scholarly journals Freeze-Fracture Enzyme Cytochemistry Reveals the Distribution of Enzymes in Biological Membranes: Enzyme Cytochemical Label-Fracture and Fracture-Label.

1997 ◽  
Vol 30 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Eiko Nakazawa ◽  
Takuma Saito
Keyword(s):  
Freeze Fracture ◽  
Biological Membranes ◽  
Enzyme Cytochemistry

Contribution of Microscopy to a Better Knowledge of the Biology ofGiardia lamblia

2004 ◽  
Vol 10 (5) ◽  
pp. 513-527 ◽  
Author(s):  
Wanderley de Souza ◽  
Adriana Lanfredi-Rangel ◽  
Loraine Campanati
Keyword(s):  
Golgi Complex ◽  
Laser Scanning ◽  
Diarrheal Disease ◽  
Freeze Fracture ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Cytochemistry ◽  
Morphological Studies ◽  
Transmission Electron ◽  
Microscopic Techniques

Giardia lambliais a flagellated protozoan of great medical and biological importance. It is the causative agent of giardiasis, one of the most prevalent diarrheal disease both in developed and third-world countries. Morphological studies have shown thatG. lambliadoes not present structures such as peroxisomes, mitochondria, and a well-elaborated Golgi complex. In this review, special emphasis is given to the contribution made by various microscopic techniques to a better knowledge of the biology of the protozoan. The application of video microscopy, immunofluorescence confocal laser scanning microscopy, and several techniques associated with transmission electron microscopy (thin section, enzyme cytochemistry, freeze-fracture, deep-etching, fracture-flip) to the study of the cell surface, peripheral vesicles, endoplasmic reticulum–Golgi complex system, and of the encystation vesicles found in trophozoites and during the process of trophozoite-cyst transformation are discussed.

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.

Membrane alterations during thylakoid formation in rhodospirillum rubrum

1978 ◽  
Vol 36 (2) ◽  
pp. 148-149
Author(s):  
E. Yokomura ◽  
M. Sugimoto ◽  
C Gomi-Baba
Keyword(s):  
Cell Membranes ◽  
Rhodospirillum Rubrum ◽  
Freeze Fracture ◽  
Biological Membranes ◽  
Model Systems ◽  
Forming Process ◽  
Biochemical Studies

Thylakoid formation in RhodospiriHum rubrum is one of the model systems adequate for studying the differentiation of the biological membranes. It is well established that thylakoids are formed from the invaginations of cell membranes. This is based on electronmicroscopic observations and biochemical studies. However, the alterations in the architecture of the cell membranes during thylakoid- forming process is ambiguous. Therefore, in the present study, thylakoid formation in R.. rubrum was investigated with freeze-fracture technique and DAB-histochemistry.For inducing thylakoid formation, the cells grown at 25° C in an aerobic-dark (AD) environment were transferred to an anaerobic-light (NL) environment. At various times after transferring from AD to NL, the cells were immersed in 30 % glycerol without any chemical prefixation and then immediately frozen in liquid Freon to be prepared for the freeze-fracture replicas. The materials were handled at 25° C throughout before freezing.

Molecular cytochemistry of freeze-fractured cells

1986 ◽  
Vol 44 ◽  
pp. 894-897
Author(s):  
Pedro Pinto da Silva
Keyword(s):  
Membrane Proteins ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Biological Membranes ◽  
Bilayer Membrane ◽  
Integral Membrane Proteins ◽  
Distilled Water ◽  
Intramembrane Particles ◽  
Freeze Etching

I will describe four approaches that combine cytochemistry with freeze-fracture: 1) FREEZE-ETCHING; 2) FRACTURE-LABEL; 3) FRACTURE-PERMEATION; and 4) LABEL-FRACTURE. These techniques, in particular fracture-label, involve delicate points of interpretation and numerous validating controls. In the publications listed at the end, these issues have been addressed in detail.1. FREEZE-ETCHING. I developed freeze-etching as a cytochemical approach to prove that membranes were split by freeze-fracture and to show that biological membranes were comprised of a bilayer membrane continuum interrupted by integral membrane proteins.1 - 4 In freeze-etching, the distribution of the marker over the membrane surface exposed by sublimation is compared to that of the intramembrane particles exposed by fracture. It is often required to aggregate the particles into domains larger than the labeling molecules (Fig. 1). This, and the need for freezing in distilled water, severely limits the application of freeze-etching.

Pore-like structures in biological membranes

1977 ◽  
Vol 25 (1) ◽  
pp. 157-161
Author(s):  
L. Orci ◽  
A. Perrelet ◽  
F. Malaisse-Lagae ◽  
P. Vassalli
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Gap Junction ◽  
Membrane Permeability ◽  
Freeze Fracture ◽  
Biological Membranes ◽  
Cell Types ◽  
Intramembrane Particles ◽  
Similar Images ◽  
Specific Array

In freeze-fracture replicas, biological membranes appear as smooth surfaces interrupted by random globular protrusion, the intramembrane particles. Smooth areas correspond to the membrane phospholipidic domain, while intramembrane particles are the morphological counterpart of membrane proteins. In the present work, examination of membranes in a variety of cell types reveals that a number of intramembrane particles contain an electron-dense spot. The spot is thought to correspond to a minute pit in the particle, filled by the platinum used in the freeze-fracture procedure. Similar images, described previously in intramembrane particles forming the specific array of the gap junction, were interpreted as hydrophilic channels bridging the interior and the exterior of the plasma membrane. Comparison between the gap junction particles and the non-junction particles containing a dense spot suggests that these latter may too contain hydrophilic channels. The channels in random intramembrane particles would represent the morphological counterparts of the water-filled pores described in models of membrane permeability.

scholarly journals Freeze-fracture Cytochemistry: A New Method Combining Immunocytochemistry and Enzyme Cytochemistry on Replicas

1998 ◽  
Vol 46 (1) ◽  
pp. 11-17 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Takuma Saito ◽  
John M. Robinson
Keyword(s):  
Freeze Fracture ◽  
Phosphatidyl Inositol ◽  
Human Neutrophils ◽  
Initial Examination ◽  
Enzyme Cytochemistry ◽  
Double Labeling ◽  
Octyl Glucoside ◽  
Additional Approach ◽  
Cytochemical Technique ◽  
The Relationship

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphatase activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.

Ultrastructural Investigations of the Contractile Filaments of Amoebae

1974 ◽  
Vol 32 ◽  
pp. 188-189
Author(s):  
P.L. Moore
Keyword(s):  
Cytoplasmic Streaming ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Amoeboid Movement ◽  
Different Types ◽  
Chemical Conditions ◽  
Complete Interpretation

Previous freeze fracture results on the intact giant, amoeba Chaos carolinensis indicated the presence of a fibrillar arrangement of filaments within the cytoplasm. A complete interpretation of the three dimensional ultrastructure of these structures, and their possible role in amoeboid movement was not possible, since comparable results could not be obtained with conventional fixation of intact amoebae. Progress in interpreting the freeze fracture images of amoebae required a more thorough understanding of the different types of filaments present in amoebae, and of the ways in which they could be organized while remaining functional.The recent development of a calcium sensitive, demembranated, amoeboid model of Chaos carolinensis has made it possible to achieve a better understanding of such functional arrangements of amoeboid filaments. In these models the motility of demembranated cytoplasm can be controlled in vitro, and the chemical conditions necessary for contractility, and cytoplasmic streaming can be investigated. It is clear from these studies that “fibrils” exist in amoeboid models, and that they are capable of contracting along their length under conditions similar to those which cause contraction in vertebrate muscles.

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