Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.     

On-grid immunogold labeling of glial intermediate filaments in epoxy-embedded tissue

On-grid immunogold labeling of structures like intermediate filaments has been difficult to achieve. Presumably this is because such structures are thinner than the thin sections themselves and because gold-labeled reagents remain on the surface and do not penetrate epoxy resins. Many pathologic and other tissues, however, are primarily available as epoxy-embedded blocks, and a postembedding gold procedure capable of detecting such thin structures would be useful. This study aimed to investigate the astrocytic intermediate filament antigen glial fibrillary acidic protein (GFAP) in glutaraldehyde-fixed, epoxy-embedded brain biopsy tissue from a child with Alexander's disease. A protocol was developed for performing on-grid immunogold labeling which minimized nonspecific deposition of gold reagent. The method utilized ovalbumin and skim milk in the washes and diluent for the gold reagent and the same solution with added Tween-20 and high sodium chloride in the diluent for antibodies and normal serum. In grids etched with metaperiodate and hydrogen peroxide, the astrocytic intermediate filaments were only occasionally and sparsely labeled. When an etching procedure with sodium ethoxide was employed, however, extensive labeling was obtained on the astrocytic intermediate filaments. In contrast, the larger, pathological Rosenthal fibers characteristic of Alexander's disease were labeled after both etching procedures, but labeling was enhanced after ethoxide etching. Postosmicated material showed much less labeling. The findings demonstrate that postembedding procedures can be used with epoxy-embedded material to immunolabel thin structures like intermediate filaments.     

Ultrastructural localization of the circulating anodic antigen and the circulating cathodic antigen in the liver of mice infected with Schistosoma mansoni: a sequential study

The present study describes the ultrastructural localization of two important circulating schistosome antigens--the circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA)--in livers of mice at various time intervals after infection with Schistosoma mansoni. For the demonstration of these antigens at the electron microscope level use was made of a direct, double immunogold labeling procedure, in which CAA-specific monoclonal antibodies, labeled with 5-nm gold particles, and CCA-specific monoclonal antibodies, labeled with 15-nm gold particles, were used. Both antigens were localized in granules and in inclusion bodies of Kupffer cells and granuloma macrophages and it was found that in these compartments the degree of 5- and 15-nm gold labeling increased with the duration of the infection. Sometimes gold particles were also encountered on the cell surface and in endocytotic vesicles of these cells, in endothelial cells, and in the space of Disse. From these data it was concluded that in the liver CAA and CCA were primarily accumulated in granules and inclusion bodies of Kupffer cells and granuloma macrophages. It is discussed whether at these locations both antigens are degraded by lysosomal enzymes and whether these antigens are complexed with antibodies.     

Morphology and distribution of the synapses to the spinal motoneuron of the frog

Summary Neurosecretory axons and their dilatations in the pars nervosa of the human neurohypophysis were studied electron microscopically. The axons are of two different types based on their content of neurosecretory granules (NSGs): (i) NSGs of Type A are 100–300 nm, and (ii) NSGs of type B are 50–100 nm in diameter.While fibers (or axons) of type B were scarce, showing simple swellings and terminal formations, fibers of type A were ubiquitous in the human pars nervosa, exhibiting numerous dilatations with a diversity of internal structure, apparently representing the ultrastructural manifestation of intraaxonal turnover of neurohypophysial hormones. Based on the predominating aspect of their internal structure, dilatations of type A-fibers were classified into six different types, with various transitional forms: Type I is characterized by abundant NSGs; type II by prominent mitochondria; type III by abundant lysosomal bodies; type IV by an electron-lucent matrix with few organelles; type V by prominent tubuloreticular profiles; and type VI by numerous microvesicles. The functional significance of each type is discussed and a scheme of possible interrelationships between these dilatations is proposed.     

Internalization of pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells

We investigated the uptake of surfactant by isolated alveolar type II cells by using native pulmonary surfactant complexed with colloidal gold. Internalization to lamellar bodies (LB) occurred via vesicles (mainly coated) and the endosomal system. The highest labeling density was found in the endosomal system: vacuoles and the electron-lucent multivesicular bodies (MVB), which were labeled within 10 min. The labeling of electron-dense MVB (D-MVB) and LB was time dependent, reaching a plateau after 120 min, at which time approximately 30% and 70% of the LB and D-MVB were labeled, respectively. Internalization of surfactant-gold was inhibited by the addition of native surfactant or treatment of the gold complex with antibody against surfactant apoproteins. The internalization pathway of lectin from Macula pomifera (MPA) complexed with gold was compared to that of surfactant. Both pathways were found to be similar, except that mainly smooth vesicles rather than coated ones were involved in the process of MPA-G internalization. The partial labeling of the LB, the possible routing to lysosomes, and the endosomes as junction between the biosynthetic and endocytic pathways are discussed.     

Ultrastructure of lipid bodies in Tilletia caries teliospores.

The ultrastructure of lipid bodies within developing, dormant, and germinating Tilletia caries (DC). Tul. (race T-16) teliospores was studied by freeze-etching and thin-sectioning techniques. When teliospores were prefixed in sodium cacodylate-buffered glutaraldehyde-acrolein for 24 h before further processing, most of the lipid bodies appeared to have a uniformly osmiophilic matrix. Some of these lipid bodies were surrounded by thin electron-dense lines that appeared to be half-unit membranes. Occasionally this membrane seemed to be absent, allowing for a direct interface between lipid and cytosol. Irregular electron-dense patterns were occasionally observed in lipid bodies of developing, dormant, and germinating teliospores. A lamellar substructure with 6- to 10-nm center-to-center spacing was visible in the electron-dense patterns at high magnifications. Irregular fracture patterns were visible in freeze-etch replicas.     

The molecular organization of myosin in stress fibers of cultured cells

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.     

Ultrastructural immunogold labeling of glial filaments in osmicated and unosmicated epoxy-embedded tissue

On-grid (post-embedding) immunolabeling methods with epoxy resins have been difficult to apply to thin structures such as intermediate filaments, which may remain inaccessible within the plastic. In this study, glial fibrillary acidic protein (GFAP), the major protein of astrocyte intermediate filaments, was localized with a post-embedding immunogold method, using both unosmicated and osmicated material embedded in epoxy resin. The tissue studied was from a diagnostic brain biopsy on a child with Alexander's disease. This disorder is characterized by proliferation of astrocyte intermediate filaments and formation of Rosenthal fibers. With unosmicated tissue, as in a previous study, extensive labeling of the glial filaments was achieved only when ultra-thin sections were pre-treated with dilute sodium ethoxide, an agent that dissolves plastic. Fifteen-nm gold could be used. With osmicated tissue, localization to glial filaments required pre-treatment with sodium ethoxide and with the oxidizing agent sodium metaperiodate, followed by the use of small (5 nm) colloidal gold. That 5-nm gold was required for labeling filaments in osmicated material suggested that osmication increases problems of penetrability and antigen accessibility within ultra-thin sections. The large Rosenthal fibers were labeled by 15-nm gold in both unosmicated and osmicated material. The methods employed may be useful for electron immunolocalizations to other thin structures in material embedded in epoxy resin.     

Some improvement in tissue preparation and colloidal-gold immunolabeling for electron microscopy

The application of freeze-substitution (FS) and freeze-drying (FD) techniques and the protein A-gold-antibody complex immunocytochemical methods are described. The two tissue-preparation techniques produced excellent ultrastructure and topographical fixation of antigens when compared with conventional tissue-preparation techniques. In the FS preparation, however, occasional extragranular immunolabeling was recognized. This may suggest the leakage of antigens from the secretory granules. The FD procedure was considered the best, since such labeling was almost negligible. The protein A-gold-antibody complexes are easily prepared and label the antigens clearly. If the protein A-coated gold particles are saturated with antibodies, there is no interaction between gold particles. Thus, multiple antigens can be determined even in single secretory granules. In fact, we demonstrated intragranular colocalization of immunoreactive oxytocin, labeled with 50-nm gold particles, and immunoreactive methionine-enkephalin, labeled with 15-nm gold particles, in the axonal terminals of the FD-prepared rat neurohypophysis. This study demonstrates the value of the use of gold-antibody complexes for immunocytochemical labeling on FS- or FD-treated tissues.     

Immunoelectron Microscopy of Giardia lamblia Cytoskeleton Using Antibody to Acetylated α-Tubulin

Giardia lamblia trophozoites contain acetylated α-tubulin but lack detectable levels of tyrosinolated α-tubulin, as demonstrated in immunoblots with monoclonal antibodies specific for these tubulin forms. By immunofluorescence microscopy, acetylated α-tubulin is localized in axonemes, median bodies and in the adhesive disk. Post-embeddment immunogold labeling of thin sections of cells was used to evaluate acetylation at the level of individual microtubules by electron microscopy. Cells were fixed with glutaraldehyde and embedded in the acrylic resin LR Gold. Results indicate all microtubules in adhesive disk, axonemes, basal bodies, funis and the median bodies contain acetylated α-tubulin. Unlike immunofluorescence labeling, all microtubules of the adhesive disk and the funis could be gold labeled. No nonspecific labeling of the cytoplasm or of structures other than microtubules was observed. Acetylated microtubules in G. lamblia do not appear to be a subset of microtubules and acetylation appears uniform along the entire length of individual microtubules. Acetylation and the tyrosinolation state of microtubules in Giardia are discussed in the context of microtubule stability and crosslinked features of the cytoskeleton.     

拟南芥菜(Arabidopsis thaliana)子叶中蛋白体形成的超微结构和免疫电镜定位研究

本文对拟南芥菜(Arabidopsis thaliana)种子发育过程中贮藏蛋白的积累和蛋白体的形成进行了超微结构和免疫电镜定位的研究。常规超薄切片的电镜观察表明,在开花后第10天(10 DAF),高电子密度的蛋白质物质开始在子叶细胞的液泡中沉积。这一过程一直延续到种子接近成熟(14 DAF),这时液泡中充满了蛋白质物质,转变成为大的蛋白体。利用了该种植物主要种子贮藏蛋白之一的12 s球蛋白的单克隆抗体作为免疫探针,以蛋白质A-胶体金电镜技术对12 s种子蛋白进行了细胞内定位,证实了在液泡中积累的物质为种子贮藏蛋白。实验结果表明在拟南芥菜中,子叶细胞中的液泡是蛋白体的前体,肯定了蛋白体的发生起源于液泡的观点。本文还对应用胶体金电镜技术进行细胞内定位的某些问题作了初步探讨。     

Immunoelectron microscopic double labeling of alkaline phosphatase and penicillinase with colloidal gold in frozen thin sections of Bacillus licheniformis 749/C.

The subcellular distribution of alkaline phosphatase and penicillinase was determined by double labeling frozen thin sections of Bacillus licheniformis 749/C with colloidal gold-immunoglobulin G (IgG). Antipenicillinase and anti-alkaline phosphatase antibodies were used to prepare complexes with 5- and 15-nm colloidal gold particles, respectively. The character of the labeling of membrane-bound alkaline phosphatase and penicillinase was different: the immunolabels for alkaline phosphatase (15-nm particles) were bound to a few sites at the inner surface of the plasma membrane, and the gold particles formed clusters of various sizes at the binding sites; the immunolabels for penicillinase (5-nm particles), on the other hand, were bound to the plasma membrane in a dispersed and random fashion. In the cytoplasm, immunolabels for both proteins were distributed randomly, and the character of their binding was similar. The labeling was specific: pretreating the frozen thin sections with different concentrations of anti-alkaline phosphatase or penicillinase blocked the binding of the immunolabel prepared with the same antibody. Binding could be fully blocked by pretreatment with 800 micrograms of either antibody per ml.     

Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.     

LIGHT AND ELECTRON MICROSCOPIC CHARACTERIZATION OF TWO TYPES OF PYRENOIDS IN GONIUM (GONIACEAE,CHLOROPHYTA)1

The single, basal pyrenoids of Gonium quadratum Pringsheim ex Nozaki and G. pectorale Müller (Goniaceae, Chlorophyta) differed in appearance when vegetative colonies were cultured photoheterotrophically in medium containing sodium acetate. Chloroplasts of G. quadratum had distinct pyrenoids when grown in medium without major carbon compounds. However, the pyrenoids degenerated and were markedly reduced in size when such cells were inoculated into a medium containing 400 mg·L^?1 of sodium acetate. No pyrenoids were visible under the light microscope; however, with electron microscopy small pyrenoids and electron-dense bodies were visible within the degenerating chloroplasts, which had only single layers of thylakoid lamellae at the periphery. The chloroplasts subsequently developed distinct pyrenoids and several layers of thylakoid lamellae as the culture aged. In contrast, vegetative cells of G. pectorale always showed distinct pyrenoids when cells were inoculated into medium containing sodium acetate, sodium pyruvic acid, sodium lactate, and/or yeast extract. Therefore, we propose two terms, “unstable pyrenoids” and “stable pyrenoids,” for pyrenoids of G. quadratum and G. pectorale, respectively. Chloroplasts of the colonial green flagellates should thus be examined under various culture conditions in order to determine whether their pyrenoids are unstable or stable when pyrenoids are used as taxonomic indicators. Immunogold electron microscopy showed that the ratios of gold particle density of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) between pyrenoid matrix and chloroplast stroma in G. quadratum grown in medium with or without sodium acetate were lower than those of G. pectorale. Heavy labeling by anti-RuBisCO was observed in both the electron-dense bodies and pyrenoid matrix of G. quadratum. This is the first electron microscopic demonstration of degeneration and development of both pyrenoids and thylakoid lamellae in the chloroplast as a function of culture condition in green algae.     

Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.     

Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins

We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.     

Intracellular localization and endocytosis of brush border enzymes in the enterocyte-like cell line Caco-2.

Immunoelectron microscopy was used to localize the brush border hydrolases sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPPIV) in the human colon carcinoma cell line Caco-2. Both enzymes were detected at the microvillar membrane, in small vesicles and multivesicular bodies (MVBs), and in lysosomal bodies. In addition, DPPIV was found in the Golgi apparatus, a variety of apical vesicles and tubules, and at the basolateral membrane. To investigate whether the hydrolases present in the lysosomal bodies were endocytosed from the apical membrane, endocytic compartments were marked with the endocytic tracer cationized ferritin (CF). After internalization from the apical membrane through coated pits, CF was first recovered in apical vesicles and tubules, and larger electronlucent vesicles (early endosomes), and later accumulated in MVBs (late endosomes) and lysosomal bodies. DPPIV was localized in a subpopulation of both early and late endocytic vesicles, which contained CF after 3 and 15 min of uptake, respectively. Also, internalization of the specific antibody against DPPIV and gold labeling on cryosections showed endocytosed DPPIV in both early and late endosomes. However, unlike CF, no accumulation of DPPIV was seen in MVBs or lysosomal bodies after longer chase times. The results indicate that in Caco-2 cells the majority of brush border hydrolases present in lysosomal bodies are not endocytosed from the brush border membrane. Furthermore, the labeling patterns obtained, suggest that late endosomes may be involved in the recycling of endocytosed DPPIV to the microvilli.     

Immunocytochemical localization of the major surfactant apoproteins in type II cells, Clara cells, and alveolar macrophages of rat lung

The adsorptive properties of phospholipids of pulmonary surfactant are markedly influenced by the presence of three related proteins (26-38 KD, reduced) found in purified surfactant. Whether these proteins are pre-assembled with lipids before secretion is uncertain but would be expected for a lipoprotein secretion. We performed indirect immunocytochemistry on frozen thin sections of rat lung to identify cells and intracellular organelles that contain these proteins. The three proteins, purified from lavaged surfactant, were used to generate antisera in rabbits. Immunoblotting of rat surfactant showed that the IgG reacted with the three proteins and a 55-60 KD band which may be a polymer of the lower MW species. Specific gold labeling occurred over alveolar type II cells, bronchiolar Clara cells, alveolar macrophages, and tubular myelin. In type II cells labeling occurred in synthetic organelles and lamellar bodies, which contain surfactant lipids. Lamellar body labeling was increased fivefold by pre-treating tissue sections with a detergent. Multivesicular bodies and some small apical vesicles in type II cells were also labeled. Secondary lysosomes of alveolar macrophages were immunoreactive. Labeling in Clara cells exceeded that of type II cells, with prominent labeling in secretory granules, Golgi apparatus, and endoplasmic reticulum. These observations clarify the organelles and pathways utilized in the elaboration of surfactant. After synthesis, the proteins move, probably via multivesicular bodies, to lamellar bodies. Both lipids and proteins are present in tubular myelin. Immunologically identical or closely similar proteins are synthesized by Clara cells and secreted from granules which appear not to contain lipid. The role of these proteins in bronchiolar function is unknown.     

Subcellular localization of type I thionins in the endosperms of wheat and barley

Summary Thionins are cysteine-rich polypeptides of about 5,000 Da. Localization at the subcellular level of type I endosperm thionins has been carried out by immunogold labeling, using an antibody that recognizes type I thionin variants. In developing wheat and barley caryopses, sectioned at different times between 13 and 24 days after flowering, this type of thionins was only detected around protein bodies from cells of the starchy endosperm, using light microscopy. Electron microscopy revealed that these proteins were located in electron-dense spheroids in the periphery of protein bodies, at the earlier stages, whereas later the label appeared also as a thin layer around these organelles.Abbreviations DAF days after flowering - RER rough endoplasmic reticulum     

ULTRASTRUCTURE AND IMMUNOLOCALIZATION OF PHYCOBILIPROTEINS AND RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE IN THE MARINE CYANOBACTERIUM TRICHODESMIUM THIEBAUTII1

Trichodesmium thiebautii Gomont, a marine planktonic diazotrophic cyanobacterium, has an unusual subcellular arrangement. To identify subcellular structures related to photosynthesis, antibodies against phycoerythrin, phycocyanin, and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used together with an immuno-gold labeling technique and electron microscopy. Thylakoid membranes, identified by transmission electron microscopy and phycobiliprotein labeling, were arranged as a loose network throughout all cells. Rubisco showed a particularly intense localization in medium electron-dense polyhedral bodies, therefore identified as carboxysomes. The average density of the carboxysomal Rubisco label was about five times higher than that in the cytoplasm. The carboxysomes (4–11 per cell section) were scattered throughout the cytoplasm. These data, together with those obtained from double immunolabeling experiments using nitrogenase (Fe-protein) and Rubisco antibodies, revealed that Trichodesmium contains both N₂- and CO₂-fixing proteins within the same cell. This is in contrast to the previous concept of a spatial segregation of the two processes in Trichodesmium and demonstrate that nitrogenase-containing cells are not comparable to heterocysts in this context.