Structural stress responses and degradation of dictyosomes in algae analysed by TEM and FIB-SEM tomography.

Stress-induced physiological deficiencies in cells are reflected in structural, morphological and functional reactions of organelles. Although numerous investigations have focused on chloroplasts and mitochondria as main targets of different stressors in plant cells, there is insufficient information on the plant Golgi apparatus as stress sensor. By using the advantages of field emission scanning electron microscopy tomography in combination with classical ultrathin sectioning and transmission electron microscopic analyses, we provide structural evidence for common stress responses of the large and highly stable dictyosomes in the algal model system Micrasterias. Stress is induced by different metals such as manganese and lead, by starvation in 9 weeks of darkness or by inhibiting photosynthesis or glycolysis and by disturbing ionic homeostasis via KCl. For the first time a stress-induced degradation pathway of dictyosomes is described that does not follow "classical" autophagy but occurs by disintegration of cisternae into single membrane balls that seem to be finally absorbed by the endoplasmic reticulum (ER). Comparison of the morphological features that accompany dictyosomal degradation in Micrasterias to similar reactions observed during the same stress application in Nitella indicates an ubiquitous degradation process at least in algae. As the algae investigated belong to the closest relatives of higher land plants these results may also be relevant for understanding dictyosomal stress and degradation responses in the latter phylogenetic group. In addition, this study shows that two-dimensional transmission electron microscopy is insufficient for elucidating complex processes such as organelle degradation, and that information from three-dimensional reconstructions as provided by field emission scanning electron microscopy tomography is absolutely required for a comprehensive understanding of the phenomenon.

Cell death upon H(2)O(2) induction in the unicellular green alga Micrasterias.

In the present study, we investigated whether the unicellular green alga Micrasterias denticulata is capable of executing programmed cell death (PCD) upon experimental induction, and which morphological, molecular and physiological hallmarks characterise this. This is particularly interesting as unicellular freshwater green algae growing in shallow bog ponds are exposed to extreme environmental conditions, and the capacity to perform PCD may be an important strategy to guarantee survival of the population. The theoretically 'immortal' alga Micrasterias is an ideal object for such investigations as it has served as a cell biological model system for many years and details on its growth properties, physiology and ultrastructure throughout the cell cycle are well known. Treatments with low concentrations of H(2)O(2) are known to induce PCD in other organisms, resulting in severe ultrastructural changes to organelles, as observed in TEM. These include deformation and part disintegration of mitochondria, abnormal dilatation of cisternal rims of dictyosomes, occurrence of multivesicular bodies, an increase in the number of ER compartments, and slight condensation of chromatin. Additionally, a statistically significant increase in caspase-3-like activity was detected, which was abrogated by a caspase-3 inhibitor. Photosynthetic activity measured by fast chlorophyll fluorescence decreased as a consequence of H(2)O(2) exposure, whereas pigment composition, except for a reduction in carotenoids, was the same as in untreated controls. TUNEL positive staining and ladder-like degradation of DNA, both frequently regarded as a hallmark of PCD in higher plants, could only be detected in dead Micrasterias cells.

Pectin-like carbohydrates in the green alga Micrasterias characterized by cytochemical analysis and energy filtering TEM.

Pectins are the major matrix polysaccharides of plant cell walls and are important for controlling growth, wall porosity and regulation of the ionic environment in plant cells. Pectic epitopes recognized by the monoclonal antibodies JIM5, JIM7 and 2F4 could be localized in the primary wall during development of the green alga Micrasterias. As the degree of pectin esterification determines the calcium-binding capacity and thus the physical properties of the cell wall, chemical and enzymatic in situ de-esterification was performed. This resulted in displacement of epitopes recognized by JIM5, JIM7 and 2F4, respectively, in changes in the intensity of the antibody labelling as visualized in CLSM. In addition, calcium-binding capacities of cell walls and components of the secretory apparatus were determined in transmission electron microscopy by electron energy loss spectroscopy and electron spectroscopic imaging. These analyses revealed that pectic polysaccharides are transported to the cell wall in a de-esterified form. At the primary wall, pectins get methyl-esterified at the inner side, thus allowing flexibility of the wall. At the outer side of the wall they become again de-esterified and bind high amounts of calcium which leads to cell wall stiffening. Mucilage vesicles possess the highest calcium-binding capacity of all structures observed in Micrasterias, indicating that the pectic polysaccharides of mucilage are secreted in a de-esterified, compact form. When mucilage is excreted through the cell wall, it loses its ability to bind calcium. The esterification of pectins involved is obviously required for swelling of mucilage by water uptake, which generates the motive force for orientation of this unicellular organism in respect to light. Incubation of Micrasterias in pectin methylesterase (PME), which de-esterifies pectic polymers in higher plants, resulted in growth inhibition, cell shape malformation and primary wall thickening. A PME-like enzyme could be found in Micrasterias by PME activity assays.

Organelle interactions and possible degradation pathways visualized in high-pressure frozen algal cells.

Summary Organelle interactions, although essential for both anabolic and catabolic pathways in plant cells have not been examined in detail so far. In the present study the structure of different organelle-organelle, organelle-vesicle and organelle-membrane interactions were investigated in growing and nongrowing cells of the green alga Micrasterias denticulata by use of high pressure freeze fixation and energy filtering transmission electron microscopy. It became clear that contacts between mitochondria always occur by formation of a cone-shaped protuberance of one of the mitochondria which penetrates into its fusion partner. In the same way, structural interactions between mitochondria and mucilage vesicles and between microbodies and mucilage vesicles are achieved. Lytic compartments contact mitochondria or mucilage vesicles again by forming protuberances and by extending their contents into the respective compartment. Detached portions of mitochondria are found inside lytic compartments as a consequence of such interactions. Mitochondria found in contact with the plasma membrane reveal structural disintegration. Our study shows that interactions of organelles and vesicles are frequent events in Micrasterias cells of different ages. The interactive contacts between lytic compartments and organelles or vesicles suggest a degradation pathway different from autophagy processes described in the literature. Both the interactions between vesicles and organelles and the degradation pathways occur independently from cytoskeleton function as demonstrated by use of cytochalasin D and the microtubule inhibitor amiprophos-methyl.

Use of energy-filtering transmission electron microscopy for routine ultrastructural analysis of high-pressure-frozen or chemically fixed plant cells.

In the present study energy-filtering transmission electron microscopy by use of an in-column spectrometer is employed as a powerful tool for ultrastructural analysis of plant cells. Images of unstained very thin (50 nm) and thick (140 nm) sections of the unicellular green alga Micrasterias denticulata, as a model system for a growing plant cell, taken by conventional transmission electron microscopy are compared to those obtained from filtering at zero energy loss (elastic bright field) and to those generated by energy filtering below the carbon-specific absorption edge at about 250 eV. The results show that the high-contrast images produced by the latter technique are distinctly superior in contrast and information content to micrographs taken at conventional transmission electron microscopy mode or at elastic bright field. Post- or en bloc staining with heavy metals, which is indispensable for conventional bright-field transmission electron microscopy, can be completely omitted. Delicate structural details such as membranous or filamentous connections between organelles, organelle interactions, or vesicle and vacuole contents are clearly outlined against the cytoplasmic background. Also, immunoelectron microscopic localization of macromolecules benefits from energy-filtering transmission electron microscopy by a better and more accurate assignment of antigens and structures and by facilitating the detection of immunomarkers without renunciation of contrast.

Ozone impact on the photosynthetic apparatus and the protective role of polyamines.

One of the primary plant mechanisms protecting leaf cells against enhanced atmospheric ozone is the accumulation of polyamines, generally observed as an increase in putrescine level, and in particular its bound form to thylakoid membranes. Ozone-sensitive plants of tobacco (cultivar Bel W3) in contrast to ozone-tolerant Bel B, are not able to increase their endogenous thylakoid membrane-bound putrescine when they are exposed to an atmosphere with enhanced ozone concentration, resulting in reduction of their photosynthetic rates and consequently reduction in plant biomass formation. In comparison to the tolerant cultivar Bel B, a prolongation of ozone exposure thus can lead to typical visible symptoms (necrotic spots) in leaves of the sensitive plant. Exogenously manipulated increase of the cellular putrescine levels of the ozone-sensitive Bel W3 is sufficient to revert these effects, whereas a reduction in endogenous putrescine levels of the tolerant cultivar Bel B renders them sensitive to ozone treatment. The results of this work reveal a regulator role for polyamines in adaptation of the photosynthetic apparatus and consequently to its protection in an environment polluted by ozone.

Impairment of cytoskeleton-dependent vesicle and organelle translocation in green algae: combined use of a microfocused infrared laser as microbeam and optical tweezers.

A Nd-YAG laser at 1064 nm is used as optical tweezers to move intracellular objects and a laser microbeam to cause impairment of cytoskeleton tracks and influence intracellular motions in desmidiaceaen green algae. Naturally occurring migrations of large nuclei are inhibited in Micrasterias denticulata and Pleurenterium tumidum when the responsible microtubules are targeted with a laser microbeam generating 180 mW power in the focal plane. Impairment of the microtubule tracks appears to be irreversible, as the nucleus cannot pass the former irradiated area in Pleurenterium or remains abnormally dislocated in Micrasterias. The actin filament-dependent movement of secretory vesicles and smaller particles can be manipulated by the same IR-laser at 90 mW when functioning as optical tweezers. In Closterium lunula particles are displaced from their cytoplasmic tracks for up to 10 micro m but return to their tracks immediately after removing the light pressure gained by the optical tweezers. The cytoplasmic tracks consist of actin filament cables running parallel to the longitudinal axis of Closterium cells as depicted by Alexa phalloidin staining and confocal laser scanning microscopy. Dynamics and extensibility of the cytoplasmic strands connecting particles to the tracks are also demonstrated in the area of large vacuoles which are surrounded by actin filament bundles. In Micrasterias trapping of secretory vesicles by the optical tweezers causes irreversible malformations of the cell shape. The vesicle accumulation itself dissipates within 30 s after removing the optical tweezers, also indicating reversibility of the effects induced, in the case of actin filament-mediated processes.

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.

Cell wall secretion in the green alga micrasterias.

The monoclonal antibodies JIM 1 against non-arabinogalactan epitopes, JIM 5 and JIM 7 recognizing unesterified and methyl-esterified pectins, and JIM 8 specific for arabinogalactan proteins (AGPs) are used for investigating different stages of cell wall formation in high pressure frozen and freeze substituted Micrasterias cells by means of immunoelectron microscopy. The results show that the septum-forming vesicles and the septum wall consist mainly of methyl-esterified pectins which become only partly de-esterified in the septum wall. Arabinogalactan proteins appear at the septum rim at the end of septum growth and are main constituents of the primary cell wall, together with esterified pectins. Only the outermost layer of the primary cell wall is labelled by JIM 5, indicating the presence of unesterified pectins. AGPs, non-AGP epitopes indicated by JIM 1, and pectins are transported together in the contents of the primary wall, forming 'dark vesicles' from the site of their production at the dictyosomes to the plasma membrane. Labelling of exclusively the plasma membrane of the non-growing semicell by JIM 1 and JIM 8 points towards a regulatory mechanism of membrane glycoproteins for vesicle fusion. The secondary cell wall of Micrasterias is not labelled by any of the antibodies used. JIM 4, JIM 15, JIM 84 and MAC 207 do not produce any specific staining in Micrasterias.

Spectrin-like proteins in green algae (Desmidiaceae).

Immunochemical detection of actin as well as spectrin-like proteins have been carried out in the green algae Micrasterias denticulata, Closterium lunula, and Euastrum oblongum. In these algae, actin is detected on Western blots at 43 kDa with antibodies to actin from higher plant and animal origin. By use of antibodies to human and chicken erythrocyte spectrin a cross-reactivity with desmid proteins is found at about the molecular mass of 220 kDa, where also human erythrocyte spectrin is detected. Additional bands are present at 120 kDa and 70 kDa, which are probably breakdown products. An antibody against chicken alpha-actinin, a small protein of the spectrin superfamily, recognizes bands at 90 kDa, where it is expected, and 70 kDa, probably the same breakdown product as mentioned for spectrin. Isoelectric focusing provides staining at pI 4.6 with antibodies against spectrin. Immunogold labelling of spectrin and alpha-actinin antigens on high-pressure frozen, freeze-substituted Micrasterias denticulata cells with the same antibodies exhibits staining, especially at membranes of different populations of secretory vesicles, at dictyosomes, and the plasma membrane. However, no clear correlation to the growth pattern of the cell could be observed. Taken together, our results demonstrate the presence of spectrin-like proteins in desmid cells which are probably functional in exocytosis.