Jiro Usukura

The cytoskeleton in myelinated axons: a freeze-etch replica study.

The organization of the cytoskeleton in myelinated axons of the rat has been analyzed without chemical fixation in replicas of deep-etched materials after rapid freezing. Freeze-etch replicas of trigeminal nerves provided three-dimensional views of the well-developed cytoskeleton inside axons. In these preparations, the axonal cytoskeleton was seen to be composed of longitudinally-oriented microtubules and neurofilaments which were interconnected by slender strands. Such strands also connected membranous organelles with microtubules and neurofilaments. After Triton X-100 extraction, the neurofilament-associated interconnecting strands (cross-linking filaments) persisted, indicating that they are not artifactual products of soluble protein condensation during freeze-etching. In non-extracted axons many granular structures were closely associated with cytoskeletal components. These granular elements were not seen after Triton treatment. These findings, together with fluorographic analyses, suggest that the granular structures may represent. These findings, together with fluorographic analyses, suggest that the granular structures may represent slowly transported soluble proteins" in axoplasm. This freeze-etch replica study, without any chemical fixation, substantiates the reality of the axonal cytoskeleton which is directly involved in the axonal transport. Furthermore, using this approach ultrastructural evidence was obtained of the close association of membranous structures with the cytoskeleton.

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.

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.

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.