CELL SURFACE LOCALIZATION OF THE 60 kDa HEAT SHOCK CHAPERONIN PROTEIN (hsp60) IN MAMMALIAN CELLS

To investigate whether the 60-kDa heat shock chaperonin protein (hsp60) is present on the surface of mammalian cells, we used immunogold labeling of intact cells and backscattered electron imaging to image gold particles. Chinese hamster ovary cells and the human leukemic CD4-positive T-cell line CEM-SS on glass coverslips were labeled using affinity-purified monoclonal and polyclonal antibodies specific for hsp60 and 30 nm gold markers. Cells were imaged using the scanning mode of the conventional transmission electron microscope. Backscattered electron imaging provided definitive identification of the gold markers while secondary electron imaging gave information on surface architecture. Labeling intensity was 250–800 gold particles per cell in Chinese hamster ovary cells and 600–2000 in CEM-SS human lymphoblasts. The finding of hsp60 on the cell surface of mammalian cells may signify chaperone involvement in surface functions.     

Ultrastructural localization of concanavalin A receptors in the plasma membrane: association with underlying actin filaments

Whole-mount cell preparations of cultured rat 3Y1 cells were examined by stereo electron microscopy to identify the ultrastructural localization of concanavalin A (Con A) receptors in the plasma membrane, and to clarify the relationship between Con A receptors and cytoskeletal components. Well spread monolayer cells were extracted with saponin, briefly fixed, and then partially broken open with shearing force to facilitate the introduction of antibodies for identification of actin filaments. Stereo electron microscopy of such treated cells revealed a 3-dimensional image of filamentous structures such as fine filaments, microtubules (MT) and endoplasmic reticulum (ER) in the flattened areas of each cell. Just beneath the plasma membrane were meshworks of actin-containing fine filaments, as identified by an immunogold staining method. Microtubules and ER were observed to be either directly or indirectly associated with this meshwork. The broken open part of each cell exhibited a meshwork of filaments which were associated with the cytoplasmic surface of the plasma membrane. Some of the filaments were connected to the plasma membrane either by their ends or by their lateral surfaces. The localization of Con A receptors was examined by binding colloidal gold-labelled Con A to the surface of fixed, saponin-extracted cells. Virtually all gold particles bound externally at the same membrane sites where intracellular actin filaments attached internally. The observations strongly suggest that the distribution of Con A receptors was regulated by the underlying meshwork of actin filaments.     

Electron microscopy of DNA replication in 3-D: evidence for similar-sized replication foci throughout S-phase

DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following the in vivo incorporation of biotin-16-deoxyuridine triphosphate, discrete RS, or foci are observed throughout the S-phase. In particular, the much larger RS or foci typically observed by fluorescence microscopic approaches in mid- and late-S-phase, are found to be composed of smaller discrete foci that are virtually identical in size to the RS observed in early-S-phase. Pulse-chase experiments demonstrate that the RS of early-S-phase are maintained when chased through S-phase and into the next cell generation. Stereologic analysis demonstrates that the relative number of smaller sized foci present at a given time remains constant from early through mid-S-phase with only a slight decrease in late-S-phase. 3-D reconstruction of serial sections reveals a network-like organization of the RS in early-S-phase and confirms that numerous smaller-sized replication foci comprise the larger RS characteristic of late-S-phase.     

Localization of Fe-containing superoxide dismutase in cyanobacteria from the Baltic Sea: depth and light dependency

The abundance and cellular location of Fe-containing superoxide dismutase (Fe-SOD) in trichomes of Nodularia , Aphanizomenon and Anabaena collected from various depths in the Baltic Sea, and in trichomes of a cultured Nodularia strain, BC Nod-9427, isolated from the Baltic Sea, was examined by immunogold labelling. For trichomes collected from natural populations the areal concentration of Fe-SOD labelling decreased with depth: trichomes collected from surface accumulations had between 8 and 11 gold particles μm⁻² whereas trichomes collected from a depth of 18 m were unlabelled. When trichomes collected from a depth of 10 m (mean areal labelling density 0·5 gold particles μm⁻²) were exposed to the higher irradiances present at 1 m, the areal concentration of Fe-SOD increased to 3·5–4 gold particles μm⁻² within 4 h. When cultures of Nodularia strain BC Nod-9427, adapted to low light (10 μmol m⁻² s⁻¹), were transferred to an incident irradiance of 1350 μmol m⁻² s⁻¹, a doubling of the areal concentration of Fe-SOD gold label was observed within 1 h. Addition of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea (DCMU) to cultures immediately before their transfer to increased illumination resulted in a decrease in areal Fe-SOD concentrations whereas addition of CdCl₂ caused an increase over and above that induced by the elevated irradiance. These results suggest that Baltic Sea cyanobacteria are able to modulate their Fe-SOD content but that this might be in response to oxidative stress rather than to light per se .     

Identification of proteins in Encephalitozoon intestinalis, a microsporidian pathogen of immunocompromised humans: an immunoblotting and immunocytochemical study

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube--the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.     

Peroxisomes of soybean (Glycine max) root nodule vascular parenchyma cells contain a "nodule—specific" urate oxidase

The cortex of soybean ( Glycine max L. cv. Centennial) nodules contain an organellerich layer of vascular parenchyma tissue, which encircles the elaborate vascular tissue of the nodule. Peroxisomes with small, electron-opaque nucleoids are found in the vascular parenchyma cells. Positive cytochemical staining for catalase (EC 1.11.1.6) confirms their morphological identification as peroxisomes. Activities of both glycolate oxidase (EC 1.1.3.1) and urate oxidase (EC 1.7.3.3) were detected cytochemically in these peroxisomes. Nodule-specific urate oxidase was localized principally in the nucleoid region of these vascular parenchyma peroxisomes, as indicated by immunogold labelling using antibodies against nodulin-35, the nodule-specific urate oxidase. The density of urate oxidase immunogold labelling in the vascular parenchyma peroxisome nucleoid is similar to that of the more well-characterized interstitial cell peroxisomes of the infected zone. These results show that the induction of nodule-specific urate oxidase may be induced in tissue outside of the infected zone.     

Lipid body biogenesis and the role of microtubules in lipid synthesis in Ornithogalum umbellatum lipotubuloids

Lipid bodies present in lipotubuloids of Ornithogalum umbellatum ovary epidermis take the form of a lens between leaflets of ER (endoplasmic reticulum) membrane filled with a highly osmiophilic substance. The two enzymes, DGAT1 [DAG (diacylglycerol) acyltransferase 1] and DGAT2 (DAG acyltransferase 2), involved in this process are synthesized on rough ER and localized in the ER near a monolayer surrounding entities like lipid bodies. After reaching the appropriate size, newly formed lipid bodies transform into mature spherical lipid bodies filled with less osmiophilic content. They appear to be surrounded by a half-unit membrane, with numerous microtubules running adjacently in different directions. The ER, no longer continuous with lipid bodies, makes contact with them through microtubules. At this stage, lipid synthesis takes place at the periphery of lipid bodies. This presumption, and a hypothesis that microtubules are involved in lipid synthesis delivering necessary components to lipid bodies, is based on strong arguments: (i) silver grains first appear over microtubules after a short [3H]palmitic acid incubation and before they are observed over lipid bodies; (ii) blockade of [3H]palmitic acid incorporation into lipotubuloids by propyzamide, an inhibitor of microtubule function; and (iii) the presence of gold grains above the microtubules after DGAT1 and DGAT2 reactions, as also near microtubules after an immunogold method that identifies phospholipase D1.     

A triple bacterial endosymbiosis in a gutless oligochaete (Annelida): ultrastructural and immunocytochemical evidence

Abstract. The gutless marine oligochaete, Olavius crassitunicatus finogenova 1986 (Tubificidae), from suboxic to sulfidic sediments off Peru, consistently harbored 3 structurally distinct types of extracellular bacterial symbionts. Large, oval bacteria were labeled immunocytochemically, proving their nature as autotrophs. Spectroscopical analysis documented storage of sulfur in this numerically dominant morphotype. Small, rod-shaped bacteria attained a more peripheral position adjacent to the cuticle. The third bacterial type was represented by long, filamentous forms which were often in close contact to the oval bacteria. With their curved or undulate cells, these filiform bacteria resembled spirochetes. They were clearly distinguishable and consistently found in all investigated host specimens.
While molecular analyses could not be performed, structural and immunocytochemical evidence indicated that the oval bacteria seemed equivalent to the γ-proteobacteria in related gutless oligochaetes. On the basis of morphological similarity and indications from closely related symbiotic tubificids, the possible relationship of the two other morphoptypes must remain unsolved and needs further molecular analysis. The three bacterial morphotypes live in a consistent, elaborate, and apparently obligate coexistence with a host that has completely reduced its digestive and excretory organs.