Ultrastructural study of synovial membrane from the antebrachiocarpal joint of calves

The synovial intima from the antebrachiocarpal joint of 4-month-old calves was between 1 and 3 cells in thickness and did not have a basal lamina. Numerous areas of the intimal matrix were in direct contact with the joint lumen. The synovial membrane was comprised mainly of A-type synoviocytes usually located adjacent to the joint lumen. These cells were characterized by numerous filopodia (or lamellipodia), large, empty-appearing vacuoles, numerous lysosomes, large vacuoles containing granular material separated from the vacuolar membrane by a radiolucent band, and coated micropinocytotic vesicles. Smooth micropinocytotic vesicles were seen only rarely in these cells. In contrast, B-type cells had few filopodia, numerous smooth micropinocytotic vesicles, few coated micropinocytotic vesicles, a well-developed Golgi apparatus and rough endoplasmic reticulum, mitochondria that were longer and had a denser matrix than that of A cell mitochondria, and surprisingly, only few maturing or fully formed secretory granules. A distinct intermediate (C or AB) type synoviocyte could not be unequivocally identified. Desmosome-like structures were present between synoviocytes, although it was considered questionable if these were true intercellular junctions. No other junctions were present.     

Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin

Outer membrane derived vesicles (MVs) secreted by Actinobacillus actinomycetemcomitans JP2 contain a membranolytic leukotoxin and are toxic to human HL60 cells. To determine how MVs interact with human target cells, HL60 cells were incubated with vesicles, reacted with anti-vesicle antibodies and a FITC-labelled reporter, and visualized by confocal scanning laser microscopy. Target cells rapidly became reactive with anti-vesicle antibodies upon exposure to vesicles. Confocal microscopy showed that labelling occurred primarily in the cytoplasmic membrane and that very little internal fluorescence was observed. The cytoplasmic membrane of HL60 cells was also strongly labelled after exposure to MVs that contained the fluorescent phospholipid, SP-DiOC18. In contrast, incubation of cells with free SP-DiOC18 resulted primarily in the labelling of internal structures of HL60 cells. These results suggest that A. actinomycetemcomitans MVs associate with, or are incorporated into the cytoplasmic membrane of HL60 cells. The leukotoxin is a membranolytic cytotoxin and cells exposed to MVs were lysed by vesicle-associated toxin in a time and dose-dependent manner. However, cells became reactive with anti-vesicle antibodies when MVs were added in the presence of inhibitors of leukotoxin-mediated lysis or when sublytic doses of MVs were analysed. In addition, MVs produced by an isogenic leukotoxin-deficient strain of A. actinomycetemcomitans JP2 were non-toxic but rapidly interacted with HL60 cells. These results suggest that A. actinomycetemcomitans MVs can deliver leukotoxin to HL60 cells but that the association of vesicles with the cytoplasmic membrane occurs independently of the leukotoxin polypeptide.     

Giardia muris: ultrastructural analysis of in vitro excystation

Giardia muris cysts were examined by transmission electron microscopy before treatment, after induction, and at timed intervals during the incubation phase of in vitro excystation. Untreated G. muris cysts had a thick cyst wall composed of a fibrous outer wall and a thin, electron-dense inner membrane which extended from the trophozoite plasma membrane. The cytoplasm was devoid of endoplasmic reticulum, Golgi bodies,and mitochondria. Numerous large vacuoles were present within the ectoplasm just beneath the plasma membrane in untreated cysts. Following induction these cysts lacked ectoplasmic vacuoles. Concurrently, numerous membrane bound vesicles were seen in the peritrophic space closely adhering to the surface of the trophozoite. These vesicles appear to be of cytoplasmic origin. The cytoplasm of fully excysted trophozoites lacked ectoplasmic vacuoles but displayed well-developed ribbons of microtubular bodies, probably precursors of ventral disk, lateral flange, and median bodies and also contained extensive granular endoplasmic reticulum. No more than two nuclei were observed within each organism. The earliest excysted organisms were observed 0-5 min after incubation had begun and most organisms had excysted within 10 min. Cytokinesis occurred only after excystation was complete.     

Membrane vesicles: an overlooked component of the matrices of biofilms

The matrix helps define the architecture and infrastructure of biofilms and also contributes to their resilient nature. Although many studies continue to define the properties of both gram-positive and gram-negative bacterial biofilms, there is still much to learn, especially about how structural characteristics help bridge the gap between the chemistry and physical aspects of the matrix. Here, we show that membrane vesicles (MVs), structures derived from the outer membrane of gram-negative bacteria, are a common particulate feature of the matrix of Pseudomonas aeruginosa biofilms. Biofilms grown using different model systems and growth conditions were shown to contain MVs when thin sectioned for transmission electron microscopy, and mechanically disrupted biofilms revealed MVs in association with intercellular material. MVs were also isolated from biofilms by employing techniques for matrix isolation and a modified MV isolation protocol. Together these observations verified the presence and frequency of MVs and indicated that MVs were a definite component of the matrix. Characterization of planktonic and biofilm-derived MVs revealed quantitative and qualitative differences between the two and indicated functional roles, such as proteolytic activity and binding of antibiotics. The ubiquity of MVs was supported by observations of biofilms from a variety of natural environments outside the laboratory and established MVs as common biofilm constituents. MVs appear to be important and relatively unacknowledged particulate components of the matrix of gram-negative or mixed bacterial biofilms.     

An observation on fine structure of conidia of sphaerotheca pannosa (wallr.) lév. attacking leaves of roses

Summary Fine structures of conidia ofSphaerotheca pannosa, the causal fungus of powdery mildew of rose plants were investigated. The cell wall of conidia was relatively thin and consisted of two layers. Nuclei, mitochondria, endoplasmic reticula and vacuoles were identified in the conidia, and there were many unknown vesicles just inside the plasma membrane. Usually the conidia were uninucleate, but before the germination the nucleus divided into two. Vacuole held a number of osmiophilic globules which were located along the tonoplast, and in large magnifications they appeared as aggregation of small electron-transparent globules when fixed with KMnO₄ solution. In a germ tube one large mitochondrion and many endoplasmic reticula were found.Contribution from the Laboratory of Plant Pathology, Kyoto University, No. 174.     

Stereological analysis of the guinea pig pancreas. I. Analytical model and quantitative description of nonstimulated pancreatic exocrine cells

A stereological model which provides detailed quantitative information on the structure of the fasted, nonstimulated gland has been developed for the guinea pig pancreas. The model consists of morphologically defined space and membrane compartments which were used to describe the general composition of the tissue and the specific components of exocrine cells. The results are presented, where appropriate, relative to a cubic centimeter of pancreas, a cubic centimeter of exocrine cell cytoplasm, and to the volume of an average exocrine cell. The exocrine cells, accounting for 82% of the pancreas volume, consisted of 54% cytoplasmic matrix, 22% rough-surfaced endoplasmic reticulum (RER), 8.3% nuclei, 8.1% mitochondria, 6.4% zymogen granules, and 0.7% condensing vacuoles. Their total membrane surface area was distributed as follows: 60% RER, 21% mitochondria, 9.9% Golgi apparatus, 4.8% plasma membranes, 2.6% zymogen granules, 1.8% plasma membrane vesicles, and 0.4% condensing vacuoles. The application of this model to the study of membrane movements associated with the secretory process is discussed within the framework of an analytical approach.     

Ultrastructural changes in the gracile nucleus of the spontaneously diabetic BB rat.

The present study describes the structural changes in the gracile nucleus of the spontaneously diabetic BB rat. At 3-7 days post-diabetes, axons, axon terminals and dendrites showed electron-dense degeneration. Degenerating axons were characterized by swollen mitochondria, vacuolation, accumulation of glycogen granules, tubulovesicular elements, neurofilaments and dense lamellar bodies. Degenerating axon terminals consisted of an electron-dense cytoplasm containing swollen mitochondria, vacuoles and clustering of synaptic vesicles. These axon terminals made synaptic contacts with cell somata, dendrites and other axon terminals. Degenerating dendrites were postsynaptic to normal as well as degenerating axon terminals. At 1-3 months post-diabetes, degenerating electron-dense axons, axon terminals and dendrites were widely scattered in the neuropil. Macrophages containing degenerating electron-dense debris were also present. At 6 months post-diabetes, the freshly degenerating neuronal elements encountered were similar to those observed at 3-7 days. However, there were more degenerating profiles at 6 months post-diabetes compared to the earlier time intervals. Terminally degenerating axons were vacuolated and their axoplasm appeared amorphous. It is concluded that degenerative changes occur in the gracile nucleus of the spontaneously diabetic BB rat.     

Surface multivesicular structures associated with maturing erythrocytes in rats

Summary Surface multivesicular structures associated with the plasmalemma of erythrocytes were observed in the peripheral blood of rats which have a significant number of circulating reticulocytes. These surface structures appear as ovoid evaginations (0.2 to 0.7 in diameter) of the plasma membrane and contain numerous small vesicles ranging from 0.05 to 0.1 in diameter. The structures were present during the final stages of maturation of erythrocytes, after nuclei and mitochondria had been extruded and only a few polysomes and small vesicles remained. They appear quite distinct from the autophagic vacuoles which have been described in association with degeneration and extrusion of mitochondria from erythrocytes. The exact origin of the small internal vesicles of these surface multivesicular structures is unknown; however, similar vesicles have been observed in the cytoplasm of the maturing erythrocyte especially in the vicinity of the Golgi body. These structures suggest a process by which Golgi elements and other small cytoplasmic vesicles are extruded during the late stages of maturation of rat erythrocytes.Supported by U.S. Public Health Service Research Grant AM 12950.The author is indebted to Dr. Edward G. Rennels and Dr. William B. Winborn for their guidance.     

Membrane Dynamics at the Nuclear Exchange Junction during Early Mating (One to Four Hours) in the Ciliate Tetrahymena thermophila

Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.     

Fine structure of spermatogonia and intercellular bridges in Macaca nemestrina

Spermatogonia of the monkey, Macaca nemestrina, were studied with the electron microscope. The spermatogonial nucleus is characterized by dense homogeneous chromatin and an eccentric nucleolus with a prominent surrounding clear zone. Cytoplasm consists chiefly of free ribosomes and vesicular endoplasmic reticulum. Scattered mitochondria with closely spaced transverse cristae are arranged singly and in pairs separated by thin electron-dense bands. Binucleated spermatogonia resemble other spermatogonia in their ultrastructural characteristics, but contain an increased number of lysosome-like structures and degenerating mitochondria. Spermatogonial interconnections are of two types: broad cytoplasmic connections and narrow intercellular bridges. Connected cells are always identical in appearance and stage of maturation. Multiple connections occur. Interconnection of spermatogonia provides a syncytial type of arrangement which allows synchronization of differentiation and results in similar apperance of adjoining cells. Similarity of regressive changes in adjacent degenerating cells is explained by the presence of intercellular bridges.     

Transfer of carbonic anhydrase-positive particles from the follicle-associated epithelium to lymphocytes of Peyer's patches in foetal sheep and lambs

Summary Carbonic anhydrase cytochemistry of the ileal Peyer's patch in foetal and neonatal lambs has indicated secretion from the follicle-associated epithelium to the follicles. Reaction for carbonic anhydrase in the follicle-associated epithelium was found in the luminal plasma membrane, in cytoplasmic vesicles, and in vacuoles containing 50-nm membrane-bounded particles that seemed to be shed to the intercellular space. The lateral plasma membrane was negative for carbonic anhydrase, indicating that formation of carbonic anhydrase-positive particles was restricted to vacuoles. Administration of ferritin to ileal loops of sheep foetuses showed ferritin localized in vesicles and vacuoles of the follicle-associated epithelium followed by exocytosis, together with carbonic anhydrase-positive particles, into the indentations of the lateral cell border. The carbonic anhydrase-positive particles seemed to be transported to the centres of lymphoid follicles where many were attached to the plasma membrane of lymphocytes. Carbonic anhydrase-positive particles were also seen in vesicles and sometimes free in the cytoplasm of the lymphocytes or attached to their nuclear envelope. Light microscopically, carbonic anhydrase reactivity of the follicle-associated epithelium was associated with the early formation of the ileal Peyer's patch at about 100 days gestation. At this time the follicle-associated epithelium showed a strong luminal but at most a week lateral staining. With further foetal development there was a progressive increase in the amount of carbonic anhydrase-positive reaction product in extracellular particles, both along the lateral cell borders of the follicle-associated epithelium and among the lymphocytes of the follicle centres.     

Segregation of Ferritin in Glomerular Protein Absorption Droplets

Ferritin was used as a tracer to study the mechanism by which proteins are segregated into droplets by the visceral epithelium of glomerular capillaries. In glomeruli from both normal and aminonucleoside-nephrotic rats ferritin molecules introduced into the general circulation penetrated the endothelial openings and were seen at various levels in the basement membrane. Striking differences between nephrotic and controls were seen only in the amount of ferritin incorporated into the epithelium. In normal animals, a few ferritin molecules were seen in small invaginations of the cell membrane limiting the foot processes, within minute vesicles in the epithelium, or within occasional large vacuoles and dense bodies. In nephrotics, epithelial pinocytosis was marked, and numerous ferritin molecules were seen within membrane invaginations and in small cytoplasmic vesicles at all time points. After longer intervals, the concentration of ferritin increased in vacuoles and particularly within the dense bodies or within structures with a morphology intermediate between that of vacuoles and dense bodies. In nephrotic animals cleft-like cavities or sinuses were frequently encountered along the epithelial cell surface facing the urinary spaces. Some of these sinuses contained material resembling that filling the dense bodies except that it appeared less compact. The findings suggest that ferritin molecules—and presumably other proteins which penetrate the basement membrane—are picked up by the epithelium in pinocytotic vesicles and transported via the small vesicles to larger vacuoles which are subsequently transformed into dense bodies by progressive condensation. The content of the dense bodies may then undergo partial digestion and be extruded into the urinary spaces where it disperses. The activity of the glomerular epithelium in the incorporation and segregation of protein is similar in normal and nephrotic animals, except that the rate is considerably higher in nephrosis where the permeability of the glomerular basement membrane is greatly increased.     

Pinocytosis in the root cells of a salt-accumulating halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> and its possible involvement in chloride transport

Ultrastructure of root cells in salt-accumulating halophyte Suaeda altissima (L.) Pall. was examined with transmission electron microscopy. Plants were grown hydroponically on nutrient media containing 3, 50, 250, and 500 mM NaCl. Some plants were exposed to hypersomotic salt shock by an abrupt increase in NaCl concentration from 50 to 400 mM. Growing S. altissima plants at high NaCl concentrations induced the formation of type 1 pinocytotic structures in root cells. Type 1 structures appeared as pinocytotic invaginations of two membranes, the plasmalemma and tonoplast. These invaginations into vacuoles gave rise to freely ‘floating’ multivesicular bodies (MVB) enclosed by a double membrane layer. The pinocytotic invaginations and MVB contained the plasmalemma-derived vesicles and membranes of endosome origin. The hyperosmotic salt shock led to formation of type 2 and type 3 pinocytotic structures. The type 2 structures were formed as pinocytotic invaginations of the tonoplast and gave rise to MVB in vacuoles. Unlike type 1 MVB, the type 2 MVB had only one enclosing membrane, the tonoplast. The type 3 structures appeared as the plasmalemma-derived vesicles located in the periplasmic space. The cytochemical electron-microscopy method was applied to determine the intracellular Cl^? localization. This method, based on sedimentation of electron-dense AgCl granules in tissues treated with silver nitrate, showed that the pinocytotic structures of all types contain Cl^? ions. The presence of Cl^? in pinocytotic structures implies the involvement of these structures in Cl^? transport between the apoplast, cytoplasm, and the vacuole.     

THE MECHANISM OF MITOCHONDRIAL EXTRUSION FROM PHENYLHYDRAZINE-INDUCED RETICULOCYTES IN THE CIRCULATING BLOOD

The mechanism of mitochondrial extrusion from reticulocytes was studied in whole blood from dogs made anemic by treatment with phenylhydrazine hydrochloride. The initial stage of preparation for mitochondrial extrusion was attraction of vesicles to mitochondria. There was subsequent encirclement of the organelle and other bodies, such as ferritin, by coalesced vesicles forming double membrane-limited vacuoles. Large vacuoles were formed from the union of single vacuoles, and they were usually situated near the periphery of the cell. Fusion of the outer membrane of vacuoles with the plasmalemma of the reticulocyte provided a route for exposure and release of mitochondria and other material to an extracellular location. An extracellular mitochondrion, therefore, was confined by its original double membrane, and a third membrane was derived from the internal boundary of vacuoles.     

Vacuolar system of ungerminated Colletotrichum graminicola conidia: convergence of autophagic and endocytic pathways

Vacuoles of ungerminated Colletotrichum graminicola conidia engulf cytoplasmic structures by a process analogous to microautophagy, demonstrated by using a vacuolar membrane acid phosphatase marker. Fusion of vesicles with vacuoles, without deposition of the acid phosphatase reaction product has been observed, suggesting other pathways of material delivery to vacuoles than microautophagy. Plasma membrane invaginations, multivesicular bodies and retention of neutral red into small vesicles, which were internalized by the vacuole, were verified. These results provided evidence for endocytosis and an active endosomal system. Together, our findings with C. graminicola demonstrated that vacuoles are very dynamic compartments, playing roles in autophagy and endocytic processes.     

Ultrastructural localization of cytoplasmic phosphatases in preimplantation mouse embryos.

The appearance and localization of the cytoplasmic phosphatases [acid phosphatase (AcPase) as a marker of lysosomes, TPPase as a marker of the Golgi apparatus, and NDPase (IDPase) as enzymatic marker of the endoplasmic reticulum (ER)] were cytochemically studied on the ultrastructural level in secondary oocytes and in preimplantation mouse embryos. The detectable AcPase activity, located on the inner surface of the membrane delimiting some cytoplasmic vacuoles (lysosomes and autophagic vacuoles), appears at the eight-cell stage and grows pregressively stronger up to the blastocyst stage. Golgi-associated reaction for TPPase was detectable in oocytes, dropped in one-cell embryos and became negative in the two-cell embryos. The reaction for TPPase and IDPase was present in plasma membranes of oocytes and early embryos and appeared in the delimiting membrane of some cytoplasmic vesicles in eight-cell embryos. Some activity of IDPase was found in small segments of the ER at the morula and blastocyst stage. The observed results suggest that the lysosomes are the first organelles in early embryos showing activity of the marker enzymes of the phosphatase type, while the activity of other marker enzymes is mainly concentrated in the plasma membrane of blastomeres. It cannot be excluded, however, that positive reaction for TPPase and IDPase in the plasma membrane results from nonspecific action of other phosphatases.     

ELECTRON MICROSCOPY OF THE HUMAN SYNOVIAL MEMBRANE

The structure of the lining cells at the surface of the synovial membrane facing the joint cavity has been studied by electron microscopy. The long cytoplasmic processes of these cells appear to be oriented toward the surface of the membrane, where they overlap and intertwine. The matrix of the lining cells contains dense material but no fibers with the periodicity of collagen. The lining cells are divided into two cell types or states of activity on the basis of their cytoplasmic contents. Type A is more numerous and contains a prominent Golgi apparatus, numerous vacuoles (0.4 to 1.5 microns in diameter) containing varying amounts of a dense granular material, many filopodia, mitochondria, intracellular fibrils, and micropinocytotic-like vesicles. Type B contains large amounts of ergastoplasm with fewer large vacuoles, micropinocytotic-like vesicles, and mitochondria. The probable functions of these cells are discussed in the light of current knowledge of the metabolism and function of the synovial membrane.     

An electron microscopic study of the adrenal cortical tissue of the domestic fowl

Summary The fine structure of the adrenal cortex of the domestic fowl has been studied from the 14th day of embryonic life to 980 days after hatching, using the electron microscope.Many mitochondria and vacuoles, round or oval, are observed in all cortical cells. The cristae mitochondriales are not laminar but villous. In the embryo, the mitochondria, whose cristae are not as well developed as those in chick and hen, are very low in electron density. Cytoplasmic vacuoles, either rough or smooth-surfaced, and with a homogeneous content of low electron density, are enclosed by a similar limiting membrane. Some of them are formed by the outer nuclear membrane and are characterized by many small particles on their outer surfaces. These vacuolated structures are perhaps parts of the endoplasmic reticulum. In the 980 day old hen, a number of osmiophilic droplets, round or irregularly shaped, are seen. Some of these are formed by mitochondria, while others accumulate within or around cytoplasmic vacuoles. In the embryo and in the chick, such droplets are never seen. It is assumed that the droplets are substances related to ageing, and that the lipid implicated in cortical hormone is not osmiophilic in the young chick and the embryo. It is perhaps produced in the cytoplasmic vacuoles with the aid of mitochondria. A perisinusoidal space and interparenchymatous cell spaces similar to those described for mammals by other workers, are observed in the adrenal gland of the domestic fowl. I assume that the secretory substance of the cortical and the medullary cell is first secreted into these spaces and then infiltrates into the capillary lumen.