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Crystalline patterns of myelin lipids visualized by freeze fracture.

Freeze fracture of rat optic nerve reveals smooth, particle-free regions on the lammellar fracture faces of myelin when prepared by standard procedures. When the fixed, glycerin-impregnated tissue is incubated at 6 degrees C for two or more days, crystalline patterns indicative of a phase transition can be seen in the particle-free regions. The crystalline patterns can be destroyed by subsequent incubation at 37 degrees C and are not seen when the initial incubation is at room temperature or 37 degrees C. Butylated hydroxytoluene has no effect on the formation of the crystalline patterns. The time course of the formation of the crystalline patterns suggest that the rate-limiting step in the process is not the phase transition itself. We propose that the lipids associated with the particles in vivo are involved in the formation of the crystalline patterns.

Animals

Correlated x-ray diffraction and freeze-fracture studies on membrane model systems. Perturbations induced by freeze-fracture preparative procedures.

Lipid-water and protein-lipid-water phases have been examined by X-ray methods before and after freezing. Frozen samples have been subsequently fractured and replicated, thus permitting an evaluation of the nature of structural perturbations in samples examined by freeze-fracture electron microscopy. Important results are summarized: (1) Freezing low water content (approx. less than 25%) phases causes perturbations in the packing of hydrocarbon chains. The results suggest that freezing liquied paraffin chains produces a condensed "glass-like" packing. (2) Additional perturbations occur in high water content samples. After freezing, much smaller lamellar repeat distances, intense ice reflections, and extensive perturbation of fracture faces are consistant with the expulsion of water from between lamellae. Presence of glycerol generally relieves these perturbations but in some cases introduces additional lattice disorder. (3) Surprisingly, cooling by a stream of cold N2 gas (-140 degrees C) produces qualitatively the same results as rapid cooling in liquid Freon-22 (-160 degrees C). (4) Complex perturbations occur in phases containing integral membrane proteins. Interesting results have been obtained with cytochrome b5-lecithin lamellar associations which display both smooth and rough fracture faces without clearly defined particles.

Binding Sites

Freeze-fracturing and freeze-etching of cardiac myosin filaments.

Myofilament structure was studied in freeze-etch replicas of unfixed, glycerinated beef cardiac muscle. The information which is revealed depends upon the direction of metal shadowing in relation to the filament axis. Shadows oblique to this axis reveal that the outer surface of a longitudinal half of a thick filament comprises three, sometimes four, rows of myosin molecules. These molecules are generally assembled in a braided manner with both left and right-handed helical components. Occasionally a more parallel to the myofilament axis reveal cross-bridges linking thick and thin filaments. These bridges are readily detectable by optical diffraction techniques, giving an axial bridge spacing of approximately 40 nm. In unetched preparations cross bridges appear as vertical rows of beads. In all replicas the effects of plastic deformation of proteins must be considered.

Animals

Evaluation of membrane structure facts and artefacts produced during freeze-fracturing.

The freeze-fracture technique is now widely used in the study of membranes, but it should be stressed that it shows internal hydrophobic planes of membranes, prepared under physical conditions far removed from those prevailing in vivo. Hence there is considerable potential for artefact. Work on the membrane lipid component, the intramembrane particles, and their aggregation under certain conditions, is reviewed in the context of fact versus artefact. Particular attention is paid to the results of complementary replica experiments, performed in the author's laboratory and elsewhere, which indicate that lipid collapse and protein particle deformation contribute to the appearance of the membrane fracture face seen in the final replica. A model, showing the effects of freeze-fracturing on membranes, is presented.

Freeze Fracturing

Membrane movements and fluidity during rotational motility of a termite flagellate. A freeze-fracture study.

Freeze-fracture electron microscopy was used to examine the structure of a region of plasma membrane that undergoes continual, unidirectional shear. Membrane shear arises from the continual clockwise rotation of one part (head) of a termite flagellate relative to the rest of the cell. Freeze-fracture replicas show that the lipid bilayer is continuous across the shear zone. Thus, the relative movements of adjacent membrane regions are visible evidence of membrane fluidity. The distribution and density of intramembrane particles within the membrane of the shear zone is not different from that in other regions of the cell membrane. Also, an additional membrane shear zone arises when body membrane becomes closely applied to the rotating axostyle as cells change shape in vitro. This suggests that the entire membrane is potentially as fluid as the membrane between head and body but that this fluidity is only expressed at certain locations for geometrical and/or mechanical reasons. Membrane movements may be explained solely by cell shape and proximity to rotating structures, although specific membrane-cytoskeletal connections cannot be ruled out. The membrane of this cell may thus be viewed as a fluid which adheres to the underlying cytoplasm/cytoskeleton and passively follows its movements.

Animals

Inverted gap and other cell junctions in cockroach hemocyte capsules: a thin section and freeze-fracture study.

Freeze-fracture and thin-section studies were done on cockroach hemocytes that had encapsulated implanted pieces of Araldite. Desmosome-like junctions and 'B' type gap junction were described. Freeze-fractured gap junctions displayed fused and clustered, but not hexagonally arrayed intramembranous practicles (approximately 130 A) on the B face and pitted areas on the A face of the plasmalemma. Gap junctions were quite numerous and counts of gap and non-gap particles indicated at least a five-fold particle density increase (4000/mu2) compared with B face particle densities (approximately 800/mu2) from free circulating blood cells where gap junctions had not been formed.

Animals

Cell membrane alterations in the stria vascularis of the guinea pig after ethacrynic acid treatment studied by freeze-fracture.

A freeze-fracture examination of the stria vascularis during the first 2 h after injection of ethacrynic acid was performed. This showed a re-distribution of the particles on the membrane fracture faces of both marginal and intermediate cells. As oedematous spaces developed, particle-poor, vesicle-like structures were found associated with both cell types. The tight junctions at the apices of the marginal cells and around basal cells were unaffected.

Animals

Structural similarity of the membrane envelopes of rhizobial bacteroids and the host plasma membrane as revealed by freeze-fracturing.

The freeze-fracture technique was used to study the host plasma membrane and the membrane envelope of bacteroids in rhizobial root nodules of three host-rhizobium combinations. In all three combinations studied, the membrane envelopes of bacteroids are structurally similar to their host plasma membrane. However, the membrane appears to be reversed, because the number and arrangement of particles in the outer fractured face (face A, concave) and in the inner fractured face (face B, convex) of the host plasma membrane are seen, respectively, in the inner fractured face (face B, convex) and in the outer fractured face (face A, concave) of the membrane envelope of the bacteroids at an early stage. This reversion of the membrane surface is consistent with the hypothesis that the membrane envelopes of bacteroids are derived from the host plasma membrane during endocytotic engulfment.

Cell Membrane

The fenestrated collar of mammalian cardiac sarcoplasmic reticulum: a freeze-fracture study.

Freeze-fracture studies of papillary muscles from cat, rabbit and dog reveal the presence of a fenestrated collar of the sarcoplasmic reticulum (SR) in the region of the M band. This membrane specialization is structurally similar to that observed previously in skeletal muscle. This report includes mammalian cardiac muscle on the list of those muscles containing this SR membrane structure.

Animals

Nuclear membrane changes in herpes simplex virus-infected BHK-21 cells as seen by freeze-fracture.

The freeze-fracture technique, which produced high-resolution replicas of large internal faces of membranes, was used for an ultrastructural study of the nuclei of herpes simplex virus-infected BHK-21 cells and mock-infected controls. Crystalline arrays of viral nucleocapsids were found in the nucleoplasm of infected cells, and numerous nuclear membrane "blebs" and protrusions were observed. The numerous areas of membrane distortions were not found to contain nuclear pores. In addition, specific areas of normal protein intramembranous particles are deleted from certain areas of the nuclear membrane as a result of herpes simplex virus, type 2, infection.

Cell Line

Fenestrations in endothelium of rat liver sinusoids revisited by freeze-fracture.

The freeze-fracture appearance of fenestrations of sinusoidal endothelial cells has been reassessed in perfusion-fixed rat liver. Fenestrations, limited to the attenuated portions of the endothelial cell cytoplasm are usually round or oval in shape, with diameters ranging from 40-600 nm. Within a given fenestrated region, they are disposed in clusters forming sieve plates. In addition, the presence of wide, irregularly shape openings suggest that adjacent fenestrations may coalesce and result in the larger openings. Our data thus confirm the existence of both small and large fenestrae in the endothelial wall, in contradistinction to previous studies showing only regular, medium sized (100 nm) openings.

Animals

Fat droplet formation in rat lactating mammary gland and mammary carcinomas viewed by freeze-fracture.

The freeze-fracture morphology of the endoplasmic reticulum and Golgi apparatus membranes was analyzed in lactating rat mammary glands and in mammary carcinomas induced by 7,12-dimethylbenzanthracene or N-nitrosomethylurea. Membranes in close proximity with fat droplets present a spectrum of transformations, from the normal, wavy, particle-rich appearance of cytoplasmic membranes to rigid, particle-free bilayers. The following sequence of events is proposed for the biogenesis of fat droplets: (1) formation of particle-free areas in the endoplasmic reticulum and Golgi membranes; (2) apposition of the bilayered lipid membranes to the growing fat droplet; and (3) progressive conversion of the membrane lipids (or amphypathic lipid precursors) into triglycerides at the periphery of the fat droplet. Our results suggest that membranes are not only involved in the synthesis and secretion processes, but that some of their components are incorporated in the fat droplets and contribute to the secretory product itself.

9,10-Dimethyl-1,2-benzanthracene

Ultrastructure of Anaplasma marginale after freeze-fracture.

Stained thin sections and freeze-fractured replicas of Anaplasma marginale-infected bovine erythrocytes were examined by electron microscopy. Freeze-fracture replication not only verified basic Anaplasma ultrastructure, but also allowed visualization of structures not previously reported. Because of the partial 3-dimensional views obtained with freeze-fracture replication, a new structure that appears as a protruding tip was discernible. Also, the surface of Anaplasma's limiting membrane was less granular than the fractured surface of host erythrocyte. A corrugated surface with a periodicity of 10.5 nm was seen when the limiting membrane was fractured.

Anaplasma

Structural states of myelin observed by x-ray diffraction and freeze-fracture electron microscopy.

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.

Animals

Preliminary cutaneous pathology observations with the freeze-fracture technique.

The freeze-fracture technique was used to study acantholytic cells of untreated pemphigus patients and the cells of histocytosis X. On the membranes of acanthocytosis cells, the particles are randomly distributed and lack the typical desmosome organization, while tight and gap junctions are still found. The study of the cells of histocytosis X provided a better understanding of the structure of the Langerhans granule. The paracrystalline organization of the inner face of the disc-shaped portion does not seem to involve the membranes limiting the granule, but rather to be confined to a periodically arranged, possibly proteic substance contained between them.

Acanthocytes