High Speed Cinemicrography of the Direct Photophobic Response of Euglena and the Mechanism of Negative Phototaxis*

SYNOPSIS. The shock reaction of Euglena gracilis strain Z to a sudden increase in light intensity (the “direct photophobic response”) was examined by high speed cinemicrography. The response is expressed as a turning reaction toward the dorsal side of the cell, after a transduction time of 0.1–0.5 sec after the onset of stimulation. Transduction times, turning rates, and flagellar beat frequencies were measured by analyzing the filmed sequences. The experimental data are consistent with a mechanism of directional homeostasis in negative phototaxis that is based upon shading of the photoreceptor by the cell's posterior end.     

Rhodopsin: A Photopigment for Phototaxis in Euglena gracilis

For over 100 years, a major focus of photobiological studies has been the unicellular flagellate, Euglena gracilis, an organism well suited for such investigations by its special complement of organelles that may be considered an ancient, yet complete “visual” system. The possible photoreceptive roles of the cytoplasmic stigma and the photoreceptor (paraflagellar swelling) of E. gracilis are still under debate, because of conflicting interpretations of the results produced so far by the different research groups working on this microorganism. This article deals with our hypothesis, first put forward in the late 1980s, that rhodopsin-like proteins are responsible for photo-detection and that the paraxial rod is involved in the control of flagellar movements. This hypothesis uses oriented dipole and electroconformational coupling mechanisms as the physical phenomena that produce signal transduction. A model for phototaxis is presented.     

Cyclic Changes in Chloroplast Structure in Synchronized Euglena gracilis*

SYNOPSIS. In populations of Euglena gracilis strain Z synchronized by cultivation on a repetitive light-dark cycle, chloroplasts undergo cyclic changes in structure. During most of the light period chloroplasts are relatively compact with closely appressed lamellae; during the dark (division) period the chloroplasts become quite distended. This change persists for at least one cycle even when the cells are left in continuous light, suggesting that the periodicity may be related more to the age of the cell than to a direct effect of light. In addition, the pyrenoid in synchronized cells has a transient existence, being present only in the first half of the light period.     

The Differentiation of Herpetomonas megaseliae: Ultrastructural Observations*

Ultrastructure of both undifferentiated (promastigote and paramastigote) and differentiated (opisthomastigote) forms of Herpetomonas megaseliae is described. There is a posterior migration of the kinetoplast at the end of the exponential growth phase. The posterior extension of the flagellar pocket precedes migration of the kinetoplast. Opisthomastigotes have an electron-translucent mitochondrial matrix in comparison with undifferentiated forms. The Golgi body changes from a stack of flattened sacs to an aggregation of vesicles. Several structures previously reported from Trypanosomatidae, e.g. subpellicular organelles, pellicular microtubules, membrane whorls, stored metabolic products, surface blebs, and an intraflagellar body are also present in H. megaseliae.     

The median body of Giardia lamblia: an ultrastructural study

Giardia lamblia is an intestinal parasite of several mammals. The most striking feature of Giardia is the presence of a complex and unique cytoskeleton, and among its components the median body (MB) is the least defined microtubular structure. In the present study, we used a technique that allowed the removal of the plasma membrane and observation of cytoskeletal structures by both routine scanning electron microscopy (SEM) and field emission high resolution SEM. This technique permitted new observations such as details and insights of the median bodies, not previously described or controversial in the literature. Light microscopy after Panotic staining, immunofluorescence microscopy using several antibodies, and thin sections were also used to better characterized the Giardia MB. The new observations concerning the median bodies were : (1) they are not one or two structures, but varied in number, shape and position ; (2) they were found in mitotic and interphasic trophozoites, in disagreement with previous works ; (3) they were present in about 80 % of the cells, and not in 50 % of the cells, as previously described ; (4) they could be connected either to the plasma membrane, to the adhesive disc, and caudal flagella, and thus they are not completely free in the cells, as published before ; (5) they can protrude the cell surface ; (6) their microtubules react with several anti-tubulin and -beta giardin antibodies. These observations add new data on the scarce literature and to this largely understudied cell structure.     

Proteolytic cleavage and structural transformation: Their relationship in bacteriophage T4 capsid maturation

Giant T4 phage capsoids formed in canavanine-treated cultures infected by phage mutants in genes 21 and 17, respectively, differ with regard to cleavage of the major capsid protein, gp 23, and in the fine structure of their hexagonal surface lattices. Quantitative computer processing of electron micrographs shows that the significant differences in capsomer morphology amount to six symmetrically placed features present in the uncleaved hexamer but absent after cleavage. These features may be related with the N-terminal portions of gp 23 monomers excised by phage-specific proteolysis. Cleaved 17^? giants can be induced to undergo a further structural transformation (expansion). Structural characteristics of partially transformed giant particles give clues about the dynamics of the cleavage and expansion transformations. Both processes appear to be polar, initiating in one cap and propagating along the particle. The transition zone of partial cleavage is diffuse, whereas the transition between unexpanded and expanded areas is confined to a narrow band of some 20 nm width.     

A Structural Model for Apolipoprotein C-II Amyloid Fibrils: Experimental Characterization and Molecular Dynamics Simulations

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple ‘letter-G-like’ β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops.     

The RecB Nuclease Domain Binds to RecA-DNA Filaments: Implications for Filament Loading

The E. coli RecBCD enzyme facilitates the loading of RecA onto single-stranded DNA produced by the combined helicase/nuclease activity of RecBCD. The nuclease domain of RecB protein, RecB^nuc, has been previously shown to bind RecA. Surprisingly, RecB^nuc also binds to phage and eukaryotic homologs of RecA, leading to the suggestion that RecB^nuc interacts with the polymerization motif that is present in all three proteins. This mode of interaction could only be with monomeric RecA, as this motif would be buried in filaments. We show that RecB^nuc binds extensively to the outside of RecA-DNA filaments. Three-dimensional reconstructions suggest that RecB^nuc binds to the ATP-binding core of RecA, with a displacement of the C-terminal domain of RecA. Solution experiments confirm that the interaction of RecB^nuc is only with the RecA core. Since the RecA C-terminal domain has been shown to be regulatory, the interaction observed may be part of the loading mechanism where RecB displaces the RecA C-terminal domain and activates a RecA monomer for polymerization.     

The Permanence of the Infraciliature in Suctoria: An Electronmicroscopic Study of Pattern Formation in Tokophrya infusionum*

SYNOPSIS. The adult Tokophrya infusionum does not possess cilia, but has 20–30 barren basal bodies arranged in 6 short rows adjacent to the contractile vacuole pore. During reproduction, which is by internal budding, the contractile vacuole sinks into the parent along with the invaginating membranes that form the embryo and the wall of the brood pouch. The 6 rows of basal bodies radiate away from the pore and elongate to form 5 long ciliary rows, that encircle the anterior half of the embryo, and 1 short row at the posterior end. The contractile vacuole pore, along with several barren basal bodies, remains in the parent when the embryo is completed. The pore rises to the surface when the embryo is born. New basal bodies are then formed in the parent to replace those which were incorporated into the embryo, and formation of another embryo may begin. The cilia of the embryo are partially resorbed 10 min after the start of metamorphosis, with depolymerization of the ciliary microtubules. Later, the cilia and most of the basal bodies disappear completely, except for a group of barren basal bodies near the embryo's contractile vacuole pore, which form 6 rows and serve as an anlage for the basal bodies and cilia that arise during embryogenesis. There is, therefore, an organized infraciliature in Suctoria throughout their life cycle, and a distinct continuity of basal bodies across the generations.     

The future is cold: cryo-preparation methods for transmission electron microscopy of cells

Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.     

The endexine: a frequently overlooked pollen wall layer and a simple method for detection

For the detection of pollen wall layers, the use of different staining methods for one and the same species is highly recommended. The usage of standard transmission electron microscopy (TEM) staining methods showed that the ektexine-layers have always the same contrast behaviour, while the endexine changes its electron opaqueness depending on the method used. However, the endexine can often not be discriminated from the other wall layers. A simple method to detect the endexine is the use of potassium permanganate, which stains the layer electron dense, producing a distinct contrast.     

The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

BACKGROUND: Analysis of plant cell dynamics over time, or four-dimensional imaging (4-DI), represents a major goal of plant science. The ability to resolve structures in the third dimension within the cell or tissue during developmental events or in response to environmental or experimental stresses (i.e. 4-DI) is critical to our understanding of gene expression, post-expression modulations of macromolecules and sub-cellular system interactions. SCOPE: Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. However, certain realities of light and electron microscopy, choice of specimen and specimen preparation techniques limit the scope of readily attaining 4-DI. Today, the plant microscopist must use a combinatorial strategy whereby multiple microscopy-based investigations are used. Modern fluorescence, confocal laser scanning, transmission electron and scanning electron microscopy provide effective conduits for synthesizing data detailing live cell dynamics and highly resolved snapshots of specific cell structures that will ultimately lead to 4-DI. This review provides a synopsis of such technologies available.     

The Casparian Strip of Clivia miniata Reg. Roots: Isolation,Fine Structure and Chemical Nature*

The root endodermis of Clivia miniata Reg. was successfully isolated using the cell wall degrading enzymes cellulase and pectinase. The enzymes did not depolymerize those regions of the primary cell walls of anticlinal endodermal root cells where the Casparian strips were located. Since the endodermis of C. miniata roots remained in its primary developmental state over the whole root length, endodermal isolates essentially represented Casparian strips. Thus, sufficient amounts of isolated Casparian strips could be obtained to allow further detailed investigations of the isolates by microscopic, histochemical and analytical methods. Scanning electron microscopy revealed the reticular structure of the Casparian strips completely surrounding the central cylinder of the roots. Whereas in younger parts of the root only the anticlinal cell walls of the endodermis remained intact in the isolates, in older parts of the root the periclinal walls also restricted enzymatic degradation due to the deposition of lignin. Extracts of the isolates with organic solvents did not reveal any wax-like substances which might have been deposited within the cell wall forming a transport barrier, as is the case with cutin and suberin. However, several histochemical and analytical methods (elemental analysis and FTIR spectroscopy) showed that the chemical nature of the Casparian strips of C. miniata roots can definitely be a lignified cell wall. These findings are in complete agreement with studies carried out at the beginning of this century on the chemical nature of the Casparian strips of several other plant species. The implications of these results concerning apoplasmatic transport of solutes and water across Casparian strips are discussed.     

The pheromonal gland of Lymantria dispar: Morphology and evidence for its innervation

The morphological features of the glandular epithelium that secretes pheromone in the polyphagous pest gypsy moth Lymantria dispar are described by light and electron microscopy. The monolayered gland cells are covered by the folded cuticle of the intersegmental membrane between the 8th and 9th abdominal segments showing neither sites of discontinuity nor distinct openings on its external surface. The cells bear a large, often irregularly shaped nucleus, and contain granules of variable amount and electron‐density. These granules are mostly located in the basal compartment of the cytoplasm, in a labyrinthine zone laying on a basement membrane. The apical membrane of the gland cells bear microvilli and cell–cell contact is established by different junctional structures. Nerve fibers enwrapped in glia are found beneath the basement membrane, in close contact with the secretory cells. This latter finding represents the first evidence of the innervation of the pheromonal gland in L. dispar. J. Morphol. 2009. © 2008 Wiley‐Liss, Inc.     

The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.     

The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria

The contractile tail of bacteriophage T4 is a molecular machine that facilitates very high viral infection efficiency. Its major component is a tail sheath, which contracts during infection to less than half of its initial length. The sheath consists of 138 copies of the tail sheath protein, gene product (gp) 18, which surrounds the central non‐contractile tail tube. The contraction of the sheath drives the tail tube through the outer membrane, creating a channel for the viral genome delivery. A crystal structure of about three quarters of gp18 has been determined and was fitted into cryo‐electron microscopy reconstructions of the tail sheath before and after contraction. It was shown that during contraction, gp18 subunits slide over each other with no apparent change in their structure.     

The Mode of Attachment of Trypanosoma vivax in the Proboscis of the Tsetse Fly Glossina fuscipes: an Ultrastructural Study of the Epimastigote Stage of the Trypanosome*

SYNOPSIS. The ultrastructure of attached Trypanosoma vivax epimastigote clusters in the proboscis of the tsetse fly Glossina fuscipes is described from electron micrographs of thin sections. Some flagellates are attached directly to the lining of the insect's labrum by their flagella, most of which are aligned along the long axis of the proboscis. Other trypanosomes are attached indirectly, their flagella adhering to those of flagellates which are directly attached. Junctional complexes similar to those described from metazoan epithelia are found on the flagellar membrane. A long zonular hemidesmosome attaches the flagellum to the proboscis wall and a series of closely set macular desmosomes link the flagellar membranes of adjacent flagellates. Unlike the trypomastigote stages of T. vivax, more than one row of macular desmosomes may be present along the flagellum-body junction of the trypanosome. It is suggested that all these Junctional complexes serve to buttress the flagellate's attachment to its insect host and so maintain anchorage of the parasite during the fly's blood meals. The ability of the flagellum of trypanosomatids to form Junctional complexes may be a factor contributing to their success as parasites, this adaptation enabling them to multiply while attached to host surfaces.     

The Vacuolar ATPase of Red Beet Storage Tissue: Electron Microscopic Demonstration of the “Head-and-Stalk” Structure*

Tonoplast membranes were prepared from tissue homogenates and from vacuoles isolated from beetroot storage tissue (Beta vulgaris L., ssp. conditiva) for transmission electron microscopic analysis of the structure of the beetroot vacuolar ATPase using the negative staining technique. By comparison of the specific inhibitor sensitivities of the ATPase activity, i.e. ATP hydrolysis and H⁺-pumping, the purity of the tonoplast preparations with respect to contamination with mitochondrial inner membranes was assessed to avoid confusion with mitochondrial F₁F₀-ATPase. Membranes prepared in Hepes/Tris or BTP/Mes-containing media rarely showed typical head-and-stalk structures although characteristic nitrate- and bafilomycin A₁-sensitive ATP-hydrolysis and H⁺-pumping could be measured. However, typical head-and-stalk structures were observed regularly when these buffers were replaced by K-phosphate buffer. Under these conditions, the beetroot vacuolar ATPase is characterized by a large head group with a central cleft, a thin stalk, connecting it to the membrane and by basal components projecting from the base of the stalks near the vacuolar membrane and forming a distinct layer of electron-light particles between the vacuolar membrane and the layer of non-stained head groups.