scholarly journals Physarum plasmodia do contain cytoplasmic microtubules!

1991 ◽  
Vol 100 (3) ◽  
pp. 509-520 ◽  
Author(s):  
I. Salles-Passador ◽  
A. Moisand ◽  
V. Planques ◽  
M. Wright
Keyword(s):  
Electron Microscopy ◽  
Cold Treatment ◽  
Three Dimensional ◽  
Immunofluorescent Staining ◽  
Microtubule Network ◽  
Cytoplasmic Microtubule ◽  
Thin Sections ◽  
Cytoplasmic Microtubules ◽  
Oriented Parallel ◽  
Physarum Plasmodia

It has been claimed that the plasmodium of the myxomycete Physarum polycephalum constitutes a very unusual syncytium, devoid of cytoplasmic microtubules. In contrast, we have observed a cytoplasmic microtubule network, by both electron microscopy and immunofluorescence in standard synchronous plasmodia, either in semi-thin sections or in smears, and in thin plasmodia, used as a convenient model. Cytoplasmic microtubules could be seen after immunofluorescent staining with three different monospecific monoclonal anti-tubulin antibodies. The immunolabelling was strictly restricted to typical microtubules as shown by electron microscopy. These cytoplasmic microtubules were entirely and reversibly disassembled by cold treatment and by either of two microtubule poisons: methyl benzimidazole carbamate and griseofulvin. The microtubule network, present in all strains that have been studied, contains single microtubules and microtubule bundles composed of two to eight microtubules. Cytoplasmic microtubules form a dense and complex three-dimensional network, distinct from the microfilamentous domains and from the nuclei. The orientation of the microtubule network varies according to the plasmodial domain examined. Generally microtubules show no special orientation except in plasmodial veins where they are oriented parallel to the long axis of the veins. Differences between our observations and those of previous workers who failed to find cytoplasmic microtubules in plasmodia are discussed. We propose that they reflect difficulties of observation mainly due to the fluorescent background. In contrast with the previous view, the discovery of a microtubule cytoplasmic cytoskeleton in Physarum plasmodia raises several questions concerning its relationships with other cellular organelles and its dynamics during different cell cycle events.

scholarly journals Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis.

1976 ◽  
Vol 69 (1) ◽  
pp. 106-125 ◽  
Author(s):  
D L Brown ◽  
A Massalski ◽  
R Patenaude
Keyword(s):  
Anterior Part ◽  
Flagellar Apparatus ◽  
Microtubule Assembly ◽  
Immunofluorescent Staining ◽  
Body Region ◽  
Basal Bodies ◽  
Cytoplasmic Microtubule ◽  
Large Numbers ◽  
Attachment Sites ◽  
Cytoplasmic Microtubules

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.

scholarly journals Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

1991 ◽  
Vol 39 (11) ◽  
pp. 1495-1506 ◽  
Author(s):  
P M Motte ◽  
R Loppes ◽  
M Menager ◽  
R Deltour
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Spatial Organization ◽  
Higher Plants ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Thin Sections ◽  
Cytoplasmic Dna ◽  
Immunocytochemical Techniques

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

scholarly journals The chondriome of selected trypanosomatids. A three-dimensional study based on serial thick sections and high voltage electron microscopy.

1975 ◽  
Vol 66 (2) ◽  
pp. 404-413 ◽  
Author(s):  
J J Paulin
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Three Dimensional ◽  
Flagellar Pocket ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
Trypomastigote Form ◽  
High Voltage Electron ◽  
Three Dimensional Models

The unitary nature of the chondriome of two species of trypanosomatids, Blastocrithidia culicis and Trypanosoma cruzi, has been demonstrated by utilizing serial thick-sectioning techniques combined with high voltage electron microscopy. Profiles of mitochondrial elements seen in thin sections and suspected to be parts of a continuum were confirmed by serial thick sectioning (0.25-0.50 mum thick) and stereopair analysis to be parts of the same mitochondrion. Three-dimensional models obtained from tracings of mitochondrial profiles on cellulose acetate reveal the mitochondrion of B. culicis to consist of a posterior mass with six tubular extensions extending upward and terminating in the anterior apex. The kinetoplast was found suspended between two of the tubular extensions, or less frequently, protuding as a nodule from one of the extensions. A bifurcation of one of the extensions was found in some specimens. The mitochondrion of T. cruzi consists of a triangular-shaped convoluted tubule, the base being the kinetoplast portion while the apex is directed posteriorly. The mitochondrion bifurcates behind the flagellar pocket, lateral to the kinetoplast, sending two entwined extensions into the tenuous anterior apex. Whether the mitochondrion of T. cruzi is unitary in the trypomastigote form was not determined in this study, since only epimastigote forms were used.

scholarly journals Three-Dimensional Scanning Transmission Electron Microscopy of Biological Specimens

2010 ◽  
Vol 16 (1) ◽  
pp. 54-63 ◽  
Author(s):  
Niels de Jonge ◽  
Rachid Sougrat ◽  
Brian M. Northan ◽  
Stephen J. Pennycook
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Mammalian Cells ◽  
Three Dimensional ◽  
Axial Resolution ◽  
Beam Radiation ◽  
Thin Sections ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Scanning Transmission

AbstractA three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2–3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

The application of three-dimensional tomographic reconstruction methods to high-voltage electron microscopy

1987 ◽  
Vol 45 ◽  
pp. 570-573
Author(s):  
B. F. McEwen ◽  
C. L. Rieder ◽  
M. Radermacher ◽  
R. A. Grassucci ◽  
J. N. Turner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Three Dimensional ◽  
Depth Of Field ◽  
Specimen Thickness ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Reconstruction Methods

High-voltage electron microscopy (HVEM) has considerably increased the thickness limit of biological specimens that can be visualized at high resolution. Because of its increased penetration power, HVEM is potentially the most powerful tool available for obtaining three-dimensional (3D) information concerning the structure of cells. In the past, such information was primarily obtained from serial thin sections or techniques based on surface shadowing, but these methods have severe problems and limitations which can only be overcome by imaging greater depths in the samples (see refs. 1 and 2). HVEM has yet to realize its potential for 3D structural determination because of the confusion arising from the overlap of features at different depths in the sample. Due to the relatively large depth of field, which exceeds the specimen thickness, HVEM (like all electron microscopy) produces an image that is essentially a projection of the sample.

scholarly journals Subaxolemmal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability.

1978 ◽  
Vol 78 (2) ◽  
pp. 597-621 ◽  
Author(s):  
J Metuzals ◽  
I Tasaki
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Nerve Fiber ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Polarized Light ◽  
Thin Sections ◽  
Loligo Pealei ◽  
Scanning Electron

A new technique utilizing the squid giant nerve fiber has been developed which permits direct examination of the inner face of the axolemma by scanning electron microscopy. The axoplasm was removed sequentially in a 15-mm long segment of the fiber by intracellular perfusion with a solution of KF, KCl, Ca++-containing seawater, or with pronase. The action potential of the fibers was monitored during these treatments. After brief prefixation in 1% paraformaldehyde and 1% glutaraldehyde, the perfused segment was opened by a lne could be related to information on the detailed morphology of the cytoplasmic face of the axolemma and the ectoplasm. The results obtained by scanning electron microscopy were further substantiated by transmission electron microscopy of thin sections. In addition, living axons were studied with polarized light during axoplasm removal, and the identification of actin by heavy meromyosin labeling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was accomplished. These observations demonstrate that a three-dimensional network of interwoven filaments, consisting partly of an actinlike protein, is firmly attached to the axolemma. The axoplasmic face of fibers in which the filaments have been removed partially after perfusion with pronase displays smooth membranous blebs and large profiles which sppose the axolemma. In fibers where the excitability has been suppressed by pronase perfusion, approximately one-third of the inner face of the axolemma in the perfusion zone is free of filaments. It is hypothesized that the attachment of axoplasm filaments to the axolemma may have a role in the maintenance of the normal morphology of the axolemma, and, thus, in some aspect of excitability.

Three-Dimensional Coherent X-Ray Diffraction Microscopy

MRS Bulletin ◽  
2004 ◽  
Vol 29 (3) ◽  
pp. 177-181 ◽  
Author(s):  
Ian K. Robinson ◽  
Jianwei Miao
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Real Space ◽  
Objective Lens ◽  
X Rays ◽  
X Ray Diffraction ◽  
Space Images ◽  
Thin Sections ◽  
X Ray ◽  
Penetration Ability

AbstractX-rays have been widely used in the structural analysis of materials because of their significant penetration ability, at least on the length scale of the granularity of most materials. This allows, in principle, for fully three-dimensional characterization of the bulk properties of a material. One of the main advantages of x-ray diffraction over electron microscopy is that destructive sample preparation to create thin sections is often avoidable. A major disadvantage of x-ray diffraction with respect to electron microscopy is its inability to produce real-space images of the materials under investigation—there are simply no suitable lenses available. There has been significant progress in x-ray microscopy associated with the development of lenses, usually based on zone plates, Kirkpatrick–Baez mirrors, or compound refractive lenses. These technologies are far behind the development of electron optics, particularly for the large magnification ratios needed to attain high resolution. In this article, the authors report progress toward the development of an alternative general approach to imaging, the direct inversion of diffraction patterns by computation methods. By avoiding the use of an objective lens altogether, the technique is free from aberrations that limit the resolution, and it can be highly efficient with respect to radiation damage of the samples. It can take full advantage of the three-dimensional capability that comes from the x-ray penetration. The inversion step employs computational methods based on oversampling to obtain a general solution of the diffraction phase problem.

scholarly journals Tea2p Is a Kinesin-like Protein Required to Generate Polarized Growth in Fission Yeast

2000 ◽  
Vol 151 (1) ◽  
pp. 15-28 ◽  
Author(s):  
Heidi Browning ◽  
Jacqueline Hayles ◽  
Juan Mata ◽  
Lauren Aveline ◽  
Paul Nurse ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Specific Marker ◽  
Polarized Growth ◽  
Microtubule Network ◽  
Cytoplasmic Microtubule ◽  
Morphological Defects ◽  
Morphological Mutants ◽  
Cytoplasmic Microtubules ◽  
Microtubule Growth ◽  
Punctate Pattern

Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity. Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2+, that is required for cells to generate proper polarized growth. Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase. They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2Δ cells, indicating that Tea2p function is necessary for proper localization of Tea1p. Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules. These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell.

Methodology for determining the three-dimensional crystal structure of myosin Sl from electron microscopy of orthogonal thin sections

1986 ◽  
Vol 44 ◽  
pp. 26-29
Author(s):  
T. S. Baker ◽  
D. A. Winkelmann
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
Single Unit ◽  
Three Dimensional ◽  
Detailed Knowledge ◽  
Thin Sections ◽  
X Ray ◽  
X Ray Crystallography ◽  
Density Map ◽  
Dimensional Crystal

Detailed knowledge of the structure of myosin is essential for understanding the mechanism of muscle contraction. The discovery of conditions for crystallizing the head of myosin (Sl = subfragment 1) has led to systematic studies of the SI structure by x-ray crystallography and electron microscopy. We describe the method used to determine the structure of the myosin Sl molecule by electron microscopy (Fig. 3). This method involved independently reconstructing the three-dimensional density of several thin sections obtained from oriented crystals and then combining these reconstructions to produce a final, averaged density map of a single unit cell.

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