scholarly journals A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

1969 ◽  
Vol 43 (1) ◽  
pp. 16-31 ◽  
Author(s):  
C. J. Arntzen ◽  
R. A. Dilley ◽  
F. L. Crane
Keyword(s):  
Freeze Fracture ◽  
Internal Surface ◽  
Coupling Factor ◽  
Negative Staining ◽  
Fracture Plane ◽  
Chloroplast Membranes ◽  
Chloroplast Membrane ◽  
Membrane Surfaces ◽  
Large Particles ◽  
Freeze Etching

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.

scholarly journals Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro.

1976 ◽  
Vol 71 (1) ◽  
pp. 136-158 ◽  
Author(s):  
L A Staehelin
Keyword(s):  
Plasma Membranes ◽  
Structural Changes ◽  
Freeze Fracture ◽  
Chloroplast Membranes ◽  
Ps Ii ◽  
Membrane Particles ◽  
Intramembranous Particles ◽  
Membrane Surfaces ◽  
Ps Ii Activity

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.

scholarly journals Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

1977 ◽  
Vol 75 (3) ◽  
pp. 619-634 ◽  
Author(s):  
N Deguchi ◽  
PL Jorgensen ◽  
AB Maunsbach
Keyword(s):  
Membrane Surface ◽  
Freeze Fracture ◽  
Negative Staining ◽  
Intramembranous Particle ◽  
Freeze Fracturing ◽  
Intramembranous Particles ◽  
Membrane Surfaces ◽  
Thin Sectioning ◽  
Surface Particle ◽  
Protein Components

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.

Path of Fracture Planes Along Membrane Surfaces in Frozen-Etched Tissue

1969 ◽  
Vol 27 ◽  
pp. 334-335
Author(s):  
L. V. Leak
Keyword(s):  
Fracture Process ◽  
Membrane Surface ◽  
Fracture Plane ◽  
Additional Information ◽  
Membrane Surfaces ◽  
Fracture Planes ◽  
En Face ◽  
Freeze Etching ◽  
Conventional Electron Microscopy ◽  
Cellular Organelles

The course of the fracture plane through frozen tissue may very often follow surfaces of membranes for long distances before cross fractures are made, exposing the interior of cells and cellular organelles. From studies on frozen-etched cellular membranes Moor and Muhlethaler suggested that fractures occur along external surfaces of membranes, while Branton proposed that the fracture process splits the membrane in half, revealing either of the two internal membrane faces. Our earlier studies suggested that most of the en face views of membranes represented fractures along the membrane surface. The present study combines the technique of freeze-etching with those of conventional electron microscopy in an effort to provide additional information on the precise nature and path of the fracture plane along membrane surfaces.

High-resolution, extended views of membrane surfaces revealed by fracture-flip

1989 ◽  
Vol 47 ◽  
pp. 738-739
Author(s):  
Pedro Pinto da Silva
Keyword(s):  
High Resolution ◽  
Colloidal Gold ◽  
Cell Membranes ◽  
Freeze Fracture ◽  
Surface Structures ◽  
New Method ◽  
Deep Etching ◽  
Membrane Surfaces ◽  
Routine Identification ◽  
Freeze Etching

I will describe a new method — fracture-flip — that uses commercially available equipment to produce extended views of cell and membrane surfaces. The resolution of this new method permits the routine identification of surface structures down to 5 nm diameter. Moreover, in contrast to freeze-etching/deep-etching, extended views are easily obtained.Conceptally, fracture-flip derives from label-fracture, another method developed in my laboratory. With label-fracture we showed that, after freeze-fracture, the exoplasmic (E) halves of cell membranes are stabilized by, and remain attached to, their platinum/carbon replicas. This allows the observation of co-incident views of the Pt/C replica of the E face, and of the distribution of colloidal gold labeled receptors or antigens. This is the sequence of steps in fracture-flip:

Membranes: Freeze-etching techniques applied to biological membranes

1974 ◽  
Vol 268 (891) ◽  
pp. 5-14 ◽  
Keyword(s):  
Plastic Deformation ◽  
Membrane Protein ◽  
Freeze Fracture ◽  
Biological Membranes ◽  
The Other ◽  
Fracture Plane ◽  
Small Scale ◽  
Replica Technique ◽  
Freeze Etching ◽  
Protein Particles

Basic freeze-etching methods are described. When biological membranes are freeze-fractured the fracture plane is smooth, but interrupted to a greater or lesser extent by numbers of small (8.5 nm) particles. The evidence that the fracture occurs in the interior of the membrane and that the particles represent proteins within the membrane is reviewed. A problem of interpretation of freeze-fracture replicas is that the two 'complementary’ faces, produced by the fracture of a single membrane, do not match exactly. In particular, particles on one face are often not matched by corresponding depressions on the other. Work in the author’s laboratory using the complementary replica technique is described. One conclusion from this work is that plastic deformation of the intra-membrane protein particles may occur, and that this may be responsible for the lack of small-scale complementarity.

Three Dimensional Images of Cardiovascular Elements With Freeze Etching

1967 ◽  
Vol 25 ◽  
pp. 118-119
Author(s):  
G. Oscar Kreutziger ◽  
James H. McAlear
Keyword(s):  
Cross Section ◽  
Transverse Section ◽  
Three Dimensional ◽  
Fracture Plane ◽  
Specimen Preparation ◽  
Mouse Heart ◽  
Endothelial Layer ◽  
New Device ◽  
Membrane Surfaces ◽  
Freeze Etching

Mouse heart muscle was prepared by freeze etching. This involves a new device ( McAlear and Kreutziger, this volume) and technique for specimen preparation. These have permitted the production of replicas as in the figure on the reverse side which have a three dimensional aspect similar to an artist's presentation of tissue sub structure. Muscle filaments can be seen in longitudinal and transverse section almost as a conventional section yet the capillary appears to arise out of the muscle. The sutures of the enclosing endothelial cells appear to wander over the upper and lateral surface of the capillary to the cross fracture plane where they and the lumen are seen in cross section. In the foreground the endothelium continues as a replica of the inner facing membrane of the outer endothelial layer. All possible membrane surfaces are seen in various replicas.

Particles Within Membranes: A Freeze-Etch View

1971 ◽  
Vol 9 (2) ◽  
pp. 435-441
Author(s):  
N. E. FLOWER
Keyword(s):  
Internal Structure ◽  
Membrane Surface ◽  
Small Particles ◽  
Membrane Surfaces ◽  
Number Distribution ◽  
Large Particles ◽  
Freeze Etching

Particles are commonly present on the membrane faces revealed by freeze-etching. The number, distribution and size of these particles vary considerably both between different membranes and, in many cases, between the 2 fracture faces found in individual membranes. Many of the larger particles appear to be too large to fit totally within smooth-surfaced membranes, so raising the question of how particles, especially the larger ones, are contained within membranes. This could be accomplished by a local reorganization of the membrane's internal structure such that small particles would be totally enclosed within smooth-surfaced membranes, while large particles would protrude from the membrane surface. Alternatively, all sizes of particles could be contained within membranes by a bulging of the 2 component lamellae such that protuberances, having a larger diameter than the underlying particles, would arise on the membrane surface. Evidence is presented to show that in the case of specialized particles, which are located in rows around the base of flagella in the mollusc Cominella maculosa, protuberances are present on the membrane surface. However, it is possible that particles could be accommodated within membranes from other tissues by a different mechanism, and only further work will decide whether or not the present findings can be applied to these other membrane surfaces.

scholarly journals IDENTIFICATION OF CHLOROPLAST COUPLING FACTOR BY FREEZE-ETCHING AND NEGATIVE-STAINING TECHNIQUES

1974 ◽  
Vol 63 (1) ◽  
pp. 24-34 ◽  
Author(s):  
Melvin P. Garber ◽  
Peter L. Steponkus
Keyword(s):  
Outer Surface ◽  
Complete Removal ◽  
Coupling Factor ◽  
Negative Staining ◽  
Chloroplast Coupling Factor ◽  
Freeze Etching ◽  
Staining Techniques ◽  
Spinach Leaves

Identification of chloroplast coupling factor particles, by the freeze-etching and negative-staining techniques, was made utilizing chloroplast thylakoids isolated from spinach leaves. Complete removal of particles, comparable in diameter to purified coupling factor particles, from the outer surface of freeze-etched thylakoids was achieved by treatment with 0.8% silicotungstate. Reappearance of particles, comparable in diameter to purified coupling factor particles, on the outer surface of freeze-etched thylakoids was demonstrated by combining silicotungstate-treated thylakoids with purified chloroplast coupling factor. Negative-staining results were in agreement with the freeze-etch data. The results demonstrate that the chloroplast coupling factor particles are exposed on the outer surface.

Freeze-Fracture Replication of Frozen Outer Segments of Rat Retinal Rods

1969 ◽  
Vol 27 ◽  
pp. 392-393
Author(s):  
Thomas S. Leeson ◽  
C. Roland Leeson
Keyword(s):  
Electron Microscopy ◽  
Cross Section ◽  
Outer Segment ◽  
Freeze Fracture ◽  
X Ray Diffraction ◽  
X Ray ◽  
Outer Segments ◽  
Retinal Rods ◽  
Temperature Electron ◽  
Freeze Etching

Numerous previous studies of outer segments of retinal receptors have demonstrated a complex internal structure of a series of transversely orientated membranous lamellae, discs, or saccules. In cones, these lamellae probably are invaginations of the covering plasma membrane. In rods, however, they appear to be isolated and separate discs although some authors report interconnections and some continuities with the surface near the base of the outer segment, i.e. toward the inner segment. In some species, variations have been reported, such as longitudinally orientated lamellae and lamellar whorls. In cross section, the discs or saccules show one or more incisures. The saccules probably contain photolabile pigment, with resulting potentials after dipole formation during bleaching of pigment. Continuity between the lamina of rod saccules and extracellular space may be necessary for the detection of dipoles, although such continuity usually is not found by electron microscopy. Particles on the membranes have been found by low angle X-ray diffraction, by low temperature electron microscopy and by freeze-etching techniques.

Warm and freeze-fracture of cotton seed radicles

1987 ◽  
Vol 45 ◽  
pp. 984-985
Author(s):  
E. L. Vigil ◽  
E. F. Erbe
Keyword(s):  
Thin Film ◽  
Water Vapor ◽  
Fracture Surface ◽  
Membrane Structure ◽  
Room Temperature ◽  
Freeze Fracture ◽  
Longitudinal Axis ◽  
Fracture Plane ◽  
Cotton Seeds ◽  
Condensed Water

In cotton seeds the radicle has 12% moisture content which makes it possible to prepare freeze-fracture replicas without fixation or cryoprotection. For this study we have examined replicas of unfixed radicle tissue fractured at room temperature to obtain data on organelle and membrane structure.Excised radicles from seeds of cotton (Gossyplum hirsutum L. M-8) were fractured at room temperature along the longitudinal axis. The fracture was initiated by spliting the basal end of the excised radicle with a razor. This procedure produced a fracture through the tissue along an unknown fracture plane. The warm fractured radicle halves were placed on a thin film of 100% glycerol on a flat brass cap with fracture surface up. The cap was rapidly plunged into liquid nitrogen and transferred to a freeze- etch unit. The sample was etched for 3 min at -95°C to remove any condensed water vapor and then cooled to -150°C for platinum/carbon evaporation.

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