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)

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 The permeability barrier in mammalian epidermis.

1975 ◽  
Vol 65 (1) ◽  
pp. 180-191 ◽  
Author(s):  
P M Elias ◽  
D S Friend
Keyword(s):  
Plasma Membrane ◽  
Stratum Corneum ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Water Soluble ◽  
Structural Basis ◽  
Fracture Plane ◽  
Permeability Barrier ◽  
Stratum Granulosum ◽  
Lamellar Bodies

The structural basis of the permeability barrier in mammalian epidermis was examined by tracer and freeze-fracture techniques. Water-soluble tracers (horesradish peroxidase, lanthanum, ferritin) were injected into neonatal mice or into isolated upper epidermal sheets obtained with staphylococcal exfoliatin. Tracers percolated through the intercellular spaces to the upper stratum granulosum, where further egress was impeded by extruded contents of lamellar bodies. The lamellar contents initially remain segregated in pockets, then fuse to form broad sheets which fill intercellular regions of the stratum corneum, obscuring the outer leaflet of the plasma membrane. These striated intercellular regions are interrupted by periodic bulbous dilatations. When adequately preserved, the interstices of the stratum corneum are wider, by a factor of 5-10 times that previously appreciated. Freeze-fracture replicas of granular cell membranes revealed desmosomes, sparse plasma membrane particles, and accumulating intercellular lamellae, but no tight junctions. Fractured stratum corneum displayed large, smooth, multilaminated fracture faces. By freeze-substitution, proof was obtained that the fracture plane had diverted from the usual intramembranous route in the stratum granulosum to the intercellular space in the stratum corneum. We conclude that: (a) the primary barrier to water loss is formed in the stratum granulosum and is subserved by intercellular deposition of lamellar bodies, rather than occluding zonules; (b) a novel, intercellular freeze-fracture plane occurs within the stratum corneum; (c) intercellular regions of the stratum corneum comprise an expanded, structurally complex, presumably lipid-rich region which may play an important role in percutaneous transport.

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.

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.

scholarly journals Distribution of filipin-sterol complexes on cultured muscle cells: cell-substratum contact areas associated with acetylcholine receptor clusters.

1983 ◽  
Vol 96 (2) ◽  
pp. 363-372 ◽  
Author(s):  
P C Bridgman ◽  
Y Nakajima
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Freeze Fracture ◽  
Muscle Cells ◽  
Distinct Pattern ◽  
Intramembrane Particles ◽  
Membrane Topography ◽  
Interference Contrast Microscopy ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin

Specialized areas within broad, close, cell-substratum contacts seen with reflection interference contrast microscopy in cultures of Xenopus embryonic muscle cells were studied. These areas usually contained a distinct pattern of light and dark spots suggesting that the closeness of apposition between the membrane and the substratum was irregular. They coincided with areas containing acetylcholine receptor clusters identified by fluorescence labeled alpha-bungarotoxin. Freeze-fracture of the cells confirmed these observations. The membrane in these areas was highly convoluted and contained aggregates of large P-face intramembrane particles (probably representing acetylcholine receptors). If cells were fixed and then treated with the sterol-specific antibiotic filipin before fracturing, the pattern of filipin-sterol complex distribution closely followed the pattern of cell-substratum contact. Filipin-sterol complexes were in low density in the regions where the membrane contained clustered intramembrane particles. These membrane regions were away from the substratum (bright white areas in reflection interference contrast; depressions of the P-face in freeze-fracture). Filipin-sterol complexes were also in reduced density where the membrane was very close to the substratum (dark areas in reflection interference contrast; bulges of the P-face in freeze-fracture). These areas were not associated with clustered acetylcholine receptors (aggregated particles). This result suggests that filipin treatment causes little or no artefact in either acetylcholine receptor distribution or membrane topography of fixed cells and that the distribution of filipin-sterol complexes may closely parallel the microheterogeneity of membranes that exist in living cells.

The erythrocyte plasma membrane in murine muscular dystrophy: a scanning electron microscopic and freeze fracture study

1979 ◽  
Vol 57 (5) ◽  
pp. 983-986 ◽  
Author(s):  
Burr G. Atkinson ◽  
Richard R. Shivers ◽  
Bruce Nixon ◽  
Kristine H. Atkinson
Keyword(s):  
Plasma Membrane ◽  
Muscular Dystrophy ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Plasma Membranes ◽  
Genetic Disorder ◽  
Freeze Fracture ◽  
Congenital Muscular Dystrophy ◽  
Molecular Architecture ◽  
Intramembrane Particles

The plasma membrane of red blood cells from mice afflicated with congenital muscular dystrophy exhibits a dramatic depletion of intramembrane particles. Examination of protein particles on fracture faces of erythrocyte plasma membranes from dystrophic mice revealed a 33% decrease in the number of intramembrane particles when compared with similarly prepared erythrocytes from nondystrophic animals. This alteration in the internal molecular architecture of these plasma membranes is correlated with the morphological distortion manifested by most red blood cells from mice inflicted with this genetic disorder.

scholarly journals AN INTERPRETATION OF LIVER CELL MEMBRANE AND JUNCTION STRUCTURE BASED ON OBSERVATION OF FREEZE-FRACTURE REPLICAS OF BOTH SIDES OF THE FRACTURE

1970 ◽  
Vol 47 (1) ◽  
pp. 49-60 ◽  
Author(s):  
J. P. Chalcroft ◽  
S. Bullivant
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Liver Cell ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Fracture Plane ◽  
Small Scale ◽  
Liver Cell Membrane ◽  
Mouse Liver Cell ◽  
Junction Structure

A modification of the freeze-fracturing technique to permit observation of replicas of both sides of the fracture is described. It has been used to study mouse liver cell membrane structure. Membranes break to give two faces with three-dimensional complementarity, although there is some small-scale mismatching which is discussed. Since the two distinctive sets of membrane faces are complementary sets, they cannot be the two outside surfaces. In particular, structures (such as particles) seen on these faces are within the membrane. It is not possible from this work to say precisely where the fracture plane goes with respect to a plasma membrane, only that it must be close to the interface between membrane and cytoplasm, or at that interface. Models, consistent with the appearance of the matching replicas, are derived for three regions of the plasma membrane: (a) The nonjunctional plasma membrane, which contains many scattered particles. Except for these particles, the otherwise flat fracture face is not at variance with a bimolecular leaflet structure. (b) Gap junctions. Each of the two membranes comprising a gap junction contains a close-packed array of particles. (c) Tight junctions. Here membranes have ridges within them.

scholarly journals Components of the plasma membrane of growing axons. I. Size and distribution of intramembrane particles.

1984 ◽  
Vol 98 (4) ◽  
pp. 1422-1433 ◽  
Author(s):  
R K Small ◽  
K H Pfenninger
Keyword(s):  
Plasma Membrane ◽  
Growth Cone ◽  
Freeze Fracture ◽  
Particle Diameter ◽  
Lateral Diffusion ◽  
Membrane Composition ◽  
Intramembrane Particles ◽  
Cellular Processes ◽  
Discrete Domains ◽  
Membrane Components

The plasmalemma of mature and growing olfactory axons of the bullfrog has been studied by freeze-fracture. Intramembrane particles (IMPs) of mature olfactory axons are found to be uniformly distributed along the shaft. However, during growth, a decreasing gradient of IMP density is evident along the somatofugal axis. The size histograms of axolemmal IMPs from different segments of growing nerve reveal regional differences in the particle composition. The distribution of each individual size class of particles along the growing nerve forms a decreasing gradient in the somatofugal direction; the slope of these gradients varies directly with particle diameter. These size-dependent density gradients are consistent with a process of lateral diffusion of membrane components that are inserted proximally into the plasma membrane. The membrane composition of the growth cone, however, appears to be independent of these diffusion gradients; it displays a mosaic pattern of discrete domains of high and low particle densities. The relative IMP profiles of these growth cone regions are similar to one another but contain higher densities of large IMPs than the neighboring axonal shaft. The shifting distributions of intramembrane particles that characterize the sprouting neuron give new insights into cellular processes that may underlie the establishment of the functional polarity of the neuron and into the dynamics of axolemmal maturation.

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.

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