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 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 (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 Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

1985 ◽  
Vol 101 (4) ◽  
pp. 1386-1399 ◽  
Author(s):  
F Torri-Tarelli ◽  
F Grohovaz ◽  
R Fesce ◽  
B Ceccarelli
Keyword(s):  
Freeze Fracture ◽  
Transmission Time ◽  
Fracture Plane ◽  
Hydrophobic Domain ◽  
Thin Sections ◽  
Total Transmission ◽  
Quick Freezing ◽  
Section Electron ◽  
Nerve Muscle ◽  
Stimulation Of

We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4-aminopyridine, 10 mM Ca2+, and 10(-4) M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze-fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of less than 1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15 degrees K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5 - 10 micron, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was approximately 3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released. Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well-preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed.

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 Arrest of membrane fusion events in mast cells by quick-freezing.

1980 ◽  
Vol 86 (2) ◽  
pp. 666-674 ◽  
Author(s):  
D E Chandler ◽  
J E Heuser
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Membrane Fusion ◽  
Extracellular Space ◽  
Freeze Fracture ◽  
Secretory Process ◽  
Membrane Pores ◽  
Peritoneal Mast Cells ◽  
Quick Freezing ◽  
Rat Peritoneal Mast Cells

We have used quick-freezing and freeze-fracture to study early stages of exocytosis in rat peritoneal mast cells. Mast cells briefly stimulated with 48/80 (a synthetic polycation and well-known histamine-releasing agent) at 22 degrees C displayed single, narrow-necked pores (some as small as 0.05 micrometer in diameter) joining single granules with the plasma membrane. Pores that had become as large as 0.1 micrometer in diameter were clearly etchable and thus represented aqueous channels connecting the granule interior with the extracellular space. Granules exhibiting pores usually did not have wide areas of contact with the plasma membrane, and clearings of intramembrane particles, seen in chemically fixed mast cells undergoing exocytosis, were not present on either plasma or granule membranes. Fusion of interior granules later in the secretory process also appeared to involve pores; granules were often joined by one pore or a group of 2-4 pores. Also found were groups of extremely small, etchable pores on granule membranes that may represent the earliest aqueous communication between fusing granules.

scholarly journals Glycosphingolipid GM3 is localized in both exoplasmic and cytoplasmic leaflets of Plasmodium falciparum malaria parasite plasma membrane

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shiomi Koudatsu ◽  
Tatsunori Masatani ◽  
Rikako Konishi ◽  
Masahito Asada ◽  
Hassan Hakimi ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Plasma Membrane ◽  
Malaria Parasite ◽  
Protein Transport ◽  
Vaccine Development ◽  
Parasitophorous Vacuole ◽  
Biological Membrane ◽  
Freeze Fracture ◽  
Plasmodium Falciparum Malaria ◽  
Quick Freezing

AbstractLipid rafts, sterol-rich and sphingolipid-rich microdomains on the plasma membrane are important in processes like cell signaling, adhesion, and protein and lipid transport. The virulence of many eukaryotic parasites is related to raft microdomains on the cell membrane. In the malaria parasite Plasmodium falciparum, glycosylphosphatidylinositol-anchored proteins, which are important for invasion and are possible targets for vaccine development, are localized in the raft. However, rafts are poorly understood. We used quick-freezing and freeze-fracture immuno-electron microscopy to examine the localization of monosialotetrahexosylganglioside (GM1) and monosialodihexosylganglioside (GM3), putative raft microdomain components in P. falciparum and infected erythrocytes. This method immobilizes molecules in situ, minimizing artifacts. GM3 was localized in the exoplasmic (EF) and cytoplasmic leaflets (PF) of the parasite and the parasitophorous vacuole (PV) membranes, but solely in the EF of the infected erythrocyte membrane, as in the case for uninfected erythrocytes. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was localized solely in the PF of erythrocyte, parasite, and PV membranes. This is the first time that GM3, the major component of raft microdomains, was found in the PF of a biological membrane. The unique localization of raft microdomains may be due to P. falciparum lipid metabolism and its unique biological processes, like protein transport from the parasite to infected erythrocytes.

Freeze-etch visualization of the outer surface of myoblasts in culture

1986 ◽  
Vol 64 (3) ◽  
pp. 194-204 ◽  
Author(s):  
Kathryn R. Barber ◽  
Ingrid E. Mehlhorn ◽  
Grace Párraga ◽  
Chris W. M. Grant
Keyword(s):  
Cell Membrane ◽  
Outer Surface ◽  
Cultured Cells ◽  
Freeze Fracture ◽  
Receptor Interaction ◽  
Fracture Plane ◽  
Similar Distribution ◽  
Surface Receptors ◽  
Freeze Fracture Electron Microscopy ◽  
Small Clusters

The Pfenninger device is one of several types of specimen holders designed to permit freeze-fracture electron microscopy of cultured cells growing attached to solid substrates. It achieves this by directing a fracture plane horizontally through a monolayer of cells frozen with overlying medium (without need for prior disruption of cell attachments or relationships). The end result is a platinum-shadowed replica of the cell membrane hydrophobic interior. Here we describe the features seen when this traditional fracture step is followed by a lengthy etching step, making possible views of the cell membrane outer surface with a resolution 100 × better than that of fluorescence microscopy. Because of the technical difficulties involved, such views have in past been restricted to samples which may be handled in suspension, particularly blood cells and model membranes. Thus we have been able to examine extensive regions of the myoblast outer surface (the so-called etch face) at a magnification that permits visualization of details on the order of individual macromolecules. Prominent clumps of glycocalyx material occupying some 50% of the surface can be readily resolved as a closely spaced network of uniformly distributed 20- to 60-nm irregular granular patches. Receptors for wheat germ agglutinin were found to be associated almost exclusively with these surface prominences, so that bound lectin tended to exist in a uniform distribution of small clusters corresponding to the patches described above. When cells were not fixed until 15 min after lectin addition there was visibly more binding, but in a similar distribution. The details of cell surface architecture recorded here at a resolution of 2–4 nm are well below the limit of resolution of light microscopy and complement existing studies by fluorescence techniques. The presence of surface receptors in small patches reinforces the possibility that some literature observations of receptor interaction may be explained on the basis of direct receptor–receptor contact.

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 Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic “honeycomb” surface coat

2005 ◽  
Vol 169 (2) ◽  
pp. 269-283 ◽  
Author(s):  
John Heuser
Keyword(s):  
Lipid Bilayer ◽  
Freeze Fracture ◽  
Fracture Plane ◽  
Honeycomb Lattice ◽  
Surface Coat ◽  
Membrane Bilayer ◽  
Thin Sections ◽  
Single Membrane ◽  
Quick Freezing ◽  
Infected Cells

Three-dimensional “deep-etch” electron microscopy (DEEM) resolves a longstanding controversy concerning poxvirus morphogenesis. By avoiding fixative-induced membrane distortions that confounded earlier studies, DEEM shows that the primary poxvirus envelope is a single membrane bilayer coated on its external surface by a continuous honeycomb lattice. Freeze fracture of quick-frozen poxvirus-infected cells further shows that there is only one fracture plane through this primary envelope, confirming that it consists of a single lipid bilayer. DEEM also illustrates that the honeycomb coating on this envelope is completely replaced by a different paracrystalline coat as the poxvirus matures. Correlative thin section images of infected cells freeze substituted after quick-freezing, plus DEEM imaging of Tokuyasu-type cryo-thin sections of infected cells (a new application introduced here) all indicate that the honeycomb network on immature poxvirus virions is sufficiently continuous and organized, and tightly associated with the envelope throughout development, to explain how its single lipid bilayer could remain stable in the cytoplasm even before it closes into a complete sphere.

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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