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.

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.

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).

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.

Membrane changes associated with mucus production by intestinal goblet cells

1978 ◽  
Vol 33 (1) ◽  
pp. 301-316
Author(s):  
J.G. Swift ◽  
T.M. Mukherjee
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Fusion ◽  
Thin Section ◽  
Structural Organization ◽  
Freeze Fracture ◽  
Goblet Cells ◽  
Mucus Production ◽  
Membrane Reorganization ◽  
Membrane Changes

Changes in the structural organization of membranes of mucous bodies and the plasma membrane that occur during mucus production in goblet cells of rat rectum have been studied by thin-section and freeze-fracture techniques. Immature mucous bodies are bounded by a trilaminar membrane and fracture faces of the membrane have randomly distributed intramembrane particles. During maturation, mucous bodies become packed tightly together and changes in the structure of their membranes include (1) fusion of apposing membranes of adjacent bodies to form a pentalaminar structure, (2) a reduction in the density of particles on membrane fracture faces, and (3) exclusion of particles from regions of membrane apposition. Some trilaminar membranes of mucous bodies fuse with the lumenal plasma membrane to form a pentalaminar structure. Sites of apposition between mucous body membranes and the lumenal plasma membrane are seen as particle-cleared bulges on fracture faces of the plasma membrane. Our results indicate that membrane reorganization associated with mucous production in goblet cells includes a reduction and redistribution of some membrane proteins and that membrane fusion occurs between portions of membranes from which proteins have been displaced.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

Intercellular communication between epithelial and fiber cells of the eye lens

1994 ◽  
Vol 107 (4) ◽  
pp. 799-811 ◽  
Author(s):  
S. Bassnett ◽  
J.R. Kuszak ◽  
L. Reinisch ◽  
H.G. Brown ◽  
D.C. Beebe
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Freeze Fracture ◽  
Cell Types ◽  
Lens Epithelium ◽  
Fiber Cell ◽  
Fracture Plane ◽  
Metabolic Cooperation ◽  
Fiber Cells ◽  
Resistance Pathway

Results of electrical, dye-coupling and morphological studies have previously suggested that gap junctions mediate communication between the anterior epithelium of the lens and the underlying lens fiber cells. This connection is believed to permit ‘metabolic cooperation’ between these dissimilar cell types and may be of particular importance to the fiber cells, which are thought incapable of autonomous ionic homeostasis. We reinvestigated the nature of the connection between epithelial and fiber cells of the embryonic chicken lens using fluorescence confocal microscopy and freeze-fracture analysis. In contrast to earlier studies, our data provided no support for gap-junction-mediated transport from the lens epithelium to the fibers. Fluorescent dyes loaded biochemically into the lens epithelium were retained there for more than one hour. There was a decrease in epithelial fluorescence over this period, but this was not accompanied by an increase in fiber cell fluorescence. Diffusional modeling suggested that these data were inconsistent with the presence of extensive epithelium-fiber cell coupling, even if the observed decrease in epithelial fluorescence was attributed exclusively to the diffusion of dye into the fiber mass via gap junctions. Furthermore, the rate of loss of fluorescence from isolated epithelia was indistinguishable from that measured in whole lenses, suggesting that decreased epithelial fluorescence resulted from photobleaching and leakage of dye rather than diffusion, via gap junctions, into the fibers. Analysis of freeze-fracture replicas of plasma membranes at the epithelial-fiber cell interface failed to reveal evidence of gap-junction plaques, although evidence of endocytosis was abundant. These studies were done under conditions where the location of the fracture plane was unambiguous and where gap junctions could be observed in the lateral membranes of neighboring epithelial and fiber cells. Paradoxically, tracer molecules injected into the fiber mass were able to pass into the epithelium via a pathway that was not blocked by incubation at 4 degrees C or by treatment with octanol and which excluded large (approximately 10 kDa) molecular mass tracers. Together with previous measurements of electrical coupling between fiber cells and epithelial cells, these data indicate the presence of a low-resistance pathway connecting these cell types that is not mediated by classical gap junctions.

scholarly journals A freeze-fracture study of membrane events during neurohypophysial secretion.

1978 ◽  
Vol 78 (2) ◽  
pp. 542-553 ◽  
Author(s):  
D T Theodosis ◽  
J J Dreifuss ◽  
L Orci
Keyword(s):  
Plasma Membrane ◽  
Unit Area ◽  
Freeze Fracture ◽  
Core Material ◽  
Number Of Clusters ◽  
Intramembrane Particles ◽  
En Face ◽  
Large Particles ◽  
Mean Diameter ◽  
Fracture Study

Freeze-fracture was used to study the membrane events taking place during neurosecretory granule discharge (exocytosis) and subsequent membrane internalization (endocytosis) in axons of neurohypophyses from control and water-deprived rats. En face views of the cytoplasmic leaflet (P face) of the split axolemma reveal circular depressions that represent the secretory granule membranes fused with the plasma membrane during exocytosis. These depressions often contain granule core material in the process of extrusion into the extracellular space. The membrane surrounding some of the exocytotic openings shows a decreased number of intramembrane particles (mean diameter, 8 nm) which are elsewhere more numerous and evenly distrubuted on the fracture face. Endocytotic sites appear as smaller plasma membrane invaginations, with associated intramembrane particles. Moreover, such invaginations often contain large particles (mean diameter, 12 nm) that appear as clusters on en face views of the membrane leaflet. Quantitative analysis indicates that the number of exocytotic images increases significantly in glands from water-deprived rats. Concomitantly, the number of endocytotic figures per unit area of membrane is raised as is the number of clusters of large particles. The observations demonstrate that, in the neurohypophysis, it is possible to distinguish exocytosis morphologically from endocytosis and that the two events can be assessed quantitatively.

scholarly journals (Na+ + K+)-ATPase correlated with a major group of intramembrane particles in freeze-fracture replicas of cultured chick myotubes.

1983 ◽  
Vol 97 (4) ◽  
pp. 1214-1225 ◽  
Author(s):  
D W Pumplin ◽  
D M Fambrough
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Acetylcholine Receptors ◽  
Average Particle Size ◽  
Freeze Fracture ◽  
Fracture Plane ◽  
Average Particle ◽  
Intramembrane Particles ◽  
Double Antibody ◽  
Cross Linkage

Immunofluorescence microscopy with a fluorescein-labeled monoclonal antibody was used to map the distribution of sodium- and potassium-ion stimulated ATPase [( Na,K]-ATPase) on the surface of tissue-cultured chick skeletal muscle. At this level of resolution it appeared that the (Na,K)-ATPase molecules were distributed nearly uniformly over the plasma membrane. These molecules could be cross-linked by use of the monoclonal antibody followed by a second antibody directed against the monoclonal antibody; the resulting fluorescent pattern was a set of small dots (patches) on the muscle surface. This pattern was stable over several hours, and there was little evidence of interiorization or of coalescence of the patches. Myotubes labeled with immunofluorescence were fixed in glutaraldehyde, cryoprotected with glycerin, then fractured and replicated by standard methods. Replicas of the immunofluorescence-labeled myotubes revealed clusters of intramembrane particles (IMP) only when the immunofluorescent images indicated a patching of the (Na,K)-ATPase molecules. Double antibody cross-linking of antigenic sites on myotubes with each of three other monoclonal antibodies to plasma membrane antigens likewise resulted in patched patterns of immunofluorescence, but in none of these cases were clusters of intramembrane particles found in freeze-fracture replicas. In each case it was shown that the (Na,K)-ATPase molecules were not patched. Other control experiments showed that patching of (Na,K)-ATPase molecules did not cause co-patching of one of the other plasma membrane proteins defined by a monoclonal antibody and did not cause detectable co-clustering of acetylcholine receptors. Detailed mapping showed that there was a one-to-one correspondence between immunofluorescent patches related to the (Na,K)-ATPase and clusters of IMP in a freeze-fracture replica of the same cell. We conclude that the intramembrane particles patched by double antibody cross-linkage of the (Na,K)-ATPase are caused by (Na,K)-ATPase molecules in the fracture plane. Quantification of the IMP indicated that the (Na,K)-ATPase-related particles account for up to 50% of particles evident in the replicas, or up to about 400 particles/micrometers2 of plasma membrane. Particles related to the (Na,K)-ATPase were similar to the average particle size and were as heterodisperse in size as the total population of IMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Intramembrane particles distribution in the naturally synchronous stage of Physarum polycephalum

1992 ◽  
Vol 50 (1) ◽  
pp. 866-867
Author(s):  
Randolph Taylor ◽  
Henrie Turner
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Plasma Membrane ◽  
Physarum Polycephalum ◽  
Freeze Fracture ◽  
Intramembrane Particles ◽  
Membrane Changes

Comparative ultrastructural investigations of the Physarum polycephalum intramembrane particles in the plasma membrane at different stages of the cycle has provided valuable information in relation to possible changes that occur in the plasma membrane of higher organisms. In addition, it gives insight on how plasma membrane changes correlate with gene expression and gene regulation in eukaryotes. In this report Freeze-fracture-etched techniques were utilized to study the arrangements of intramembrane particles (IMP) distribution and density at eight hours of the naturally synchronous plasmodial stage.

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.

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