Electron microscope tomography

Electron microscope tomography allows three-dimensional reconstruction of ultrastructural objects at the molecular level. The method is general and not limited to symmetric, or regularly ordered structures. Alone, or in combination with immunoelectron microscopy and electron spectroscopic imaging, electron microscope tomography is a powerful technique in cell and molecular biology.     

Homology of Balbiani Ring DNA in two closely related Chironomus species

Cytogenetic analysis indicates that Balbiani Ring 2 (BR 2) in the two sibling species Chironomus tentans and Chironomus pallidivittatus arises from identifically banded segments in the salivary gland polytene chromosomes, although chromosomal rearrangements have occurred. In situ hybridization of BR 2 RNA to the polytene chromosomes of each individual species, as well as their F1 hybrids, reveals that the repetitious BR 2 DNA in the two species has, within the limits of the technique, retained identity of nucleotide sequences and degree of repetition. The DNA of the naturally expressed BR 1 and BR 3 in both species and that ot the galactose induced BR 6 in C. pallidivittatus did not hybridize with BR 2 RNA, indicating that these BR's are different from BR 2 with regard to sequence content.     

Modeling the 3-D RNA distribution in the Balbiani Ring granule

Mature Balbiani Ring (BR) granules in situ were stained with the nucleic acid specific stain, osmium ammine-B, recorded by electron spectroscopic imaging and reconstructed by electron microscope tomography to examine the three-dimensional (3-D) distribution of BR heterogeneous nuclear RNA (hnRNA). The BR2 granules contain ca. 37 kb of mRNA. Reconstructed BR granules were selected to emphasize one of the prevalent conformations seen in the sectioned salivary glands, the en face or pin-wheel conformation. A variety of image processing and volume-rendering operations were applied to the set of reconstructed BR granules. Some of the conclusions of this study are the following: (1) RNA distribution is not uniform throughout the granule; (2) RNA is condensed into about ten particles per granule, which all appear to possess approximately the same RNA stain density; (3) heterogeneity exists in the positions and sizes of particles within the various BR granules. These data argue for the folding of a beaded ribbon, consisting of connected particulate condensations of BR mRNA, possessing considerable 3-D flexibility, even in the packaged state. A comparison of this beadedribbon model and a prior folded hnRNP fiber model is also presented.     

Electron microscope study ofLeptospira

A leptospira consists of the cytoplasmic cylinder (contained within its own wall) which winds helically anticlockwise around the axistyle; both these components are enclosed by the outer covering membrane enveloping the whole organism.     

Electron microscope tomography: further demonstration of nanocontacts between caveolae and smooth muscle sarcoplasmic reticulum

A spatial relationship between caveolae and sarcoplasmic reticulum (SR) in smooth muscle cells (SMC) was previously reported in computer-assisted three-dimensional reconstruction from transmission electron microscope serial sections. The knowledge of the three-dimensional organization of the cortical space of SMC is essential to understand caveolae function at the cellular level. Cellular tomography using transmission electron microscopy tomography (EMT) is the only available technology to reliably chart the inside of a cell and is therefore an essential technology in the study of organellar nanospatial relationships. Using EMT we further demonstrate here that caveolae and peripheral SR in visceral SMC build constantly spatial units, presumably responsible for a vectorial control of free Ca2+ cytoplasmic concentrations in definite nanospaces.     

Electron microscope studies of pH effects on assembly of tubulin free of associated proteins. Delineation of substructure by tannic acid staining

Bovine brain tubulin, purified by phosphocellulose chromatography (PC), was assembled in the presence of 10% dimethyl sulfoxide (DMSO), and the reaction was monitored turbidimetrically. Samples were fixed in glutaraldehyde-tannic acid after completion of polymerization, as indicated by no further change in absorbance, and then sectioned and studied electron microscopy, with special attention being given to the arrangement of protofilaments in the walls of formed elements. Samples of PC-tubulin were polymerized in buffer having various pH values from 6.0 to 7.7. At the lower pH values, only branched and flattened ribbons of protofilaments are formed. At intermediate values, the ribbons are unbranched, narrower, and more curved in cross section; complete microtubules are also seen. At the higher pH values, the predominate formed elements are complete microtubules. Most of the complete microtubules examined in this study had 14 wall protofilaments. The effect of pH on tubulin assembly was shown not to be an effect of DMSO. The dimers of associated protofilaments in ribbons and microtubules are conceptually viewed as having trapezoidal profiles in cross section, and, as additional dimers are added, the "C"-shaped ribbon closes to form a tube. The tilt angle of the lateral surfaces of the "trapezoidal" dimers will determine the number of wall protofilaments in the microtubules. At low pH, it is theorized that the trapezoidal profile of the dimer is shifted to a more rectangular configuration such that flat ribbons are formed by the lateral association of dimers. Also, variously shaped ribbon structures are formed at intermediate pH values, including "S"- and "W"-shaped structures, and elements shaped like a figure "6," all representing ribbons viewed in cross section. By visualizing the trapezoidal dimer in three-dimensions, and by arbitrarily indexing its six binding surfaces, it is possible to discuss interdimer binding in terms of preferred and possible binding interactions.     

Electron microscope study on spermatogenesis

Summary The morphogenesis of chromosomes at early prophase of spermatocytes I is studied in three ortoptherian species: Grillus argentinus (Grillidae), Laplatacris dispar (Acrididae) and Blaptica dubia (Blaptidae).The first chromosome component appearing at the beginning of meiosis is a thread (single elementary thread; S.E.T.) of low electron density measuring about 700 Å to 0.1 width. A group having the same width and integrated by three helically twisted ribbon-like components develops from S.E.T. and is called primary tripartite group, P.T.G. The three components are at first of the same width (about 200 Å) but the lateral arms progressively increase in thickness and in that way the group becomes coated by a layer of dense microfibrils supposed to be chromatin.Late stages of prophase were not thoroughly investigated in this study, but many evidences were however found helping to identify synaptene stage. In accordance with these evidences each homologous chromosome is integrated by an axial component (tripartite groups) coated by chromatin.The medial component of tripartite groups of Blaptica dubia is double and also shows multistranded regions which are called puffy regions.A comparative optical and electron microscope study was made in order to better understand the above described process. This study includes the comparison of the thickness of chromosomes as measured in light and electron micrographs.On the other hand, rubber models were made to illustrate the same process and pohotographs of these models are exhibited in the text.The nuclear structure of early spermatids of the same species is also studied in this paper. It was found that Blaptica and Grillus early spermatids nuclei contain groups similar to those found at the beginning of prophase of spermatocyte I, with the only difference that in many cases composite groups, formed by fusion of the lateral arms of two or more than two single groups, were found.The nuclear structure of late spermatids was also considered in this paper. Notwithstanding the study only aimed to point out that the pattern of organization of the spermatozoon chromatin greatly differs in the species examined.     

Electron microscope studies on Tomentella

Summary Tomentella bombycina andT. fuscoferruginosa were examined by routine techniques of light and electron microscopy. Special attention was paid to the ultrastructure and inter-relationships of the generative subicular hyphae and subhymenial hyphae. The cell wall consisted of three layers in the generative subicular hyphae and only one layer in the subhymenial hyphae; this corresponded to the inner layer of the first and originated from it. Cells with two nuclei, normal mitochondria, large vacuoles, sparse endoplasmic reticulum and lomasomes were observed. Sometimes the nuclear membrane had unusually large pores and larger open gaps extending over 1/4 of the circumference. Septa with central dolipore-type pores were present. Clamp connections were not frequent. Finally, certain cells of the subhymenial hyphae appeared full of globular material, presumably lipids.     

Electron microscope studies on collagen

Summary The present paper describes the effect of intensive tryptic digestion of native tropocollagen (TC) macromolecules in solution. Contrary to earlier investigations it has been found that the trypsin treatment results in a fragmentation of the TC molecules. The addition of ATP to solutions exposed to the enzyme yields SLS fragments, 2250 Å in length. Comparison between these and normal SLS type aggregates shows that the scission occurs in a well-defined locus adjacent to the -1/2-line seen after positive staining. The significance of this finding is discussed.This study was supported by grant NB-02215-04 from the National Institute of Neurological Diseases and Blindness, Public Health Service, U.S.A. and a Student research fellowship from the Norwegian Research Council for Science and the Humanities. This aid is gratefully acknowledged.I am indebted to Mrs. J. Line Vaaland and Mr. B. V. Johansen for technical assistance.     

Electron microscope study on meiosis

Summary The cycle of the nucleolus and sex chromosome was studied with the electron microscope during the following stages of spermatogenesis and spermiogenesis of Gryllus argentinus: (1) spermatogonia; (2) prophase cyte I, leptotene, part of pachytene, and end of diplotene till breakdown of the nuclear envelope; (3) division I, metaphase and anaphase; (4) cyte II, prometaphase; (5) division II, metaphase, anaphase and telophase; (6) early and late spermatids. Some observations were also carried out in the primary oocyte until beginning of the growth period.It was found that nucleolus and sex chromosome are associated, at first without mixture of their components (leptotene) and afterwards interchanging components (pachytene). The interchange takes place by the passage from one element to another of filamentous units ot low electron density, similar in appearance to those existing in the medial plane of tripartite groups (synaptinemal complexes).At pachytene the primary results of interchange are: (1) the nucleolus contains filaments of chromosomal nature; (2) the nucleolus emits a long rod-like prolongation containing a cylindrical bundle of filaments, and an axial unit of the same nature; two equidistant clear spaces separate the axial unit from the cylindrical bundle and the latter from the dark wall of nucleolar material. At the end of diplotene these components are found organized in two bodies and a prolongation. One of the bodies is formed by a number of alternatively dark and light bands, the other by a pack of tubular units each showing the structure of the former nucleolar prolongation. The prolongation is either formed as in the preceding stage or it is composed of five ribbons, two dark ones outside and three light ones between them. It is supposed that both bodies are united by the prolongation but no definite proof was obtained. It is assumed that the complex thus constituted represents the sex chromosome.The sex chromosome was found at any phase of both divisions as well as at the intermediate stages between them; at the division phase the chromosome is separated from the autosomes and moves independently of them.The element could not be traced at telophase II but it reappears within the reorganized nuclei of spermatids. Amorphous nucleolar-like material and chromosome-like material are found associated at this stage with banded complexes like those seen at the end of prophase I. All these components undergo involution during spermatid maturation. At the final step of maturation no traces of them are found.A similar association of nucleolus and chromosome was found at prophase of primary oocytes of the same species. The associated body is of the same structure as that described for primary spermatocytes. The structures existing in the primary oocytes disorganize at the beginning of growth. At this time the nucleolus has developed into a large body containing masses of chromatin-like material.This investigation was supported in part by U.S. Public Health Service, Research Grant No. GM-08337 from the National Institute of General Medical Sciences.     

Electron microscope study of Cryptostroma corticale

An ultrastructural study of Cryptostroma corticale has been carried out with scanning and transmission electron microscopes. The usual features in fungi, as well as features characteristic of the family, were shown. It was noted that lomasomes and myelin-type structures were demonstrated by the two fixatives used. Their significance is discussed.     

Electron microscope study of lens fibers

The in vitro swelling action of L-thyroxine on rat liver mitochondria as examined photometrically represents an acceleration of a process which the mitochondria are already inherently capable of undergoing spontaneously, as indicated by the identical kinetic characteristics and the extent of thyroxine-induced and spontaneous swelling, the nearly identical pH dependence, and the fact that sucrose has a specific inhibitory action on both types of swelling. However, thyroxine does not appear to be a "catalyst" or coenzyme since it does not decrease the temperature coefficient of spontaneous swelling. The temperature coefficient is very high, approximately 6.0 near 20 degrees . Aging of mitochondria at 0 degrees causes loss of thyroxine sensitivity which correlates closely with the loss of bound DPN from the mitochondria, but not with loss of activity of the respiratory chain or with the efficiency of oxidative phosphorylation. Tests with various respiratory chain inhibitors showed that the oxidation state of bound DPN may be a major determinant of thyroxine sensitivity; the oxidation state of the other respiratory carriers does not appear to influence sensitivity to thyroxine. These facts and other considerations suggest that a bound form of mitochondrial DPN is the "target" of the action of thyroxine. The thyroxine-induced swelling is not reversed by increasing the osmolar concentration of external sucrose, but can be "passively" or osmotically reversed by adding the high-particle weight solute polyvinylpyrrolidone. The mitochondrial membrane becomes more permeable to sucrose during the swelling reaction. On the other hand, thyroxine-induced swelling can be "actively" reversed by ATP in a medium of 0.15 M KCl or NaCl but not in a 0.30 M sucrose medium. The action of ATP is specific; ADP, Mn(++), and ethylenediaminetetraacetate are not active. It is concluded that sucrose is an inhibitor of the enzymatic relationship between oxidative phosphorylation and the contractility and permeability properties of the mitochondrial membrane. Occurrence of different types of mitochondrial swelling, the intracellular factors affecting the swelling and shrinking of mitochondria, as well as the physiological significance of thyroxine-induced swelling are discussed.     

Electron microscope studies of glycogen synthase

Summary Glycogen synthase from rabbit muscle was examined with the electron microscope. In preparations of the completely converted glucose-6-phosphate dependent form (GSD) and the independent form (GSI) three structures were observed: toroids, hexagons and stacks of four elements which appear to be aggregates of four toroids. Toroids can be formed from hexagons by radial inward movement of subunits which form the vertices of the hexagons. Analysis of the dimensions of these structures and comparison of the known chemistry of the enzyme to the subunits as inferred from electron microscopy suggests a model for the structure of glycogen synthase. The model allows predictions of types of subunits in the enzyme, their relation to phosphorylatable and -SH sites and the possibilities of control by small effector molecules.