Diedrik Menzel

Freeze shattering: a simple and effective method for permeabilizing higher plant cell walls

This article describes a practical technique for permeabilization of higher plant cell walls, which is usually one of the first steps required for immunolocalization of cellular components (and other cytological methods) in plant cell studies. Our strategy involves shattering the walls of cells while the tissues are frozen in liquid nitrogen. It replaces the use of wall degrading enzymes or the need to employ laborious sectioning or other mechanical means for providing access of probes to cells. Freeze‐shattering retains the integrity of whole tissues and cells surprisingly well and thus is especially useful when used in conjunction with confocal laser scanning microscopy for recording the three‐dimensional arrangement of cytoskeletal elements in relation to cell shape. In this article, we demonstrate the effectiveness of this technique for anti‐tubulin and anti‐actin immunofluorescence and for rhodamine phalloidin labelling of the cytoskeleton in various higher plant tissues including onion root tip and bulb scale epidermis, Tradescantia stamen hairs and Arabidopsis leaf epidermis and mesophyll cells.

Actin in living and fixed characean internodal cells: identification of a cortical array of fine actin strands and chloroplast actin rings

SummaryWe report on the novel features of the actin cytoskeleton and its development in characean internodal cells. Images obtained by confocal laser scanning microscopy after microinjection of living cells with fluorescent derivatives of F-actin-specific phallotoxins, and by modified immunofluorescence methods using fixed cells, were mutually confirmatory at all stages of internodal cell growth. The microinjection method allowed capture of 3-dimensional images of high quality even though photobleaching and apparent loss of the probes through degradation and uptake into the vacuole made it difficult to record phallotoxin-labelled actin over long periods of time. When injected at appropriate concentrations, phallotoxins affected neither the rate of cytoplasmic streaming nor the long-term viability of cells. Recently formed internodal cells have relatively disorganized actin bundles that become oriented in the subcortical cytoplasm approximately parallel to the newly established long axis and traverse the cell through transvacuolar strands. In older cells with central vacuoles not traversed by cytoplasmic strands, subcortical bundles are organized in parallel groups that associate closely with stationary chloroplasts, now in files. The parallel arrangement and continuity of actin bundles is maintained where they pass round nodal regions of the cell, even in the absence of chloroplast files. This study reports on two novel structural features of the characean internodal actin cytoskeleton: a distinct array of actin strands near the plasma membrane that is oriented transversely during cell growth and rings of actin around the chloroplasts bordering the neutral line, the zone that separates opposing flows of endoplasm.

An interconnected plastidom inAcetabularia: Implications for the mechanism of chloroplast motility

SummaryIn the unicellular green algaAcetabularia, the vital fluorochrome 3,3′-dihexyloxacarbocyanine (DiOC6) readily accumulates in chloroplasts and mitochondria at low concentrations, suboptimal for the visualization of the endoplasmic reticulum (ER). These organelles align along motility tracks and partially obscure each other, resulting in the loss of image information in conventional fluorescence microscopy. However, superior imaging of organelles was achieved by confocal laser scanning microscopy, which was particularly evident in areas where mitochondrial profiles overlap with chloroplasts. In addition to the tubular mitochondria, a new type of tubular membrane profiles was discovered inAcetabularia which connects the chloroplasts with each other. These tubules may either form short bridges or may stretch over hundreds of micrometers before connecting to the next chloroplast. Because staining intensity, size and overall shape of mitochondria and the connecting membrane tubules were very similar, pharmacological treatments have been applied to differentiate more clearly between the two compartments. Inhibitors of mitochondrial function are shown here to affect mitochondrial shape but not that of the chloroplast tubules. Finally, electron microscopic analysis of thin sectioned materials revealed long tubular emanations from the chloroplasts proving their plastidal origin. The function of these hitherto unknown plastidal membrane tubules is not known, but their behaviour suggests that they interact with the cytoskeleton and effectively modify chloroplast behaviour.

18S RDNA AND EVOLUTION IN THE DASYCLADALES (CHLOROPHYTA) - MODERN LIVING FOSSILS

Phylogenetic relationships were inferred from parsimony and distance analyses of nuclear small‐subunit ribosomal DNA sequences taken from 14 species representing 8 of the 11 extant genera in the Dasycladales. Of 1733 aligned positions, 412 (23.8%) were variable and 251 (61%) of those were phylogenetically informative within the Dasycladales. Secondary structure was analyzed and taken into account during all phases of data analysis. Robustness of the trees was assessed using bootstrap analysis and g1 statistics of tree‐length decay. Strongly supported branches were robust to all methods of analysis regardless of weighting schemes used. The secondary structure of the 18S within the Dasycladales agrees with that of other green algae with the exception of a shared deletion in stemloop E10‐1 (ca. 13 nucleotides long), which provides additional support for the uniqueness of this monophyletic group. A molecular clock was calibrated from the dasyclad fossil record and suggests a radiation of the Acetabulariaceae at 120 ± 30 million years (Ma) ago and the Dasycladaceae 215 ± 40 Ma ago. The split of the two lineages from a shared ancestor is estimated at 265 ± 50 Ma ago. Within the Dasycladaceae, Neomeris and Cymopolia are sister taxa, as are Batophora and Chlorocladus. Bornetella groups with the Neomeris and Cymopolia clade in 78% of the bootstrap replicates. Relationships among the Acetabulariaceae show that Acetabularia and Polyphysa do not form monophyletic groups as presently circumscribed. No evidence indicates that Acicularia is the oldest genus. Halicoryne, Chalmasia, and Dasycladus were not included in the analysis. Molecular data provide afresh background perspective from which to discuss the evolution of one of the most ancient lineages of green plants.

The anti-proliferative agent jasplakinolide rearranges the actin cytoskeleton of plant cells

In the present study, we have characterized the action of the natural cyclodepsipeptide jasplakinolide (JAS) on the cytoplasmic architecture, actin-based cytoplasmic motility, and the organization of the actin cytoskeleton in selected examples of green algae (Acetabularia, Pseudobryopsis and Nitella) and higher plant cells (Allium bulb scale cells and Sinapis root hairs). JAS was capable of influencing the actin cytoskeleton and inhibiting cytoplasmic streaming in a differential, cell type-specific manner. With the exception of Nitella, two consecutive responses were observed upon incubation with 2.5 microM JAS: In the first phase cytoplasmic streaming increased transiently alongside with minor modifications of the actin cytoskeleton in the form of adventitious actin spots and spikes appearing throughout the cell cortex in addition to the normal actin bundle system typical for each cell type. In the second phase, cytoplasmic streaming stopped and the actin cytoskeleton became heavily reorganized into shorter, straight, more and more randomly oriented bundle segments. JAS exerted severe long-term effects on the actin cytoskeleton when treatments exceeded 30min at a concentration of 2.5 microM. An in situ competition assay using equimolar concentrations of JAS and FITC-phalloidin suggested that JAS has a phalloidin-like action. Effects of JAS were significantly different from those of cytochalasin D with respect to the resulting degree of perturbance of cytoplasmic organization, the distribution of actin filaments and the speed of reversibility.

The role of the cytoskeleton in polarity and morphogenesis of algal cells

Selected algal species continue to serve as model organisms for the study of cell growth and cellular morphogenesis. Recent improvements in immunohistochemical and microinjection methods have helped to consolidate our views of the role of the cytoskeleton as a generator of spatial patterns in the cytoplasm before cellular morphogenesis. Progress has also been made in the discovery and characterization of molecular components of both the cytoskeleton and the extracellular matrix (ECM). Studies on the oocytes of fucoid brown algae have demonstrated that the ECM serves an active role in controlling cell shape and in defining the developmental fate of a cell. Actin, transmembrane proteins of the beta-integrin type, and vitronectin-like proteins in the ECM have been discussed as important elements in polar axis formation in the early steps of post-fertilization development. The mechanism of cell expansion has been investigated in the large coenocytic cells of the siphonoclad green algae. It was shown that the alignment of cell wall microfibrils in these cells depends on the degree of order in the cortical microtubule system. However, in contrast to earlier hypotheses, microtubules do not appear to function as physical boundaries guiding the paths of cellulose synthesizing terminal complexes in the plane of the plasma membrane. Recent work on the giant unicellular green alga Acetabularia has revealed dynamic reorganizations of the actin cytoskeleton during the course of apical morphogenesis. Actin has also been suggested to play a role, in more subtle ways, in the establishment of membrane prepatterns during cellular morphogenesis of the desmid green alga Micrasterias, prepatterns that predict regions of future surface expansion.

Improved visualization of F-actin in the green alga Acetabularia by microwave-accelerated fixation and simultaneous FITC-Phalloidin staining.

SummaryBy employing a new procedure we have been able to visualize a highly intense actin cytoskeleton in the unicellular green algaAcetabularia acetabulum Silva. The protocol described in this study involves microwave-accelerated simultaneous permeabilization with 10% dimethyl sulphoxide, fixation with 1% glutaraldehyde and incubation with 0.5 μM fluorescein-isothiocyanateconjugated Phalloidin. Comparison of the images of the actin cytoskeleton of the stalk, as visualized by methods used previously, with those obtained in our own experiments shows that the actin filaments were preserved completely in an excellent condition. The required time for each procedure could be reduced from 12 h for the most commonly used immunofluorescence technique to 35 min. Moreover, it has been possible to observe the actin filament system of hair whorls, rhizoid and tip. Previously, the actin cytoskeleton of these parts of the cell could not be visualized by conventional techniques. It is shown that each region of the cell-stalk, tip, rhizoid and sidebranches-displays characteristic degrees of actin bundling and regularity of actin alignment.

The perinuclear microtubule system in the green algaAcetabularia: anchor or motility device?

SummaryMigrating secondary nuclei inAcetabularia are tightly associated with actin bundles and possess a comet-like tail composed of microtubules. When secondary nuclei begin to settle in preparation for cyst morphogenesis, the tails expand into radially symmetrical arrays of microtubules. Concomitantly, nuclei become gradually dissociated from the actin bundles and eventually stop moving, even though the actin bundles remain intact and persist through this stage. If, however, the radial perinuclear microtubule arrays are destroyed by inhibitors, the nuclei reassociate with the actin bundles and regain their motile activity. Because this movement is sensitive to Cytochalasin D, we propose that actin is required for nuclear movements, whereas microtubules most likely function as a trailing anchor that begins to act as a braking device above a certain threshold in the number and length of perinuclear microtubules.

Class XIII myosins from the green alga Acetabularia: driving force in organelle transport and tip growth?

The green alga Acetabularia cliftonii (Dasycladales) contains at least two myosin genes, which already have been assigned class XIII of the myosin superfamily (Cope et al., 1996, Structure 4: 969–987). Here we report a complete analysis of their gene structure and their corresponding transcripts Aclmyol and Aclmyo2. Despite promising Northern blot data no evidence for alternative splicing could be found. Dissecting the primary structure at complementary deoxyribonucleic acid (cDNA) level we found a myosin typical organization in head, neck and variable tail region. Most striking is the extremely short tail region of Aclmyol with only 18 residues and the maximum number of 7 IQ motifs in Aclmyo2. Probing Acetabularia protein extracts with an antibody raised to a synthetic peptide derived from the amino terminal region in Alcmyol showed cross-reactivity to a polypeptide with a molecular mass of ∼100 kD. This corresponds to the predicted molecular weight of Aclmyol, which is 106 kD as deduced from the amino acid sequence. Additionally, the same cross-reactive protein is capable of binding F-actin as indicated by a co-sedimentation assay. Confocal laser scanning microscopy with raised antibody revealed co-localization with organelles, the budding region of lateral whorls and the cell apex suggesting involvement of putative Acetabularia myosin in organelle transport and tip growth.

Chromosomal architecture in giant premeiotic nuclei of the green algaAcetabularia

SummaryGiant primary nuclei of the unicellular green algaAcetabularia contain 40 small lampbrush chromosomes which have proved difficult to visualize in the light microscope in vivo by conventional fluorescent DNA staining techniques. We report here that immunofluorescence staining with the monoclonal anti-phosphoepitope antibody MPM 2 is the method of choice to study the architecture of whole chromosomes within primary nuclei fixed in situ or after hand isolation. Using confocal laser scanning microscopy, we have been able to produce images ofAcetabularia lampbrush chromosomes of hitherto unsurpassed structural detail. Particularly striking is the visualization of abundant loops extending from the periaxial chromosome core region for variable distances into the nucleoplasm. Staining of the loops can be abolished by pretreatment of isolated nuclei with alkaline phosphatase, whereas the chromosomal core remains unaffected. At meiosis, when transcription ceases and the extended chromosomes condense, MPM 2 staining is lost indicating that maintenance of the loops depends on the phosphorylation status of the MPM 2 antigen. Anti-histone antibodies, on the other hand, exclusively stain the chromosomal core in the extended lampbrush configuration as well as in the condensed metaphase configuration. This indicates that MPM 2 and anti-histone antibodies recognize different molecular components on the chromosomes. We postulate that the loops stained by MPM 2 represent actively transcribing regions of the chromosomes and that the MPM 2 antigen could be a molecular component involved either in the structural maintenance of the loops or in a process associated with transcription.