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.

Functional organization of the bovine rumen epithelium

2005 ◽  
Vol 288 (1) ◽  
pp. R173-R181 ◽  
Author(s):  
C. Graham ◽  
N. L. Simmons
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Stratum Corneum ◽  
Connexin 43 ◽  
Functional Organization ◽  
Stratum Granulosum ◽  
Bovine Rumen ◽  
Rumen Epithelium ◽  
Stratum Spinosum ◽  
Cell Layers

The functional organization of the bovine rumen epithelium has been examined by electron and light microscopy combined with immunocytochemistry to define a transport model for this epithelium. Expression of connexin 43, an integral component of gap junctions, the tight-junction molecules claudin-1 and zonula occludens 1 (ZO-1), and the catalytic α-subunit of Na+-K+-ATPase was demonstrated by SDS-PAGE and Western blotting. From the lumen surface, four cell layers can be distinguished: the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale. Both claudin-1 and ZO-1 immunostaining showed plasma membrane staining, which was present at the stratum granulosum with decreasing intensity through the stratum spinosum to the stratum basale. The stratum corneum was negative for claudin-1 immunostaining. Transmission electron microscopy confirmed that occluding tight junctions were present at the stratum granulosum. Plasma membrane connexin 43 immunostaining was most intense at the stratum granulosum and decreased in intensity through stratum spinosum and stratum basale. There was intense immunostaining of the stratum basale for Na+-K+-ATPase, with weak staining of the stratum spinosum. Both the stratum granulosum and the stratum corneum were essentially negative. Stratum basale cells also displayed a high mitochondrial density relative to more apical cell layers. We conclude that epithelial barrier function may be attributed to the stratum granulosum and that cell-cell gap junctions allow diffusion to interconnect the barrier cell layer with the stratum basale where Na+-K+-ATPase is concentrated.

scholarly journals Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections.

1988 ◽  
Vol 107 (6) ◽  
pp. 2511-2521 ◽  
Author(s):  
G Knoll ◽  
K N Burger ◽  
R Bron ◽  
G van Meer ◽  
A J Verkleij
Keyword(s):  
Plasma Membrane ◽  
Apical Cell ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Morphological Evidence ◽  
Epithelial Cell Line ◽  
Rapid Freezing ◽  
Intramembrane Particle ◽  
Local Point ◽  
Basolateral Plasma Membrane

The fusion of liposomes with the plasma membrane of influenza virus-infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processing by freeze substitution and low temperature embedding. Before fusion, radioactivity was solely detected on the apical cell surface, indicating the absence of redistribution artifacts and demonstrating the reliability of lipid autoradiography on both a light and electron microscopical level. After induction of fusion by a low pH treatment, the basolateral plasma membrane domain became progressively labeled, indicative of rapid lateral diffusion of [3H]dipalmitoylphosphatidylcholine in the plasma membrane. Analysis of individual fusion events by freeze fracture after rapid freezing confirmed the rapid diffusion of the liposomal lipids into the plasma membrane, as intramembrane particle-free lipid patches were never observed. After the induction of liposome-cell fusion, well-defined intramembrane particles were present on the otherwise smooth liposomal fracture faces and on the fracture faces of the plasma membrane. Morphological evidence thus was obtained in favor of a local point fusion mechanism with an intramembrane particle as a specific structural fusion intermediate.

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)

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 CELL JUNCTIONS IN AMPHIBIAN SKIN

1965 ◽  
Vol 26 (1) ◽  
pp. 263-291 ◽  
Author(s):  
Marilyn G. Farquhar ◽  
George E. Palade
Keyword(s):  
Stratum Corneum ◽  
Membrane Fusion ◽  
Water Soluble ◽  
Cell Junctions ◽  
Physiological Data ◽  
Junctional Complex ◽  
Stratum Granulosum ◽  
Intercellular Spaces ◽  
Outermost Layer ◽  
Zonulae Occludentes

Cell junctions have been investigated in the amphibian epidermis, a stratified squamous epithelium, and compared to those described previously in simple columnar epithelia of mammalian cavitary organs. In adult frogs and toads, and in larvae approaching metamorphosis, belts of membrane fusion or zonulae occludentes of considerable depth are regularly found between adjoining cells of the outermost layer of the stratum corneum, binding the cells together into a continuous, uninterrupted sheet. Another set of occluding zonules appears in the second cornified layer (when such a layer is present), and a third set usually occurs in the outermost layer of the stratum granulosum. Specialized elements described as "modified" and "composite" desmosomes are encountered along the lateral and basal aspects, respectively, of the cornified cells; ordinary desmosomes and maculae occludentes (i.e., spots of membrane fusion) are found in all other strata. The usual 200 A intercellular gap is generally maintained between the cells of the stratum germinativum at the basal ends of the intercellular spaces. Hence, the intercellular spaces of the epidermis form a largely continuous network, closed to the external medium and open to the dermal interstitia. The situation is comparable to that found in columnar epithelia, except that the intercellular spaces are much more extensive, and an extracellular subcompartment (or two) apparently exists in the stratum corneum and between the latter and the stratum granulosum. The last subcompartment is usually filled with a dense substance, probably derived from discharged secretory granules. The tripartite junctional complex characteristic of lumen-lining epithelia (i.e., a zonula occludens followed by a zonula adhaerens, and desmosome) is seen only in early larvae; in adults and in larvae approaching metamorphosis, the occluding zonule is followed directly by a series of modified desmosomes. Interpreted in the light of current physiological data, these findings suggest that the diffusion of water, ions, and small, water-soluble molecules is impeded along the intercellular spaces of the epidermis by zonulae occludentes while it is facilitated from cell to cell within the epidermis by zonulae and maculae occludentes.

Fenestrated vessels in human hemangioblastoma

1974 ◽  
Vol 40 (6) ◽  
pp. 696-705 ◽  
Author(s):  
Eiichi Tani ◽  
Kimiyuki Ikeda ◽  
Susumu Kudo ◽  
Shogo Yamagata ◽  
Noboru Higashi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Thin Section ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Fracture Plane ◽  
Content Type ◽  
Plasmalemmal Vesicles ◽  
Fracture Edge ◽  
Fine Print

✓ The capillaries in two cerebellar hemangioblastomas were studied by thin-section and freeze-fracture techniques. Fenestrae were found in the attenuated portions of the endothelium, and plasmalemmal vesicles in the nonfenestrated portions. In freeze-fracture preparations the fenestrae of the endothelial plasma membrane were about 450 to 550 A in diameter. They appeared as holes and “necks” in clusters of about 40 to 60 per µm2. When the fracture plane passed in a stepwise fashion from the luminal plasma membrane into the contraluminal plasma membrane, the fenestrae at the fracture edge involved both plasma membranes.

scholarly journals Metal Sandwich Method to Quick-freeze Monolayer Cultured Cells for Freeze-fracture

1997 ◽  
Vol 45 (4) ◽  
pp. 595-598 ◽  
Author(s):  
Toyoshi Fujimoto ◽  
Kazushi Fujimoto
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Gold Foil ◽  
Freeze Fracture ◽  
Fracture Plane ◽  
Lower Plasma ◽  
Confluent Monolayer ◽  
Cold Stage ◽  
Quick Freezing ◽  
Sandwich Method

We describe a simple quick-freezing method to obtain a large fractured plane of the plasma membrane from monolayer cultured cells. Cells were grown on thin gold foil, inverted on a thin layer of gelatin on thin copper foil, and frozen by a quick press between two gold-plated copper blocks precooled in liquid nitrogen. The frozen cell sandwich was mounted on the cold stage of a freeze-fracture device with the gold side up and was fractured by separating the sandwich with a cold fracture knife. When this technique was applied to confluent monolayer cells, large replicas of the E-face of the upper plasma membrane and the P-face of the lower plasma membrane were obtained. The present metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation.

scholarly journals Detection of gap junctions between the progeny of a canine macrophage colony-forming cell in vitro.

1979 ◽  
Vol 82 (2) ◽  
pp. 555-564 ◽  
Author(s):  
M Porvaznik ◽  
T J MacVittie
Keyword(s):  
Gap Junctions ◽  
Freeze Fracture ◽  
Growth Conditions ◽  
Structural Basis ◽  
Fracture Plane ◽  
Tracer Techniques ◽  
Lanthanum Tracer ◽  
Colony Forming Cell ◽  
Macrophage Colony

An in vitro monocyte-macrophage colony-forming cell (M-CFC) has been detected in canine bone marrow (BM). The colonies derived from these progenitor cells were similar to murine-derived M-CFC (MacVittie and Porvaznik, 1978, J. Cell Physiol. 97:305--314) colonies, since they showed a singular macrophage line of differentiation, a lag of 14--16 days before initiating colony formation, and they survived significantly longer in culture in the absence of colony-stimulating factor (CSF) than granulocyte-macrophage colony-forming cells (GM-CFC). Endotoxin (Salmonella typhosa lipopolysaccharide W)-stimulated dog serum was used as the CSF (7% vol/vol). Canine-derived M-CFC progeny were identified as macrophages on the basis of morphology, phagocytosis, and the presence of Fc receptors for IgG. Gap junctions were observed only in canine BM, M-CFC-derived colonies using freeze-fracture and lanthanum tracer techniques. They were not observed in any GM-CFC-derived colonies. The number of gap junctions observed in freeze-fracture replicas of BM, M-CFC-derived colonies (21 colonies from three different dogs) showed a significantly positive correlation (Kendall's tau = 0.70, P less than 0.001) with the size of the colony fracture plane area. Gap junctions were observed displaying hexagonal lattices of 9.3 nm +/- 0.08 (SE) particles with a center-to-center spacing of 10.4 nm +/- 1.0 (SE) on membrane P-fracture faces. On membrane E-fracture faces, highly ordered arrays of pits with 8.7 nm +/- 0.12 (SE) center-to-center spacing were observed. Arrays of both particles and pits were also observed in fracture-face breakthroughs within a gap junction. Thus, gap junctions can form in vitro between the cells of macrophage progeny of a canine M-CFC under appropriate growth conditions. The significance of this observation is that there may be a structural basis for cell-to-cell collaboration between BM macrophages and other capable cells that either pass into the tissue for modification or develop there into mature cell forms.

Ca-Histochemistry in slime mold plasmodia

1978 ◽  
Vol 36 (2) ◽  
pp. 130-131
Author(s):  
Ulrich Dierkes
Keyword(s):  
Physarum Polycephalum ◽  
Carbon Coating ◽  
Freeze Drying ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Slime Mold ◽  
Staining Procedure ◽  
Protoplasmic Streaming ◽  
Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

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).

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