Bridging fluorescence microscopy and electron microscopy

Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy in biology is the ability to follow specific targets on or in living cells, revealing dynamic localization and/or function of target molecules. One of the inherent limitations of fluorescence microscopy is the resolution. Several efforts are undertaken to overcome this limit. The traditional and most well-known way to achieve higher resolution imaging is by electron microscopy. Moreover, electron microscopy reveals organelles, membranes, macromolecules, and thus aids in the understanding of cellular complexity and localization of molecules of interest in relation to other structures. With the new probe development, a solid bridge between fluorescence microscopy and electron microscopy is being built, even leading to correlative imaging. This connection provides several benefits, both scientifically as well as practically. Here, I summarize recent developments in bridging microscopy.     

The Synapse-Like Interaction between Chloroplast, dictyosome, and Other Cell Compartments during Increased Ethylene Production in Leaves of Rye (Secale cereale L.)

Rye (Secale cereale L.) plants were treated with an ethylene releaser ethephon (2-chloroethylphosphonic acid) in concentration of 4×10⁻² M. We studied electron microscopically, if and how chloroplasts interact with well-documented sites of ethylene production/binding, i.e., with endoplasmic reticulum, dictyosomes, mitochondria, plasma membrane, and tonoplast. During the sharp increase of ethylene synthesis in mesophyll cells of rye leaves, the direct local continguity of chloroplast envelope or envelope protrusions with the above mentioned cell compartments was typical. Moreover, a large number and diversity of versatile chloroplast-dictyosome associations were conspicuous, in which both the chloroplast and each cisterna of dictyosome were capable to exo/endocytosis. The dictyosomes were directed towards the chloroplasts, plasma membrane, or tonoplast both with cis-face, trans-face, or with the rim, they could change their direction or shut up the trans-face, developing simultaneously several flexible chains of vesicular dispatches among chloroplasts and some other cell compartments. This reflects interaction of protein/ethylene producing, photosynthesising, DNA containing compartments, and regulated action of lysosomal system. Structural contacts and vesicular transport among compartments of symplastic system equalises concentrations of H⁺, Ca²⁺, etc. ions, as well as provide connection with an apoplast. We propose that ethylene functions in plant mesophyll cells are both as intra/intercellular signalling substance and as phytohormone that regulates gene expression in nuclei, chloroplasts, and mitochondria in a complicated synapse-like process and causes programmed death of leaves of the main stalks of rye for the sake of promoted growth of side shoots. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ultrastructural observations on the kinetosomes,and Golgi bodies during the asexual life cycle ofSaprolegnia

Summary At the onset of zoospore cleavage the centrioles ofSaprolegnia ferax reorientate, develop into kinetosomes and become associated with microtubular roots and a striate fibre. After cytoplasmic cleavage a flagellum, with a hitherto undescribed transition zone structure, develops from each kinetosome. Flagellum axonemes occur inside recently encysted primary spores. In vegetative hyphae and germinating cysts most recognizable Golgi bodies are characteristically associated with a cisternum of the endoplasmic reticulum and a mitochondrion but during sporogenesis they all lie adjacent to nuclei where they are apparently active in vesicle production. The structural details of these changes are described and their significance discussed. We wish to acknowledge the numerous helpful discussions with Dr. J. L. Gay. The senior author held a S.R.C. studentship during the course of this work, part of which was submitted in partial fulfillment of the requirements for the degree of Ph. D. at the University of London.     

Light and Electron Microscope Study of Dunaliella primolecta Butcher (Volvocida)*

SYNOPSIS. Light and electron microscope observations on Dunaliella primolecta Butcher from logarithmic and stationary phases of batch cultures are correlated. Except for the lack of a cell wall the fine structure has typical volvocid features. The transition from logarithmic to stationary phase is marked by changes in content and size of cytoplasmic vacuoles, accumulation of cytoplasmic lipid, accumulation of starch in the plastid matrix, and by the formation of autophagosome-like bodies. The organelles in stationary-phase flagellates are closely packed together because of the cytoplasmic lipid and starch-distended chloroplast. Organisms from logarithmic phase have an abundant ribosome-packed groundplasm supporting the organelles. In the cytochemical demonstration of acid phosphatase activity, Golgi cisternae and smooth and coated Golgi vesicles contain Gomori reaction product. The possible roles of the Golgi apparatus in this flagellate are discussed.     

LOCALIZATION OF CA2+-STIMULATED ATPASE IN THE COCCOLITH-PRODUCING COMPARTMENT OF CELLS OF PLEUROCHRYSIS SP. (PRYMNESIOPHYCEAE)1

Cell homogenates of Pleurochrysis sp. (CCMP299) were fractionated by means of sucrose gradients. Ca²⁺-stimulated ATPase (EC 3.6.1.3., ATP phosphohydrolase) was associated primarily with the plasma membrane, Golgi, and high density (1.21 g·cm^?3) membranous structures. Ca²⁺-stimulated ATPase was highly enriched in the latter. Based on treatments with Triton X-100 and NBD ceramide, we conclude that the high-density structures were membrane-delimited organelles. These vesicle-like organelles contained complex polysaccharides, a high concentration of calcium, and, upon microscopic examination, structures resembling coccoliths. These findings are consistent with observations on the known composition of coccoliths and the presumed mineralizing function of the sub-cellular coccolith-producing compartment. The high-density vesicles were linked to the Golgi by means of colchicine-sensitive materials, presumably microtubules. These data and prior ultrastructural observations by other investigators indicating vectorial assembly and secretion suggest that the subcellular movement of the newly formed coccoliths may be directed and/or powered by colchicine-sensitive cytoskeletal elements. We interpret the data to mean that the high-density vesicles represent the coccolith-producing compartment previously observed by others in electron micrographs.     

Some Ultrastructural and Functional Aspects of the Golgi Apparatus of Thelohania sp. (Microsporida) in the Shrimp Pandalus jordani Rathbun

SYNOPSIS. Electron microscope observations on Thelohania sp. in the shrimp Pandalus jordani support the view that the Golgi complex in Microsporida is a classical one, composed of vesicular, vacuolar, and cisternal components. During development of the sporoblast, a portion of the Golgi complex is seen as an electron-dense reticulum enmeshing the core of the polar filament. Associated with the reticulum are electron-dense bodies. The reticulum and dense bodies, reported in several previous publications, have not been well understood and have been given a variety of names. The evidence favors the view that these structures have secretory activity in which the reticulum concentrates or synthesizes material, some of which takes the form of membrane-bounded granules. It is suggested that the most appropriate name for the reticulum is reticulum golgien, and that the correct name for the dense bodies is the standard cytologic term, secretion granules. The secretion granules apparently remain in the posterior part of the spore, and may be stored there for some as yet undetermined use.     

Isonema papillatum sp. n., a New Colorless Marine Flagellate: A Light- and Electronmicroscopic Study

SYNOPSIS. Isonema papillatum sp. n. is described from observations with the light and electron microscope. It has 2 short emergent subapical flagella without appendages, a specialized ingestion apparatus, and a nonrigid naked cell body capable of pronounced changes in shape similar to euglenoid movements. It lacks a stigma and flagellar swelling and does not form paramylon granules. The nucleolus persists throughout nuclear division.     

Aspects infrastructuraux de la sécrétion dans les cellules glandulaires du canal déférent chez Porcellio scaber Latreille (Isopode terrestre)

Résumé Dans ce travail, les auteurs présentent les résultats de leurs recherches, au niveau de la microscopie électronique, concernant les processus sécrétoires dans les cellules glandulaires de la partie antérieure, réniforme, du canal déférent chez Porcellio scaber Latreille.Les observations sont dirigées spécialement sur l'appareil de Golgi qui est poursuivi dans toutes les phases d'élaboration de la sécrétion, dans laquelle il paraît avoir un rôle essentiel.Après avoir obtenu la conviction que, dans le processus délaboration de la sécrétion, les dictyosomes s'usent complètement et disparaissent, les auteurs recherchent l'origine de nouveaux dictyosomes qui participeront au cycle de sécrétion suivant.

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Infrastructural aspects of the secretion in the glandular cells of the vas deferens in Porcellio scaber Latreille (terrestial Isopoda)

Summary In this paper the authors present an investigation at infrastructural level concerning the secretory process in the glandular cells of the anterior kidney-like portion of the deferent ducts in Porcellio scaber Latreille.The observations are directed especially to the Golgi apparatus in all phases of elaboration of secretion in which it seems to have an essential role.After ascertaining that in the process of elaboration of secretion the dictyosomes waste away and disappear, the authors investigated the origin of the new dictyosomes that become involved in the following secretory cycle.

Nous remercions beaucoup les Drs Al. Petrovitch et M. Ionesco, ainsi que l'Ing. C. Dumitriu et Mme M. Russu, chercheur soientifique, qui nous ont aidé avec beaucoup d'amabilité dans nos recherches.     

Heparan Sulfate Biosynthesis: Methods for Investigation of the Heparanosome

Heparan sulfate is perhaps the most complex polysaccharide known from animals. The basic repeating disaccharide is extensively modified by sulfation and uronic acid epimerization. Despite this, the fine structure of heparan sulfate is remarkably consistent with a particular cell type. This suggests that the synthesis of heparan sulfate is tightly controlled. Although genomics has identified the enzymes involved in glycosaminoglycan synthesis in a number of vertebrates and invertebrates, the regulation of the process is not understood. Moreover, the localization of the various enzymes in the Golgi apparatus has not been carried out in a detailed way using high-resolution microscopy. We have begun this process, using well-known markers for the various Golgi compartments, coupled with the use of characterized antibodies and cDNA expression. Laser scanning confocal microscopy coupled with line scanning provides high-quality resolution of the distribution of enzymes. The EXT2 protein, which when combined as heterodimers with EXT1 comprises the major polymerase in heparan sulfate synthesis, has been studied in depth. All the data are consistent with a cis-Golgi distribution and provide a starting point to establish whether all the enzymes are clustered in a multimolecular complex or are distributed through the various compartments of the Golgi apparatus.     

Selectivity of Ingestion and Digestion in the Chrysomonad Flagellate Ochromonas malhamensis

Ochromonas malhamensis cells were allowed to feed on autoclaved Aerobacter aerogenes, biochemically inert polystyrene latex particles of comparable size, shape and density, or a combination of these to determine if this protozoan is capable of phagotrophic selectivity under defined experimental conditions. Electron microscopic examination of Gomoristained food vacuoles indicated that the type of particle ingested was determined entirely by the type available. Apparently the biochemical or physical configuration of the particle surface is not decisive in particle selection. Gomori acid-phosphatase reaction product, which indicates digestive enzyme activity, was present in all particle-containing vacuoles, suggesting that digestive activity in O. malhamensis is due to the presence of particulate material in the vacuole, but not necessarily to the nature of the particle.