Eichi Yamada

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

SummaryThe 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.

Isolation and characterization of vitamin-A-storing lung cells

Vitamin A-storing cells have been shown to be distributed among various organs and tissues, including the lungs. In order to investigate this unique type of cell, the in vitro isolation has been carried out from rat lungs. Lungs were perfused with EGTA and collagenase solution in situ, and were digested with trypsin-collagenase solution at 60-min intervals for 2 h. Then, the cell suspensions obtained were incubated at 37 degrees C in F-10 medium supplemented with 10% fetal bovine serum (FBS) for 72 h. Non-adherent cells were then removed by vigorous washing with medium, and the resultant cell monolayer was harvested with trypsin to remove the contaminating macrophages. These cell fractions were shown to contain more than 96% of vitamin A-storing cells, judged by electron and fluorescence microscopic examinations. The cells grown in vitro retained well the overall morphology characteristic of the vitamin A-storing cells found in lung tissues. The isolated cells grew well in vitro and the growth was inhibited by D-valine or cis-hydroxyproline. The progeny of the cells still contained vitamin A lipid droplets after several transfer generations. Characteristic networks of fibronectin were also demonstrated around the cells. These results have shown that vitamin A-storing cells in the lung was successfully isolated from rat lungs and the cells possessed fibroblast-like characters storing vitamin A in small lipid droplets.

Observations on the cytolemma of the olfactory receptor cell in the newt

SummaryThe fine structure of the cytolemma of olfactory receptor cells in the newt was studied by the freeze-fracture replica method. Two kinds of receptor cells were recognized, namely ciliated cells (ciliary type) and non-ciliated cells (microvilli type). The cytolemma of olfactory knobs as well as their processes from both types of receptor cells showed an abundance of large membrane particles 80∼110Å in diameter. The large square aggregation of membrane particles, 0.1×0.1 μm to 0.2×0.3 μm in size, consisting of 50∼100 cuboidal subunits, were found in the cytolemma of the dendrite. A structural model of aggregation is presented. The soma of the receptor cell revealed large pitted membrane particles about 140Å in diameter. These particles are possibly the morphologic counterpart to ionophores which have been proposed by electrophysiological studies.

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R1mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all‐trans‐retinal converted R1mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light‐dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R1mW. Electron micrographs of freeze‐fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure.

Ultrastructure of purple membrane and cell wall of Halobacterium halobium

The purple membrane and other membrane systems of Halobacterium halobium were studied by the freeze-replica method, employing rotary shadowing, and by thin sectioning combined with freeze substitution. The trimeric structure of bacteriorhodopsin in the purple membrane was observed directly under the electron microscope. The existence of distinct pits on E face of the purple membrane was confirmed. This suggests that bacteriorhodopsin is a transmembranous protein. A new type of purple membrane arrangement yielding optically triclinic diffraction patterns with center-to-center spacing of 8 nm was distinguished from the usual type, which produces hexagonal diffraction patterns with spacings of 6.2 nm. The relationship between two arrangements was discussed with reference to the formation of purple membrane. Freeze-deep etching revealed detailed structures of the cell wall in three dimensions. The true surfaces of purple membrane and red membrane were also observed.

High resolution scanning electron microscopy of frog sartorius muscle

A field emission-type scanning electron microscope (SEM) was used to study the three-dimensional ultrastructure of frog sartorius muscles. Various preparative procedures were tested to seeks better specimen preparation for high resolution SEM observation. Procedures should be chosen depending on the information desired. The cell surface and intracellular organization of muscle fibers were best visualized when the tissues were fixed with tannic acid-OsO4 and torn after critical point drying. The basal lamina appeared as a continuous felt-like layer, onto which fine filamentous materials adhered. The true outer surface of the sarcolemma was not seen, whereas the true inner surface was occasionally exposed and exhibited numerous caveolae, membraneous fragments and fine filaments attached to its surface. In freeze-fractured and dried tissues, the cleaved sarcolemma showed numerous apertures of caveolae and T-system tubules. Inside the cell, the myofibrils showed a typical branding pattern of the sarcomere. Thick myofilaments were regularly beaded except for the pseudo-H-zone. Around the myofibrils the sarcoplasmic reticulum and T-system were also clearly observed. The results are discussed with special reference to the usefulness and limitation of the high resolution SEM in studying the ultrastructure of cells and tissues.

Dense tissue and special stains.

Publisher Summary This chapter presents data concerning the selective staining method combined with high-voltage electron microscopy to analyze the three-dimensional structure within dense tissue. The Golgi impregnation technique is an excellent method of revealing the three-dimensional morphology of neurons and glia cells and it has been used extensively by light microscopists to explore the organization of the central nervous system. In contrast to light microscopy, electron microscopy of Golgi impregnated tissue has the advantage of revealing both impregnated and impregnated cells at the same time in greater detail. It is demonstrated that the whole Golgi apparatus could be stained by this double impregnation technique, using uranyl acetate followed by lead and copper. The principle of selective staining for the T-system is to fill or stain the extracellular space with molecular tracers. Selective stains or tracers successfully used are horseradish peroxidase, lanthanum, ruthenium red, and diaminobenzidine. It is found that selective staining with 3,3′-diaminobenzidine tetrahydrochloride–ferrocyanide for smooth endoplasmic reticulum was used to visualize its distribution within 0.5- to 1-pm-thick sections of mouse and frog peripheral nerves in high-voltage electron microscopy.

SOME OBSERVATIONS ON THE MORPHOGENESIS OF LATTICE STRUCTURE IN THE PURPLE MEMBRANE

Abstract— The formation process of purple membrane was studied morphologically by freeze‐fracture and etching methods. The bacteria, H. halobium R1, cultured in the medium containing 2mM nicotine, did not show the hexagonal structure of purple membrane, but a particle free area was frequently observed in the plasma membrane. However, within 24h after returning to the normal medium, hexagonal and triclinic lattices with spacings of 6 to 8nm occurred as small patches on the P face of the plasma membrane. Furthermore, it was revealed that the retinal was not required for the formation of the two‐dimensional hexagonal lattice structure of purple membrane, since the mutant bacteria, H. halobium R1mW, lacking the ability of retinal synthesis, displayed a similar lattice. The other mutant, H. halobium R1mR, lacking the ability of bacterio‐opsin synthesis, never showed any highly ordered arrangement. This evidence suggests that membrane proteins other than bacterio‐opsin or bacteriorhodopsin do not form highly ordered lattices in the membrane.