Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Summary The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.     

Fine structure of the glomerular basement membrane of the rat kidney visualized by high-resolution scanning electron microscopy

Summary The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5-to 9-nm-thick fibrils, had pores 11–30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10–24 nm wide) than those near the filtration slits (16–32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.     

Fibronectin in basement membrane of hertwig's epithelial sheath

Summary The distribution of fibronectin throughout the basement membrane of Hertwig's epithelial sheath was studied using specific antibodies with the immunoperoxidase technique in both light and electron microscopy.—Our results demonstrate that, after collagenase digestion in situ, the basement membrane was strongly labelled by antifibronectin antibodies on the lamina lucida, the lamina densa and the lamina (pars) fibroreticularis which contained aperiodic fibrils of 5–10 nm in diameter.     

The nerve fibres in the basement membrane and related structures in Saccoglossus horsti (Enteropneusta)

Summary The structure of the basement membrane of Saccoglossus horsti has been examined with the electron microscope. The membrane consists of two lamellae each of two layers. An outer amorphous layer 150 nm across and an inner fibrillar layer 1–3 m across. The fibrils of the fibrillar layer are two sizes, the majority are 5–9 nm in diameter and at least 2 m long. The thicker 30 nm fibrils occur in small patches and have striations with a 30 nm period.Within the lamellae of the basement membrane are blood spaces. The only regularly found structures in these spaces are blood particles some 12–16 nm in diameter.Nerve fibres of varying diameters traverse all the layers of basement membrane. These fibres run longitudinally and obliquely through the basement membrane, and emerge amongst the muscle cells inserted into the coelomic side of the membrane. No motor end plates have been seen. Preliminary observations suggest that many of the nerve fibres have no sheath other than the cell membrane of the fibre itself.The muscle cells are attached to the basement membrane by structures that resemble hemidesmosomes. The blood vessels of Saccoglossus have a basement membrane on the lumenal side of the endothelial cell cytoplasm.I wish to thank Professor J. Z. Young, F. R. S. for continuous encouragement and advice. To Dr. R. Newell I am indebted for the collection and identification of the specimens. I am pleased to acknowledge my debt to Dr. R. Bellairs for the use of electron microscope facilities, and to Mrs. J. Hamilton and Mr. R. Moss for skillful technical assistance.     

Fine structure of the stretch receptor in the bursa copulatrix of the butterfly,Pieris rapae crucivora

Summary A pair of multipolar stretch-receptive neurons were found in the bursa copulatrix of the female cabbage white butterfly, Pieris rapae crucivora. The cell body of each neuron, about 10 m in diameter, lies on the edge of the muscular region in the antero-lateral wall of the corpus bursae. No special accessory structure, such as a receptor muscle, is associated with the neuron. The several dendrites extend radially into the muscle layer. The dendrites are ensheathed except for their terminal tips, and, on their course, they anchor repeatedly on the epithelial cells or the muscle fibers in such a manner that their basement membranes fuse together. While the ensheathed dendrite is usually 0.1–0.2 m in diameter, it often forms 1–2 m varicosities especially at anchor sites, so that it looks like a varicose, or beaded, chain. The varicosities contain a number of mitochondria, but only microtubules are found in the fine interconnecting parts of the dendrite. The naked dendritic tips terminate in the basement membrane of the epithelial cell. The varicosities, as well as naked tips, seem to be important for stimulus transduction in the sensory cell of this type.     

Architecture of the cell wall of a green alga,Oocystis apiculata

Summary The cell wall of a green alga,Oocystis apiculata, was visualized by electron microscopy after preparation of samples by rapid-freezing and deep-etching techniques. The extracellular spaces clearly showed a random network of dense fibrils of approximately 6.4 nm in diameter. The cell wall was composed of three distinct layers: an outer layer with a smooth appearance and many protuberances on its outermost surface; a middle layer with criss-crossed cellulose microfibrils of approximately 15–17 nm in diameter; and an inner layer with many pores between anastomosing fibers of 8–10 nm in diameter. Both the outer and the inner layer seemed to be composed of amorphous material. Cross-bridges of approximately 4.2 nm in diameter were visualized between adjacent microfibrils by the same techniques. The cross-bridges were easily distinguished from cellulose microfibrils by differences in their dimensions.     

The cell wall of the chlamydomonad flagellate,Gloeomonas kupfferi (Volvocales,Chlorophyta)

Summary The large unicellular flagellate,Gloeomonas kupfferi, has recently been used as an important tool in chlamydomonad cell biology research, especially in studies dealing with the structure and function of the endomembrane system. However, little is known about the main secretory product, the cell wall. This study presents structural, chemical and immunological information about this wall. This 850–900 nm thick matrix is highly elaborate and consists of three distinct layers: an inner stratum (325 nm thick) consisting of tightly interwoven fibers, a medial crystalline layer consisting of 22–23 nm subunits and an outer wall layer (500 nm thick) of outwardlyradiating fibrils. Rapid freeze-deep etch analysis reveals that the 35–40 nm fibers of the outer layer form a quasi-lattice of 160 nm subunits. The outer wall can be removed from whole pellets using the chelator, CDTA. The medial wall complex can be solubilized by perchlorate. SDS-gel electrophoresis reveals that the perchlorate soluble-material consists of five high molecular weight glycoproteins and five major low molecular weight glycoproteins. The electrophoretic profile is roughly similar to that ofChlamydomonas reinhardtii. Antibodies were successfully raised against the outer wall component and were shown to label the outer wall layer.     

Cells that emerge from embryonic explants produce fibers of type IV collagen

Double immunofluorescence staining experiments designed to examine the synthesis and deposition of collagen types I and IV in cultured explants of embryonic mouse lung revealed the presence of connective tissue-like fibers that were immunoreactive with anti-type IV collagen antibodies. This observation is contrary to the widely accepted belief that type IV collagen is found only in sheet-like arrangements beneath epithelia or as a sheath-like layer enveloping bundles of nerve or muscle cells. The extracellular matrix produced by cells that migrate from embryonic mouse lung rudiments in vitro was examined by double indirect immunofluorescence microscopy. Affinity-purified monospecific polyclonal antibodies were used to examine cells after growth on glass or native collagen substrata. The data show that embryonic mesenchymal cells can produce organized fibers of type IV collagen that are not contained within a basement membrane, and that embryonic epithelial cells deposit fibers and strands of type IV collagen beneath their basal surface when grown on glass; however, when grown on a rat tail collagen substratum the epithelial cells produce a fine meshwork. To our knowledge this work represents the first report that type IV collagen can be organized by cells into a fibrous extracellular matrix that is not a basement membrane.     

Demonstration of microfibrils in Bruch's membrane of the eye

The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruch's membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruch's membrane may depend on both the elastin and microfibrillar components.     

Formation of the basement membrane during regeneration of the corneal epithelium

Summary The epithelium regenerating after a surface lesion of the cornea forms a new basement membrane. This process begins 6–8 days after the lesion when the wound is completely covered, and epithelial cells have ceased to migrate. Only that part of the epithelial cells facing the stroma is involved. First, tufts of fine filamentous structures (about 30 Å thick) appear on the internal side of the plasma membrane, and apparently penetrate it reaching the extracellular space where they form a loose network. This then differentiates into two discrete layers, a very thin discontinuous one, restricted to areas with tufts, very close to the plasma membrane (juxtamembranous layer), and a thick continuous layer, the basement membrane proper, parallel to and much further away from the plasma membrane. The basement membrane appears to be the product of cytoplasmic secretion by epithelial cells, and there is no evidence for connective tissue cells taking part in this process.Supported by the Deutsche Forschungsgemeinschaft.     

Three dimensional observation of the nerve fibers along the cerebral blood vessels

Summary Three dimensional observation of the nerve fibers along the cerebral blood vessels was investigated by scanning electron microscopy. Electron probe X-ray microanalysis was also performed in the cerebral blood vessels treated with peroxidase-antiperoxidase immunohistochemistry intensified by nickel ammonium sulfate.Nerve fibers (2–8 m in diameter) formed a plexus on the outer surface of the adventitia. After branching, the nerve fibers penetrated the blood vessel adventitia. Substance P-immunoreactive nerve fibers showed a meshwork pattern in the outer layer of the adventitia, and vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers revealed a spiral running pattern in the inner layer of the adventitia. Taken together with previous studies, these findings suggest that substance P nerve fibers in the cerebral arteries may not be related to arterial dilatation or constriction, but VIP nerve fibers may be vasodilative.     

Chondroitin sulfate proteoglycan is a constituent of the basement membrane in the rat embryo parietal yolk sac

Summary In addition to containing Type IV collagen, laminin and entactin, basement membranes contain small amounts of proteoglycans substituted primarily with heparan sulfate chains. We have previously shown, however, that parietal yolk sacs in organ culture synthesize predominantly chondroitin sulfate proteoglycan. In the present study, we have used histochemical and immunohistochemical techniques coupled with chondroitinase ABC digestion to provide evidence for the presence of chondroitin sulfate proteoglycan in the basement membrane (Reichert's membrane) of the 14.5-day rat embryo parietal yolk sac. The results revealed numerous cuprolinic blue-positive filaments and granules, 20–30 nm in greater length or diameter, dispersed throughout the thickness of the basement membrane. Both structures were removed by preincubating freshly isolated parietal yolk sacs with chondroitinase ABC. A similar labeling pattern was also obtained with immunoelectron microscopy using gold-labeled monoclonal anti-bodies directed against the three major isomers of protein-bound chondroitin sulfate. In contrast, coarser cuprolinic blue granules, 40–100 nm in diameter, were neither sensitive to chondroitinase ABC digestion nor labeled by the monoclonal antibodies. These results thus indicate that Reichert's membrane contains chondroitin sulfate proteoglycan in addition to heparan sulfate proteoglycan.     

Laminar ultrastructure of the dorsal cochlear nucleus in rats

The laminar ultrastructure of the dorsal cochlear nucleus was studied in ultrathin wide frontal sections, passing through all layers of the nucleus, placed on blinds with a Formvar film. The ultrastructural characteristics of cells corresponding to the cell types distinguished previously by light microscopy are described. The laminar distribution of the axon terminals was studied. In the surface and middle layers of the neuropil, by contrast with the deep layer, large branching terminals measuring 6–8 µ with spherical synaptic vesicles 40–50 nm in diameter, small terminals measuring 1–3 µ with spherical synaptic vesicles 45–60 nm in diameter, and thin unmyelinated fibers running perpendicularly to the plane of the section were predominant. On transition from the middle to the deep layer there was a corresponding increase in the number of myelinated axons and large oval-shaped terminals measuring 4–6 µ, with central mitochondria and neurofilaments, and also with spherical synaptic vesicles 50–60 nm in diameter, in the neuropil. In the surface and middle layers granular cells also were more numerous than in the deep layer. The functional significance of terminals of each type is discussed.N. A. Semashko Moscow Medical Stomatologic Institute. Translated from Neirofiziologiya, Vol. 10, No. 4, pp. 368–374, July–August, 1978.     

The structure of Gloeobacter violaceus and its phycobilisomes

The fine structure of the atypical cyanobacterium Gloeobacter violaceus has been studied on frozen-etched replicas and compared to that of a typical unicellular strain: Synechocystis 6701. The complementary fracture faces of G. violaceus cytoplasmic membrane contain particles less numerous and more heterogenous in size than either the cytoplasmic membrane or the thylakoid membranes of Synechocystis. The most frequently observed particles of the exoplasmic fracture (EF) face of the G. violaceus cytoplasmic membrane are 11 nm in diameter and occasionally form short alignments. This particle class is similar in appearance to the numerous, aligned EF particles of Synechocystis thylakoid membranes. In replicas of cross-fractured G. violaceus, a layer 50–70 nm thick, composed of rod-like elements, underlies the inner surface of the cytoplasmic membrane. The rods, 12–14 nm in diameter, are oriented perpendicularly to the cytoplasmic membrane and show a 6 nm repeat along their length.Isolated phycobilisomes of G. violaceus appear, after fixation and negative staining, as bundles of 6 parallel rodshaped elements connected to an ill-defined basal structure. The bundles are 40–45 nm wide and 75–90 nm long. The rods are 10–12 nm in width; their length varies between 50 and 70 nm. These rods are morphologically similar to those observed at the periphery of hemidiscoidal phycobilisomes of other cyanobacteria, with a strong repeat at 6 nm intervals and a weaker one at 3 nm intervals along their length.The calculated molar ratio of phycobiliproteins in isolated G. violaceus phycobilisomes corresponds to 1:3.9:2.9 for allophycocyanin, phycocyanin and phycoerythrin respectively. When excited at 500 nm, isolated phycobilisomes exhibit a major fluorescence emission band centered at 663 nm.Abbreviations PBS phycobilisome(s) - PBP phycobiliprotein(s) - AP allophycocyanin - PC phycocyanin - PE phycoerythrin - K–PO₄ buffer KH₂PO₄ titrated with KOH to a given pH     

Lipid detection by malachite green-aldehyde in the dental basement membrane in the rat incisor

Summary Developing rat incisors were treated with malachite green-aldehyde fixative solution (MGA), which retains and stains lipids. We observed positive staining occurring as dots in the basement membrane. Most of these dots (2–3.5 nm in diameter) were grouped in the lamina densa but some were also present in the lamina lucida and the lamina fibroreticularis. These data provide evidence for the existence of lipids in the dental basement membrane and suggest that they are distributed together with the various groups of proteins so far detected.     

Atomic-force microscopy imaging of plasma membranes purified from spinach leaves

Summary Plasma membranes purified from spinach leaves by aqueous two-phase partitioning were examined by atomic-force microscopy (AFM) in phosphate buffer, and details on their structure were reported at nanometric scale. Examination of the fresh membrane preparation deposited on mica revealed a complex organization of the surface. It appeared composed of a first layer of material, about 8 nm in thickness, that practically covered all the mica surface and on which stand structures highly heterogeneous in shape and size. High-resolution imaging showed that the surface of the first layer appeared relatively smooth in some regions, whereas different characteristic features were observed in other regions. They consisted of globular-to-elliptical protruding particles of various sizes, from 4–5 nm x-y size for the smallest to 40–70 nm for the largest, and of channel-like structures 25–30 nm in diameter with a central hole. Macromolecular assemblies of protruding particles of various shapes were imaged. Addition of the proteolytic enzyme pronase led to a net roughness decrease in regions covered with particles, indicating their proteinaceous nature. The results open fascinating perspectives in the investigation of membrane surfaces in plant cells with the possibility to get structural information at the nanometric range.Abbreviations AFM atomic-force microscopy - EM electron microscopy - TMAFM tapping-mode atomic-force microscopy     

Accelerating repaired basement membrane after bevacizumab treatment on alkali-burned mouse cornea

To understand the corneal regeneration induced by bevacizumab, we investigated the structure changes of stroma and basement membrane regeneration. A Stick soaked in 0.5 N NaOH onto the mouse cornea and 2.5 mg/ml of bevacizumab was delivered into an alkali-burned cornea (2 μl) by subconjunctival injections at 1 hour and 4 days after injury. At 7 days after injury, basement membrane regeneration was observed by transmission electron microscope. Uneven and thin epithelial basement membrane, light density of hemidesmosomes, and edematous collagen fibril bundles are shown in the alkali-burned cornea. Injured epithelial basement membrane and hemidesmosomes and edematous collagen fibril bundles resulting from alkali-burned mouse cornea was repaired by bevacizumab treatment. This study demonstrates that bevacizumab can play an important role in wound healing in the cornea by accelerating the reestablishment of basement membrane integrity that leads to barriers for scar formation. [BMB Reports 2013; 46(4): 195-200]     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Zur positiven Reaktivität der Retikulumfasern des Lymphknotens bei der Perjodat-Leukofuchsinreaktion (PAS-Reaktion)

Zusammenfassung Die positive Perjodatreaktion der Retikulumfasern des Lymphknotens findet sich nur an der Grenzschicht der Retikulumfasern zu den Retikulumzellen. Dies entspricht weitgehend der Lokalisation der Argyrophilie. Diese perjodatpositive Oberschicht der Retikulumfasern geht an der Kapsel und an den Trabekeln direkt in die perjodatpositive Basalmembran des Sinusendothels über, so daß die oberflächliche Schicht der Retikulumfasern ebenso als Basalmembran gelten kann.

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Summary The periodic acid-Schiff reaction of the reticular fibers is examined in the cervical lymph node of a dog. The reticular fibers possess PAS reactivity especially at their surface layer bounding the reticular cells. This greatly resembles the site of their argyrophilic staining. The surface layer of the reticular fibers runs directly into the PAS positive basement membrane of the endothelial cells of lymphatic sinuses at the inner surface of capsule and trabeculae. Accordingly the PAS reactive layer of the reticular fibers can be regarded — as far as light microscopy is concerned — as a basement membrane.

     

Possible sites of ultrafiltration in Tubifex tubifex Müller (Annelida,Oligochaeta)

Summary The endothelia of Tubifex tubifex Müller consist of myoendothelial cells, chloragocytes, or podocytes. The latter seem to occur only as windows on the ventral vessel which has an endothelium of myoendothelial cells elsewhere. The podocytes are large cells, with several processes on the inner side which ramify into several pedicels. These are aligned upon the outside of the basement membrane which lines the inside of the endothelium. The gaps between adjacent pedicels are about 40 nm wide. In capillaries fenestrated endothelia occur with irregular spacings measuring up to 0.4–1 m. A diaphragm in podocytes or capillary fenestrations do not seem to exist. The basement membrane is the only continuous layer lining the blood vessels and capillaries of Tubifex with a rather uniform diameter in the range of 50 nm. It is the only permeability barrier between blood and coelomic fluid.