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.

Image Analysis of Intramembrane Particles in Freeze-Fractured Biomembranes Using Computer Simulation Techniques

1975 ◽  
Vol 33 ◽  
pp. 214-215
Author(s):  
D.J. Benefiel ◽  
R.S. Weinstein
Keyword(s):  
Membrane Proteins ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Systematic Evaluation ◽  
Intramembrane Particles ◽  
Modeling Methods ◽  
Simulation Techniques ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Computer Simulation Techniques

Intramembrane particles (IMP or MAP) are components of most biomembranes. They are visualized by freeze-fracture electron microscopy, and they probably represent replicas of integral membrane proteins. The presence of MAP in biomembranes has been extensively investigated but their detailed ultrastructure has been largely ignored. In this study, we have attempted to lay groundwork for a systematic evaluation of MAP ultrastructure. Using mathematical modeling methods, we have simulated the electron optical appearances of idealized globular proteins as they might be expected to appear in replicas under defined conditions. By comparing these images with the apearances of MAPs in replicas, we have attempted to evaluate dimensional and shape distortions that may be introduced by the freeze-fracture technique and further to deduce the actual shapes of integral membrane proteins from their freezefracture images.

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.

Local shadow angle and heights of intramembrane particles in freeze-fracture replicas of proteoliposomes

1986 ◽  
Vol 44 ◽  
pp. 214-215
Author(s):  
M. J. Costello ◽  
G. Gomez
Keyword(s):  
Electron Microscope ◽  
Membrane Proteins ◽  
Inclination Angle ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Irregular Surfaces ◽  
Intramembrane Particles ◽  
Aqueous Suspensions ◽  
Microscope Stage

The heights of intramembrane particles (IMPs) produced by integral membrane proteins are difficult to obtain because the local shadow angle in the vicinity of the IMPs is not known for typical freeze-fracture experiments. Even though the average shadow inclination angle is set by the operator (usually 20°-45°), fractures normally produce very irregular surfaces. We have devised a procedure for determining the local shadow angle in selected spherical proteoliposomes from which IMP heights can be calculated. The procedure is an extension of a method to determine the true diameter of vesicles in freeze-cleaved aqueous suspensions and relies on the use of a tiltrotation electron microscope stage.

Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

1977 ◽  
Vol 35 ◽  
pp. 596-597
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Synthetic Medium ◽  
Freeze Fracture ◽  
Translational Diffusion ◽  
Yeast Cells ◽  
Distilled Water ◽  
Intramembrane Particles ◽  
The Matrix ◽  
Water Cell

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

scholarly journals Fracture faces of frozen membranes: 50th anniversary

2016 ◽  
Vol 27 (3) ◽  
pp. 421-423
Author(s):  
Daniel Branton
Keyword(s):  
Electron Microscopy ◽  
Lipid Bilayers ◽  
Fracture Process ◽  
Relative Proportion ◽  
50Th Anniversary ◽  
Biological Membranes ◽  
Bilayer Membrane ◽  
Biological Cells ◽  
Membrane Surfaces ◽  
Freeze Etching

In 1961, the development of an improved freeze-etching (FE) procedure to prepare rapidly frozen biological cells or tissues for electron microscopy raised two important questions. How does a frozen cell membrane fracture? What do the extensive face views of the cell’s membranes exposed by the fracture process of FE tell us about the overall structure of biological membranes? I discovered that all frozen membranes tend to split along weakly bonded lipid bilayers. Consequently, the fracture process exposes internal membrane faces rather than either of the membrane’s two external surfaces. During etching, when ice is allowed to sublime after fracturing, limited regions of the actual membrane surfaces are revealed. Examination of the fractured faces and etched surfaces provided strong evidence that biological membranes are organized as lipid bilayers with some proteins on the surface and other proteins extending through the bilayer. Membrane splitting made it possible for electron microscopy to show the relative proportion of a membrane’s area that exists in either of these two organizational modes.

A gradient in the density of intramembrane particles is formed during capping induced by concanavalin A

1986 ◽  
Vol 83 (1) ◽  
pp. 61-76
Author(s):  
H. Bennett ◽  
J. Condeelis
Keyword(s):  
Cell Polarity ◽  
Concanavalin A ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Asymmetric Distribution ◽  
Numerical Density ◽  
Ion Currents ◽  
Ion Pumps ◽  
Intramembrane Particles ◽  
The Difference

During capping of concanavalin A (ConA) by amoebae of Dictyostelium discoideum, each cell becomes polarized, with the ConA at one end and newly extended pseudopodia at the opposite end of the cell. This new polarity is stable until the cap is shed or internalized. Intramembrane particles (IMPs) are widely believed to represent large integral membrane proteins, many of which are ion pumps and channels. Since asymmetric ion currents have been implicated in the development of cell polarity, we have used morphological landmarks associated with the capped cells in freeze-fracture to make a morphometric analysis of the IMP distribution relative to the axis of polarization of the capped cell. Untreated cells in suspension extend pseudopodia randomly from their surfaces. In these cells the numerical density of IMPs is random. However, capped cells demonstrate a density gradient of IMPs with the lowest density usually in the pseudopodia and the highest in the cap. The difference in density between the cap and other regions of the cell is two- to threefold for all IMPs, but can be as much as sevenfold for greater than 12 nm IMPs. This study is the first to document that the numerical density of IMPs is altered in response to ligand-induced capping and demonstrates that the distribution of IMPs in a capped cell is related to the axis of polarization of the cell. These results suggest that the development of cell polarity during capping in Dictyostelium amoebae may be due to the asymmetric distribution of IMPs, which may cause asymmetric ion currents across the cell.

scholarly journals Modeling Membrane-Protein Interactions

2018 ◽  
Author(s):  
Haleh Alimohamadi ◽  
Padmini Rangamani
Keyword(s):  
Membrane Proteins ◽  
Protein Interactions ◽  
Current Model ◽  
Bilayer Membrane ◽  
Integral Membrane Proteins ◽  
Elastic Membrane ◽  
Protein Distribution ◽  
Peripheral Membrane Proteins ◽  
Membrane Models ◽  
Generation Mechanisms

In order to alter and adjust the shape of the membrane, cells harness various mechanisms of curvature generation. Many of these curvature generation mechanisms rely on the interactions between peripheral membrane proteins, integral membrane proteins, and lipids in the bilayer membrane. One of the challenges in modeling these processes is identifying the suitable constitutive relationships that describe the membrane free energy that includes protein distribution and curvature generation capability. Here, we review some of the commonly used continuum elastic membrane models that have been developed for this purpose and discuss their applications. Finally, we address some fundamental challenges that future theoretical methods need to overcome in order to push the boundaries of current model applications.

On the Nature of Intramembrane Particles

1977 ◽  
Vol 35 ◽  
pp. 680-683
Author(s):  
J. David Robertson
Keyword(s):  
Chemical Nature ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Purple Membrane ◽  
Halobacterium Halobium ◽  
Polar Regions ◽  
Hydrophobic Core ◽  
Intramembrane Particles ◽  
Protein Molecules ◽  
Complete Report

The chemical nature of the ubiquitous ∽8-10nm intramembrane particles (IMP's) observed with freeze-fracture-etch (FFE) techniques in biological membranes has not been unequivocally established. Some believe that they represent directly metal coated solid globular protein macromolecules residing within the hydrophobic core of the lipid bilayer. The particles are clearly related to protein molecules in the membrane surface but the extent to which the protein extends into the replicated particles has not been established. The particles at least in some instances could be partly or even entirely lipid bound specifically to the protein with the polypeptide in the polar regions of the membrane. We have used two membranes to study this problem: the purple membrane of Halobacterium halobium and the fusiform vacuole membrane of urothelial epithelial cells (1). A complete report of these studies will be published with collaborators W. Schreil in the case of the purple membrane and J. Vergara on the urothelial membrane. One preliminary report on the purple membrane has already been published with W. Schreil (2).

Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes

1995 ◽  
Vol 108 (11) ◽  
pp. 3443-3449 ◽  
Author(s):  
K. Fujimoto
Keyword(s):  
Membrane Proteins ◽  
Tight Junction ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Immunogold Labeling ◽  
Integral Membrane Proteins ◽  
Tissue Slices ◽  
Electron Microscopic ◽  
Junction Protein ◽  
Junction Proteins

We propose a new electron microscopic method, the sodium dodecylsulphate (SDS)-digested freeze-fracture replica labeling technique, to study the two-dimensional distribution of integral membrane proteins in cellular membranes. Unfixed tissue slices were frozen with liquid helium, freeze-fractured, and replicated in a platinum/carbon evaporator. They were digested with 2.5% SDS to solubilize unfractured membranes and cytoplasm. While the detergent dissolved unfractured membranes and cytoplasm, it did not extract fractured membrane halves. After SDS-digestion, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for cytochemical labeling, followed by electron microscopic observation. As an initial screening, we applied this technique to the immunogold labeling of intercellular junction proteins: connexins (gap junction proteins), occludin (tight junction protein), desmoglein (desmosome protein), and E-cadherin (adherens junction protein). The immunogold labeling was seen superimposed on the image of a fracture face visualized by platinum/carbon shadowing. The immunoreaction was specific, and only the structures where the proteins were expected were labeled. For instance, anti-occludin immunogold complexes were observed immediately adjacent to the tight junction strands on the protoplasmic and exoplasmic fracture faces. No significant levels of gold label were associated with non-tight-junctional regions of plasma membranes. The procedures of the SDS-digested freeze-fracture replica labeling and its potential significance are discussed.

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