Three-dimensional structure of endosomes in BHK-21 cells.

Abstract

The organization of the endosome compartment in BHK-21 cells was studied by using horseradish peroxidase as a fluid-phase marker and Semliki Forest virus as an adsorptive marker. Stereo pairs of semithin sections (0.2-0.5 ,sm) and computer-aided reconstruction and tracing of serial thin sections (CARTOS) were used to obtain three-dimensional images of the labeled compartments. Two types of labeled organelles could be observed: (i) small vesicles and tubules (=z50 nm in diameter) and (i) large complex structures consisting of central vesicular elements (with diameters up to 0.5 ,um) and associated systems of radiating tubules. The large endosomes were located either in the peripheral cytoplasm or in the perinuclear region, and, importantly, they existed as independent organelles and not as an interconnected reticulum. Each endosomal vacuole had two to seven tubules oriented in random directions from the central vesicle. The tubules were 60-100 nm in diameter and up to 4 Im in length. Morphometric estimates indicated that 60-70% of the endosomal membrane was in the tubules, in contrast to 30-40% of the volume. No structural continuity between endosomes and Golgi cisternae was observed, although elements of the two systems were frequently found in close proximity.Endosomes constitute a ubiquitous prelysosomal compartment with critical functions in the endocytic pathway (1-3). They serve as intermediates in the transport of endocytosed ligands, fluid-phase components, and membrane components to lysosomes; they are involved in the dissociation of ligand-receptor complexes and the recycling ofreceptors and other plasma membrane components; and they constitute the major site for molecular sorting in the endocytic pathway (2-9). In some cell types, they also are thought to be involved in storage and processing of incoming ligands, regulation of receptor expression on the cell surface, and the maintenance of plasma membrane polarity (10,11). Endosomes are, in addition, the sites of entry for certain enveloped viruses and bacterial toxins (12-15). Many of these functions depend on the acidic endosomal pH generated, at least in part, by proton-translocating ATPases (16)(17)(18).Available data from thin-section and whole-mount preparations indicate that endosomes comprise an extensive system of vacuoles and membrane-bounded tubules (4-9, 19, 20). Here we have investigated the three-dimensional structure of endosomes and their overall organization in the cytoplasm by a combination of semithin-section electron microscopy and by computer-aided reconstruction of serial thin sections (CARTOS). MATERIALS AND METHODSCells, Labeling, and Electron Microscopy. Baby hamster kidney (BHK-21) cells were maintained in minimal essential medium, Glasgow variation (G-MEM), with 5% fetal calf serum and 10%6 tryptose phosphate broth as described (12). For electron microscopy, monolayers of cells were grown to confluence (>48 hr) on 35-mm plastic tissue culture dishes.Horseradish peroxidase (HRP; type II, Sigma) was used to label BHK-21...

Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway

“…Secondly, the early endosome network is not morphologically well characterized. Those studies that have been performed indicate that what is commonly referred to as the early endosome is in fact highly complex and heterogeneous and dependent on cell type (Marsh et al, 1986;Hopkins et al, 1990;Tooze and Hollinshead, 1991). In most cell types, this early endosome network is composed of at least two separate compartments, the sorting endosome and the recycling compartment, which are morphologically and functionally distinct (Dunn et al, 1989;Presley et al, 1993;Marsh et al, 1995).…”

Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.

“…They consist of a few narrow diameter (•50-60 nm) tubules attached to spherical vesicles ~250-500 nm in diameter. Estimates of the relative surface areas in the tubules of the sorting endosome vary from 50 to 80% while the majority (60-70%) of the volume is located in the spherical portion of the endosome Marsh et al, 1986). Although the budding off of tubules and their direction towards the recycling pathway in a single step would not account for efficient recycling of membrane components (Linderman and Lauffenburger, 1988), this feature can be explained by an iterative fractionation process taking advantage of the geometry of sorting endosomes (Dunn et al 1989).…”

“…Sorting endosomes have been characterized in BHK cells (early endosomes; Griffiths et al, 1989;Marsh et al, 1986) and CHO cells (Raub et al, 1990a;Yamashiro et al, 1984). They consist of a few narrow diameter (•50-60 nm) tubules attached to spherical vesicles ~250-500 nm in diameter.…”

Evidence of a Tubular System for Transendothelial Transport in Arterial Capillaries of the Rete Mirabile

“…They have been reported in other cell types, including the secreting parietal cells in the gastric mucosa (Sugai et al 1985;Pettitt et al 1995Pettitt et al ,1996. They have been involved in endocytic processes as part of the endosomal compartment in several epithelial cells (Marsh et al 1986;Kobayashi and Robinson 1991;Tooze and Hollinshead 1991;Luo and Robinson 1992;Myers et al 1993;Sandborn and Bendayan 1996;van Deurs et al 1996). In this context, one should keep in mind that endothelial cells are a specialized form of epithelial cell in which endocytosis is mainly targeted to transcytosis.…”