus, a single chloroplast, and only one mitochondrion. This unicellular marine green alga, which has diverged at the base of the green lineage, shows the minimal yet complete set of core cell cycle genes described to date. It has only one homolog of CDKA, CDKB, CDKD, cyclin A, cyclin B, cyclin D, cyclin H, Cks, Rb, E2F, DP, DEL, Cdc25, and Wee1. We have also added the APC and SCF E3 ligases to the core cell cycle gene set. We discuss the potential of genome-wide analysis in the identification of divergent orthologs of cell cycle genes in different lineages by mining the genomes of evolutionarily important and strategic organisms.     

Control of cell division in a green alga, Ankistrodesmus gracilis

The proliferation pattern of Ankistrodesmus gracilis, a speciesof Chlorococcales, is described. Under standard conditions,various proliferation patterns; di-, tri-, tetra-, or multichotomicalcell division were observed. Light-dark diurnal rhythms, LD12 : 12 and 14 : 10 induced growth patterns which formed two-to several-ten-celled colonies, whereas the rhythms LD 16 :8 to 20 : 4 induced only the formation of two-celled colonies.These inductions were observed at a cell density of 4.0?10⁶cells/ml. Dichotomical cell division occured at a cell densityof more than 1.5?10⁷ cells/ml. No influence of self-shadingon the pattern of colony formation was detected. (Received May 30, 1974; )     

Architecture of the Golgi apparatus of a scale-forming alga: biogenesis and transport of scales

Mechanisms of transport of secretory products across the Golgi apparatus (GA) as well as of scale formation in prymnesiophytes have remained controversial. We have used a quantitative morphological approach to study formation and transport of scales across the GA in haploid cells of Pleurochrysis sp. The GA of these cells differs from the GA of higher plants in at least six morphological characteristics. Our results show that scales form in the trans-Golgi network (TGN) and transit the TGN in heretofore unrecognized prosecretory vesicles. Prosecretory vesicles differentiate into secretory vesicles prior to exocytosis of scales to the cell surface. Because prosecretory vesicles are only fragments of TGN cisternae, the classical model of cisternal progression is not a valid mechanism of transport in this alga. TGN transport vesicles are also involved in scale formation; however, the role of tubular connections between cisternae of a single stack-TGN unit is not clear. The relationship of two morphological types of cisternal dilations to a membrane-associated, bottlebrush-shaped macromolecule of novel morphology suggests a new hypothesis for the biogenesis of scales.