scholarly journals Label-fracture: a method for high resolution labeling of cell surfaces.

1984 ◽  
Vol 99 (3) ◽  
pp. 1156-1161 ◽  
Author(s):  
P Pinto da Silva ◽  
F W Kan
Keyword(s):  
High Resolution ◽  
Cell Shape ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Cell Surfaces ◽  
Surface Distribution ◽  
Membrane Surfaces ◽  
A Cell ◽  
Dense Marker

We introduce here a technique, "label-fracture," that allows the observation of the distribution of a cytochemical label on a cell surface. Cell surfaces labeled with an electron-dense marker (colloidal gold) are freeze-fractured and the fracture faces are replicated by plantinum/carbon evaporation. The exoplasmic halves of the membrane, apparently stabilized by the deposition of the Pt/C replica, are washed in distilled water. The new method reveals the surface distribution of the label coincident with the Pt/C replica of the exoplasmic fracture face. Initial applications indicate high resolution (less than or equal to 15 nm) and exceedingly low background. "Label-fracture" provides extensive views of the distribution of the label on membrane surfaces while preserving cell shape and relating to the freeze-fracture morphology of exoplasmic fracture faces. The regionalization of wheat germ agglutinin receptors on the plasma membranes of boar sperm cells is illustrated. The method and the interpretation of its results are straightforward. Label-fracture is appropriate for routine use as a surface labeling technique.

scholarly journals Label-fracture of plasma membranes isolated from measles virus-infected cells.

1993 ◽  
Vol 41 (7) ◽  
pp. 1085-1091 ◽  
Author(s):  
G Rutter ◽  
H Hohenberg
Keyword(s):  
Cell Surface ◽  
Measles Virus ◽  
Plasma Membranes ◽  
Protein A ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Immune Serum ◽  
Outer Cell ◽  
Membrane Surfaces ◽  
Transmission Electron

We present a method that permits correlation of the intramembrane architecture of plasma membrane fracture faces with the distribution of specific molecules at the corresponding cytoplasmic or exoplasmic membrane surfaces. HeLa cells infected with measles virus were used as a model system. Large fragments of the dorsal membrane were isolated after the virus glycoproteins were tagged at the outer cell surface with immune serum and protein A-gold markers. In a second step, different virus polypeptides at the inner cell surface were also identified by a smaller gold label. Thereafter, the isolated plasma membranes were frozen and freeze-fractured. The complementary fracture faces were shadowed with heavy metals and carbon and examined in the transmission electron microscope without cleaning of remaining biological material. Thus, the micromorphology of the replicated fracture faces and the topochemistry of virus components localized at the corresponding leaflets of the plasmalemma could be seen on the same image at high resolution. Of note is that the freeze-fracture morphology of the protoplasmic face is related to the molecular composition of the cytoplasmic surface, as revealed by antibody tagging.

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 Anchorage-dependent surface distribution and partition during freeze-fracture of viral transmembrane glycoproteins.

1990 ◽  
Vol 38 (10) ◽  
pp. 1421-1426 ◽  
Author(s):  
M R Torrisi ◽  
A Pavan ◽  
L V Lotti ◽  
G Migliaccio ◽  
M C Pascale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Viral Proteins ◽  
Low Ph ◽  
Surface Distribution ◽  
Transfected Cells ◽  
Cytosolic Side ◽  
Infected Cells

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.

scholarly journals Cryo-Fracture or Freeze-Fracture, a Method to Expose Internal Tissue Surfaces and Cell Surfaces for Viewing in the Scanning Electron Microscope

2008 ◽  
Vol 16 (4) ◽  
pp. 56-59 ◽  
Author(s):  
Jeannette Taylor
Keyword(s):  
Membrane Surface ◽  
Freeze Fracture ◽  
Internal Tissue ◽  
Cell Surfaces ◽  
Naturally Occurring ◽  
Critical Point Drying ◽  
Tissue Surfaces ◽  
A Cell ◽  
Bowman's Capsule ◽  
Scanning Electron

Cryo-fracture, in conjunction with critical point drying is a method used to prepare biological samples in order to expose, for viewing via scanning electron microscopy, those naturally occurring surfaces which might otherwise remain obscure. For example, the Bowman’s capsule and tubules of a kidney, tiny blood vessels on any organ, inter-cellular spaces in liver or alveoli in the lungs. Also, some surfaces, not normally exposed at all such as the membrane surface of a nuclear envelope, mitochondria or chloroplasts or the cytoplasm of a cell, can be brought to light with this method. Herein is a review of the development of cryo-fracture and how it is currently used at our facility.

A Freeze Fracture Preparation Chamber Attached to the SEM

1977 ◽  
Vol 35 ◽  
pp. 588-589
Author(s):  
J. B. Pawley ◽  
T. L. Hayes
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Chemical Element ◽  
Fracture Process ◽  
Freeze Fracture ◽  
Element Analysis ◽  
Actual Sample ◽  
Membrane Surfaces ◽  
Subsequent Fracture ◽  
Standard Tool

The freeze fracture technique has developed into a standard tool of biological electron microscopy particularly for the study of membrane surfaces. While permitting high resolution (2.5 nm) study of replicated specimens, freeze fracture has always had certain technical limitations: 1) The carbon and platinum replicas are extremely fragile and much care and patience are required to prepare them for EM observation. This is particularly true when large areas must be replicated or when complementary replicas are required. 2) The carbon and platinum replica despite its very “real” appearance is not the actual sample. The sample itself has been completely destroyed in preparing the replica and can neither be subjected to chemical element analysis nor reprocessed for any sort of subsequent study. 3) The investigator can neither monitor the fracture process nor make a subsequent fracture of the same sample.

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:

Fracture-flip: new high-resolution images of cell surfaces after carbon stabilization of freeze-fractured membranes

1988 ◽  
Vol 90 (4) ◽  
pp. 531-541 ◽  
Author(s):  
C. Andersson Forsman ◽  
P. Pinto da Silva
Keyword(s):  
High Resolution ◽  
Cell Surface ◽  
Cell Surfaces ◽  
Carbon Stabilization ◽  
Membrane Surfaces ◽  
High Resolution Images

We introduce fracture-flip, a method of obtaining high-resolution views of membrane surfaces. The method, a corollary of label-fracture, is based on the stabilization of the exoplasmic halves of membranes by carbon evaporation. Inversion of these casts followed by Pt/C imaging leads to new views of the cell surface at macromolecular resolution.

Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells

1981 ◽  
Vol 296 (1080) ◽  
pp. 47-54 ◽  
Keyword(s):  
Plasma Membrane ◽  
Secretory Granule ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Cholesterol Content ◽  
Intramembrane Particles ◽  
Exocrine Cells ◽  
Luminal Membrane

The freeze-fracture morphology of intracellular and plasma membranes in endocrine and exocrine polypeptide-secreting cells has been studied to detect changes while these membranes interact during secretion. A qualitative and quantitative evaluation of intramembrane particles and filipin binding as indicators of protein and cholesterol content of the membranes, respectively, reveals the following changes. From the forming of the maturing pole of the Golgi complex, membranes lose morphologically detectable protein and gain morphologically detectable cholesterol. The protein-poor, cholesterol-rich secretory granule membrane then interacts with a richly particulate plasma membrane in endocrine cells and with a moderately particulate luminal membrane in exocrine cells. The site of interaction between secretory granule and plasma membrane is characterized by a local clearing of intramembrane particles; by contrast, filipin-binding sites revealing cholesterol are present in this area. In exocrine cells, the fused secretory granule, which is initially rich in filipin-cholesterol complexes and poor in particles, appears to lose progressively its filipin labelling to resemble the poorly labelled luminal membrane. These findings, although they cannot be interpreted definitely at present, clearly show impressive changes of membrane structure along the secretory pathway and suggest that a corresponding degree of functional specialization is needed for proper interaction to occur.

scholarly journals Label-fracture of cell surfaces by replica staining.

1988 ◽  
Vol 36 (11) ◽  
pp. 1413-1418 ◽  
Author(s):  
C A Forsman ◽  
P Pinto da Silva
Keyword(s):  
Freeze Fracture ◽  
Cell Surfaces ◽  
Membrane Surfaces ◽  
Simultaneous Processing ◽  
Cell Biol ◽  
New Variant

We introduce replica-staining label-fracture, a method for the cytochemical mapping of membrane surfaces. This method is a corollary of the rationale of label-fracture (Pinto da Silva and Kan, 1984: J Cell Biol 99:1156). After freeze-fracture the exoplasmic halves of the membrane remain attached to the replica. We show that cytochemical labeling of cell surfaces can be performed by direct post-fracture staining of freeze-fracture replicas. This new variant of label-fracture leads to miniaturization of labeling procedures and allows standardization of labeling conditions and simultaneous processing of different specimens.

scholarly journals High-resolution three-dimensional views of membrane-associated clathrin and cytoskeleton in critical-point-dried macrophages.

1983 ◽  
Vol 97 (5) ◽  
pp. 1452-1458 ◽  
Author(s):  
J Aggeler ◽  
R Takemura ◽  
Z Werb
Keyword(s):  
Plasma Membrane ◽  
High Resolution ◽  
Critical Point ◽  
Plasma Membranes ◽  
Cytoplasmic Membrane ◽  
Three Dimensional ◽  
Ventral Surface ◽  
Experimental Conditions ◽  
Membrane Surfaces ◽  
Critical Point Drying

We obtained high-resolution topographical information about the distribution of clathrin and cytoskeletal filaments on cytoplasmic membrane surfaces of macrophages spreading onto glass coverslips by both critical-point drying of broken-open cells and preparation of rotary platinum replicas. Irregular patches of the adherent ventral surface of the plasma membrane were exposed in these cells, and large areas of these exposed membranes were covered with clathrin-coated patches, pits, and vesicles. Various amounts of cytoskeleton were attached to the plasma membranes of these spreading cells, either as distinct starlike foci, or as individual filaments and bundles radiating out from the cytoskeletal meshwork. In newly adherent cells a well developed Golgi-GERL complex, characterized by smooth, dish-like cisternae associated with rough endoplasmic reticulum, was observed. There were many coated vesicles budding off from the Golgi cisternae, and these were predominantly of the large type (150 nm) usually associated with the plasma membrane. In critical-point-dried samples, both cytoskeleton and membranes were preserved in detail comparable to that of quick-frozen samples, after appropriate fixation. Rotary replication of critical-point-dried cells provides a rapid, easily controlled, and generally easy to perform method for obtaining samples of exposed membrane large enough to permit quantification of membrane-associated clathrin and cytoskeleton under various experimental conditions.

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