E.G. van Donselaar

Recycling Compartments and the Internal Vesicles of Multivesicular Bodies Harbor Most of the Cholesterol Found in the Endocytic Pathway

We employed our recently developed immuno‐electron microscopic method (W. Möbius, Y. Ohno‐Iwashita, E. G. van Donselaar, V. M. Oorschot, Y. Shimada, T. Fujimoto, H. F. Heijnen, H. J. Geuze and J. W. Slot, J Histochem Cytochem 2002; 50: 43–55) to analyze the distribution of cholesterol in the endocytic pathway of human B lymphocytes. We could distinguish 6 categories of endocytic compartments on the basis of morphology, BSA gold uptake kinetics and organelle marker analysis. Of all cholesterol detected in the endocytic pathway, we found 20% in the recycling tubulo‐vesicles and 63% present in two types of multivesicular bodies. In the multivesicular bodies, most of the cholesterol was contained in the internal membrane vesicles, the precursors of exosomes secreted by B cells. Cholesterol was almost absent from lysosomes, that contained the bulk of the lipid bis(monoacylglycero)phosphate, also termed lysobisphosphatidic acid. Thus, cholesterol displays a highly differential distribution in the various membrane domains of the endocytic pathway.

Cytochrome P-450 monooxygenase system and benzo(a)pyrene metabolism in echinoderms

A comparative study on mixed-function oxygenase (MFO) systems was carried out on four echinoderm species: the asteroidsAsterias rubens andMarthasterias glacialis, a holothurianHolothuria forskali and an echinoidEchinus esculentus. Cytochromes P-450 andb5 and the MFO system-associated NADH-ferricyanide reductases NADH-cytochromec reductase, NADPH-cytochromec reductase and benzo(a)pyrene (BP) hydroxylase activities were present in microsomal fractions of pyloric caeca ofA rubens andM. glacialis and in the haemal plexus ofH. forskali. In contrast, cytochrome P-450 and BP hydroxylase activity were not detectable in the gonads ofE. esculentus. The tissue and subcellular distribution of the MFO system was studied inA. rubens. MFO system components were found in the stomachs and gonads, although detection of cytochrome P-450 in the latter tissue was difficult. Sex-related differences were not significant. The contents of the MFO system components and BP hydroxylase activities were highest in the microsomal (100 000 ×g) fractions, but MFO system components were also found in the mitochondrial (12 000 ×g) and cytosolic fractions. The BP hydroxylase activity in pyloric caeca microsomes ofA. rubens was NADPH-dependent and was inhibited by several agents known to be inhibitors of vertebrate cytochromes P-450. In the former respect, the characteristics of the MFO system were more like those of vertebrates and crustaceans than that of molluscs.

Aclar discs: a versatile substrate for routine high‐pressure freezing of mammalian cell monolayers

High‐pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze‐substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high‐pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high‐pressure freeze, freeze‐substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high‐pressure freezing, freeze‐substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.

Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers, by TEM and FIB–SEM

Aclar, a copolymer film with properties very similar to those of tissue culture plastic, is a versatile substrate to grow cells for light (including fluorescence) and electron microscopic applications in combination with both chemical fixation and cryoimmobilization. In this paper, we describe complete procedures to perform correlative light and electron microscopy using Aclar as substrate for the culture of cell monolayers to be finally embedded in plastic. First, we developed straightforward, efficient and flexible ways to mark the surface of the Aclar to create substrates to locate cells first at the light microscopy and then the electron microscopy level. All the methods enable the user to self‐design gridded Aclar pieces, according to the purpose of the experiments, and create a large number of substrates in a short time. Second, we confirmed that marked Aclar supports the normal growth and morphology of cells. Third, we validated the correlative light and electron microscopy procedure using Aclar. This validation was done for the high‐resolution analysis of endothelial cells using transmission electron microscopy and focused ion beam–scanning electron microscopy in combination with the use of fluorescence, phase contrast and/or bright field microscopy to map areas of interest at low resolution. The methods that we present are diverse, easy to implement and highly reproducible, and emphasize the versatility of Aclar as a cell growth substrate for diverse microscopic applications.

Cytopathological investigations of digestive tract and storage cells in Daphnia magna exposed to cadmium and tributyltin.

Histopathological effects on the ultrastructural organization of digestive tract and storage cells of Daphnia magna were examined after a 7-day exposure to cadmium or tributyltin chloride. No distinct cytological changes were noticed in the above-mentioned tissues of D. magna exposed to 10 μg Cd/l. In animals exposed to 20 μg Cd/l, however, there was a slight decline in the glycogen content of the storage cells and some structural changes in the mitochondria of these cells. By contrast, in daphnids exposed to 1 or 5 μg TBTC/l the ultrastructural integrity of storage cells appeared to be seriously affected. In addition, conspicuous lipid accumulations were observed in posterior gut epithelium and in a specific haemolymphatic cell-type of TBTC-exposed D. magna. The results of this histopathological investigation are discussed within the framework of our toxicity studies on the effects of cadmium exposure in Daphnia.

Effects of cadmium in freshwater clams. II. Ultrastructural changes in the renal system ofAnodonta cygnea

An ultrastructural study was made of the renal system of freshwater clams,Anodonta cygnea, that had been exposed to cadmium chloride (50 μg Cd/L) for 12 weeks. By stereological analysis an extended lysosomal system and a decreased number of mitochondria was apparent in the epithelial cells lining the proximal compartment of the kidney. The increase of the lysosomal system was mainly accountable to the appearance of a distinct type of lysosome, that accumulated in the apical cell region. The decrease of the mitochondrial population was accompanied by a considerable swelling of the individual mitochondria. Finally, a severe reduction of the glycogen stores was noticed. Similar, but less obvious, changes occurred in the distal kidney compartment. The results suggest that long-term exposure ofAnodonta cygnea to cadmium stimulates the lysosomal system and disturbs the function of organelles involved in the energy metabolism of resorptive kidney cells.

Effects of cadmium on gametogenesis in the sea star Asterias rubensL.

Abstract The effects of cadmium on gametogenesis in the sea star Asterias rubens were studied after short-term exposure to 200 μg Cd/l or after long-term exposure to 25 μg Cd/l under semi-field conditions. Short-term exposure of female sea stars to cadmium caused a reduction in ovary growth. Part of the oocytes obtained from Cd-exposed females showed no germinal vesicle breakdown (GVBD) upon stimulation with 1-methyladenine, while the other part needed more time to complete GVBD than oocytes from unexposed sea stars. Histological study of the ovaries of exposed females revealed a growth delay in part of the oocytes. No effect of short-term cadmium exposure was found on growth of testes. Long-term exposure of sea stars to 25 μg Cd/l caused a delay in ovary growth. This effect was clear after five months of exposure, but at the end of the reproductive cycle the difference had become smaller. No effects were observed on the overall biochemical composition of the ovaries or oocytes. The cadmium concentration in oocytes of exposed sea stars was only about 30% lower than the level found in whole gonads. Gonadal growth in male sea stars was not affected by long-term cadmium exposure. The cadmium level in testes of Cd-exposed males was about a factor 6 lower than in ovaries. The cadmium concentration in spermatozoa was comparable to that of whole testes.

Ultrastructural changes in the storage cells of the pyloric caeca of the sea star Asterias rubens L. (Echinodermata: Asteroidea) during the reproductive cycle

At six week time intervals between August and June, samples were taken from the pyloric caeca of female sea stars, Asterias rubens, in order to study the ultrastructural organization of the storage cells. In August, at the start of the phase of gonadal growth, the storage cells are characterized by the presence of a large number of storage granules, an elaborate RER in the perinuclear region, and lipid droplets located in the basal part of the cells. The strongly dilated cisternae of the RER are filled with a fine granular glycoproteinous material. From October onwards, during the phase of gonadal growth, the size of the RER gradually decreases, while the Golgi system becomes increasingly active. The Golgi system produces dense-core vesicles which accumulate near the basal membrane, indicating that their content is transferred to the mesenterial connective tissue layer. From December onwards the number of storage granules decreases and their content becomes more electron-lucent, suggesting a remobilization of stored material. In April-May, just before spawning, the Golgi system is strongly reduced and remaining elements of RER and storage granules are found in autophagic vacuoles. In June, one month after spawning, the cells again have an elaborate RER and numerous storage granules, indicating that new reserves have been built up already. No morphological evidence has been found for a release of nutrients from the caeca by destruction of complete storage cells. It is concluded that the reduction of pyloric caeca weight during the gonadal development is caused by a gradual release of substrates from storage granules, RER and lipid droplets, resulting in a decrease in cell volume.

A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.

Extremely thin layer plastification for focused-ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells: EXTREMELY THIN LAYER PLASTIFICATION

Since the recent boost in the usage of electron microscopy in life‐science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused‐ion beam scanning electron microscopy (FIB‐SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold‐labelled breast cancer SKBR3 cells was to visualise gold‐labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 μ m2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back‐scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB‐SEM. Cross‐sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.