Freeze-fracture Replication Research Articles

Brain Edema in Delayed Radiation Necrosis: Study of Capillary Ultrastructure

A 42-year-old male was found to have cerebral radiation necrosis 2 years after a split course of radiation therapy following subtotal excision of a left parasagittal meningioma. The surgical specimens were studied by means of conventional ultrathin sectioning and freeze-fracture replication techniques. The electron density of the capillary endothelium was decreased, as was the degree of surface infolding. The width was irregular, and there was an unusually large number of pinocytotic vesicles (37/micron 2). The endothelium was also discontinuous, and the basal laminae were irregular in thickness. The pericapillary space was markedly enlarged, and accumulation of a fibrin-like substance was noted in the extravascular space. The pericapillary zone contained inflammatory cells. However, the tight junctions were preserved and the intercellular junctions were composed of five strands that appeared to consist of continuous particles. The authors conclude that activated pinocytotic vesicles play an important role in increasing vessel permeability in delayed cerebral radiation necrosis and that leakage may occur through interruptions in the endothelium.

Nectar secretion inAbutilon: a new model

Nectary trichomes ofAbutilon striatum secrete copious amounts of sucrose, fructose and glucose. The nectar emerges from transient pores in the cuticle overlying the trichome tip cells. Calculations of the required transmembrane fluxes, either across the tip cell plasmalemma or across the cell membrane of the whole trichome, give very high rates compared with those obtained from other situations in plants and, therefore, cast doubt on the possibility that nectar secretion inAbutilon is an eccrine process. Quantitative evaluation of the possibility of granulocrine secretion, by successive fusion of vesicles with the cell membrane, suggests that this is an even less probable mechanism of secretion. Rapid freezing followed by freeze-substitution or freeze-fracture replication reveals that an extensive “secretory reticulum” (SR) is present within the hair cells. As similar micrographs are obtained from conventional, chemical fixation it is argued that the secretory reticulum is a relatively stable endomembrane system. Freeze-fracture and freeze-substitution micrographs show that this internal membrane system is closely associated with the plasmalemma. Taken together with other structural information, as well as physiological data, it is concluded that prenectar is actively loaded into the secretory reticulum of all trichome cells. Increase in hydrostatic pressure within this compartment leads to the opening of “sphincters” which connect the cisternal space of the SR to the outside of the plasmalemma. Thus a pulse of nectar is forcibly expelled into an apoplastic compartment sealed to the outside by the impermeable cuticle and on the inside by the plasmalemma. As this apoplastic compartment is also sealed at the stalk cell, the only route for pressure release is via the transient pores which overlay the tip cell. Distension renders these patent so that, again, pulsed secretion is observed. This hypothesis overcomes the necessity for envisaging excessively high transmembrane fluxes or rates of vesicle fusion. It would imply the need for a continuing supply of prenectar to the hair cells accompanied by active loading into the SR. This loading process may well be supported by the hydrolysis of sucrose to glucose and fructose and is probably the site where ions and other low molecular weight solutes are filtered from the nectar.

Cryo-SEM of particulate suspensions

Advances in particulate coatings for paper products and high-density recording media require an understanding of orientation, clustering, and cluster aggregation of submicron particles suspended in high volume fractions in liquid. Particles for paper coatings are typically platelets; for magnetic coatings, acicular, tabular, or compact metal oxide or composite grains. One goal is to visualize the dynamic microstructures behind the rheological properties of these suspensions.Fixation of liquid calls for cryo-techniques. Replication for TEM examination is difficult because clusters and cluster aggregates adhere to carbon support films in such a way as to interfere with imaging and even disrupt replicas. The best technique we have found is cryo-SEM with freeze-fracture and deep etching.A procedure similar to that for freeze-fracture replication for TEM was used. For each sample, a small drop was flattened between two copper sheets (Balzers BU 012 056-T and BU 012 054-T).

51] Electron microscopy: Assays involving freeze-fracture and freeze-etching

Publisher Summary This chapter discusses the assays involving freeze-fracture and freeze-etching. The freeze-fracture replication technique, developed by Moor and Muhlethaler has two unique features. First, it exposes structures residing within the lipid bilayer matrix for electron microscopic examination, and second, it permits the study of biological structures “fixed” only by freezing, thus circumventing a number of artifacts linked to other preparation methods for electron microscopy. Though the latter advantage is the main impetus for development of the technique, it is rarely fully exploited because of the major difficulties encountered in freezing native biological fluids without introducing ice-crystal artifacts. The standard geometry of shadowing is unidirectional at an angle of 45° relative to the average fracture plane. The inclination of a particular small area of the fracture face is not known, however, and it can be quite different from the average plane. Therefore, calculations of heights based on the lengths of shadows are very approximate.

Ação da cardioplegia na proteção miocárdica: estudo experimental através das réplicas de criofraturas

Evaluation of myocardial protection by cardioplegic solutions and hipothermia in anoxic hearts. An experimental study. This investigation was performed in a group of 20 dogs submitted to myocardial anoxia by cross-clamping of the ascending aorta for sixty minutes under extracorporeal circulation, followed by reperfusion for 360 minutes. Ultrastructural changes of the myocardium were studied. The animals were divided into 4 groups of 5 dogs each. In the first group (control), myocardial anoxia was induced in normothermia and no cardioplegic solution was administered. In the second group, the body temperature was reduced to 28oC before the onset of myocardial anoxia, and no cardioplegic solution was used. In the third and fourth groups, in addition to 28oC systemic hypothermia, coronary infusion of 4oC cardioplegic solutions were performed immediately and after 30 minutes of clamping of the aorta. The cardioplegic solutions utilized have NaCl, KCl, CaCl2, Lidocaine, Verapamil, NaHCO3, MgSO4 and the infusion vehicle was 5% glucose solution in Group III, and blood in Group IV. Biopsies for freeze-fracture replication were obtained from the anterior wall of the left ventricle, before and after 60 minutes of myocardial anoxia, and 360 minutes after reperfusion. The alterations in sarcolemma and celular organeles due to anoxia or myocardial reperfusion was observed, specially through the diminution and aggregation of intramembranous particles in its P surface. We may conclude that both cardioplegic solutions used, associated to hipothermia, protect efficiently the myocardial cells against anoxia. It was not possible, however, to demonstrate any superiority of the blood solution over an acellular solution. Systemic hypothermia as a single method was not sufficient to offer good protection to the anoxic myocardium. The animals of Group I (control) showed severe compromise of the structure and function of the myocardium.

Epidermal Permeability Barrier: Transformation of Lamellar Granule-Disks into Intercellular Sheets by a Membrane-Fusion Process, a Freeze-Fracture Study

Freeze-fracture replication of lamellar granules and intercellular sheets of the horny layer in mouse, chicken, and snake epidermis reveals a pattern of serial fracture faces which is highly suggestive of polar lipids in a bilayer configuration. The occurrence of alternating wide and narrow fracture faces separated by intervening steps supports the view that epidermal barrier bilayers display lipid asymmetry similar to membranes. Within the lamellar granules, bilayers arrange to form disks which in fact are equivalent to flattened unilamellar liposomes. Stacking of the disks in turn gives rise to the lamellar pattern. After exocytosis into the intercellular space, the disks are arranged parallel to the cell membranes. In tangentially fractured specimens, the cleavage plane jumps back and forth from the plasma membrane to a disk-bilayer, thereby giving rise to the known phenomenon of EF-ridges (on the extracellular fracture face) and PF-grooves (in the plasmatic fracture face) which both represent the level of the plasma membrane sur- or subjacent to the aisles between disks. Concomitantly with the upward movement of the keratinocytes, the ridges and grooves become narrower until they fade away by the second or third cell layer of the stratum corneum. This phenomenon is explained by the fusion of adjacent disks at their highly curved brims due to a mechanism similar to the process of membrane fusion which causes the formation of wide, uninterrupted sheets.

A study of post-secretory maturation ameloblasts in the cat by transmission and freeze-fracture electron-microscopy

Permanent canine and molar tooth germs of kittens were processed for thin-sectioning and freeze-fracture replication. Maturation ameloblasts were divided into ruffle-ended (RA), smooth-ended (SA) and intermediate (IA). RA had an extensive distal ruffled border consisting of deep membrane invaginations, forming complicated extracellular channels. Adjacent RA were connected by extensive distal junctional complexes (zonulae occludentes). The SA had flattened distal cell surfaces and few coated and smooth vesicles in the distal cytoplasm. Adjacent SA were connected by proximal zonulae occludentes, but had only maculae occludentes at their distal ends so permitting broad lateral extracellular spaces to communicate directly with the enamel surface. IA near the RA layer had a ruffled border consisting of deep and narrow membrane invaginations and pinosomes filled with granular material. IA next to the SA layer had no ruffled border but had pinosomes that seemed to originate directly from the distal cell surfaces. IA were polarized and connected by proximal and distal junctional complexes consisting of either zonulae or fasciae occludentes and associated gap junctions.

Structure of Lipid Tubules Formed from a Polymerizable Lecithin

We have studied tubules formed from a polymerizable lipid in aqueous dispersion using freeze-fracture replication and transmission electron microscopy. The polymerizable diacetylenic lecithin 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine converts from liposomes to hollow cylinders, which we call tubules, on cooling through its chain melting phase transition temperature. These tubules differ substantially from cochleate cylinders formed by phosphatidylserines on binding of calcium. The tubules have diameters that range from 0.3 to 1 mum and lengths of up to hundreds of micrometers depending on conditions of formation. The thickness of the walls varies from as few as two bilayers to tens of bilayers in some longer tubules. Their surfaces may be either smooth, gently rippled, or with spiral steps depending on sample preparation conditions, including whether the lipids have been polymerized. The spiral steps may reflect the growth of the tubules by rolling up of flattened liposomes.

Freeze-fracture study of the mechanoreceptive digital corpuscles of mice.

The freeze-fracture replication technique was used to study the mechanoreceptive digital corpuscles in toe pads of mice. The axon terminal plasmalemma had intramembranous particles (IMPs) at a density of 2367 +/- 517 microns-2 (mean +/- S.E.M.) in the P-face and 84 +/- 4 microns-2 in the E-face. Particles were 10 +/- 1.8 nm in diameter in the P-face and 10 +/- 1.5 nm (mean +/- S.D.) in the E-face. Particle-rich and particle-free areas were noted in the P-face. The lamellar cell plasmalemma had IMPs at a density of 3359 +/- 224 microns-2 in the P-face and 265 +/- 95 microns-2 in the E-face. Particles were 10 +/- 1.4 nm in diameter in the P-face and 10 +/- 1.6 nm in the E-face. Non-terminal unmyelinated fibres in the connective tissue compartment of toe pads were also examined: the P-faces of the axolemma and Schwann cell plasmalemma had IMPs at a density of 1356 +/- 283 microns-2 and 1514 +/- 514 microns-2, respectively, while the E-face of these membranes had only a few particles. Particles were 9 +/- 1.2 nm and 10 +/- 1.6 nm in diameter in the P-faces of axon and Schwann cell plasmalemmata, respectively. The results show that the IMPs in terminal axolemma and in lamellar cell plasmalemma have a much higher density than those of non-terminal axons or Schwann cells in myelinated and unmyelinated fibres. In addition, IMPs in the terminal axolemma are larger than those in non-terminal axolemma except for the nodal axolemma. It can be said that plasmalemmata of both the axon terminals and lamellar cells of digital corpuscles are specialized in terms of IMPs, suggesting that they have specific physiological properties in mechanoreceptive functions including mechano-electric transduction.

Cytological damage and cell wall modification in cultured apple cells following exposure to pectin lyase from Monifinia fructigena

Cultured apple cells were rapidly injured by pectin lyase (PL) from Monilinia fructigena. Changes in the staining of the plasmalemma and cytoplasm were observed concomitant with the loss of vital staining. Paramural bodies, containing vesicles apparently derived from the plasmalemma, were present at an early stage of injury. Further injury was indicated by pseudoplasmolysis, increased vacuolation and cytoplasmic collapse. Methylated pectin was rapidly lost from the walls of treated cells. However, the gross integrity of the cell walls appeared little changed during the early stages of injury. Cell wall degradation appeared to accompany rather than precede severe injury. ATPase activity was localized by a lead capture method at the plasmalemma of cultured cells. A considerable increase in lead stain within the cytoplasm occurred after PL treatment but it still remained localized at the plasmalemma even in apparently severely injured cells. Freeze fracture replication did not reveal any major modification of the plasmalemma after PL treatment even when considerable injury had occurred.

Cell-matrix interrelation and cell-to-cell connection in the secretory ameloblast layer of kitten teeth.

To investigate the cell-matrix interrelation and the structure and permeability of the junctional complexes of secretory ameloblasts, molar tooth germs from kittens were examined by means of scanning electron microscopy, routine thin sections and freeze-fracture replication. Scanning electron microscopy showed remarkably dissolved growth fronts of enamel in materials that had been fixed with glutaraldehyde and then subjected to EDTA perfusion for 10 min. By the action of EDTA, intercrystallite spaces in rod and interrod enamel were prominently widened, and their longitudinal ends of crystallites displayed irregular and extremely sparse structures. In enamel rods surrounded entirely by interrod enamel, and in enamel rods of the typical key hole shape with successive interrod enamel participation, the most striking dissolution of crystallites occurred at the boundaries between rod and interrod enamel, where broad expanses of rod-sheath spaces were observed. In thin sections, the Tomes processes of secretory ameloblasts occupying the above rods were rectangular or variations of a rectangular shape, respectively; and interameloblast spaces opened to the enamel growth fronts, which corresponded to the junction between rod and interrod enamel. In enamel rods standing in regular rows and showing the typical arcade shape, the centers of the rods were drastically dissolved and exhibited single and deep slits, whereas the boundaries between rod and interrod enamel showed no wide furrows. The Tomes processes occupying such arcade-shaped rods were typically triangular, and the interameloblast space always joined the type-1 face of process, which is responsible for enamel rod formation. Secretory ameloblast possessed two sets of junctional complexes at the proximal and distal ends of the cell body. The distal one was situated proximally to the Tomes process. Freeze-fracture replication demonstrated the functional structures of these junctions: the proximal junction was fascia occludens, and the distal one incomplete zonula occludens with many free-ending tight junctional strands and interstrand spaces or a less developed irregular junction.

Complementary freeze fracture replication: An example of its use in the study of horizontal cell gap junctions of the carp retina

AbstractDark‐adapted carp retinas were fixed with glutaraldehyde, frozen in liquid nitrogen slush, and their complementary replicas were harvested using a bipolar‐vacuum evaporater. On montage pictures, plasmic (PFF) and ectoplasmic fracture faces (EFF) of the external horizontal cells (EHC) were marked, and PFF of gap junction (GJ) areas were scrutinized by virtue of particulate aggregation visible at relatively low magnification. Summation of digitized values obtained from a complementary pair of replicas was taken to represent all existent GJ areas in an examined EHC surface. A similar method was applied to large horizontal processes (LHP) that are located in the inner‐most part of the internal nuclear layer and currently accepted to be extensions of EHC. The occurrence of fractured faces of plasma membrane was smaller in EHC than LHP. The percentage of GJ areas was about ten times larger in EHC than LHP. Examining complementary replicas at relatively low magnification appears to be useful for facilitating quantitative analysis of intercellular coupling by GJ structures.

Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs.

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.

A freeze-fracture study of ruffle-ended post-secretory ameloblasts.

The post-secretory portion of the rat incisor enamel organ was prepared for routine transmission electron microscopy and freeze-fracture replication in order to define further the structural surface features of the ruffle-ended ameloblasts. Surface views of the distal plasma membrane of the ruffle-ended ameloblasts revealed a well-developed zonula occludens junction with from six to ten rows of tight junctional strands. Gap junctions were also observed just proximal to the tight junctional strands. The membranes of the ruffled border contained a rich supply of intramembrane particles (IMP). The IMPs were approximately 7 to 8 nm in diameter and preferentially located on the P-face profiles of the membrane. The density of IMPs on the membranes of the ruffled border was higher than that on the lateral borders of the cell. It is suggested that the IMPs of the ruffled border may represent enzymatic proteins in the basal cell membrane of absorptive ameloblasts. In addition, the large, highly-developed zonula occludens appeared structurally capable of sealing the intercellular spaces between the ruffle-ended ameloblasts.

Morphological Analysis of the Golgi Apparatus in Rat Amelogenesis as Revealed by the Ur-Pb-Cu Block Staining Method and Freeze-Fracture Replication

Morphological Analysis of the Golgi Apparatus in Rat Amelogenesis as Revealed by the Ur-Pb-Cu Block Staining Method and Freeze-Fracture Replication

Intercellular junctions in the cells of the human enamel organ as revealed by freeze-fracture

Examined by thin sections and freeze-fracture replication techniques, secretory ameloblasts possessed two sets of the junctional complexes at both proximal and distal ends of the cell bodies, which consisted of tight junctions and occasional gap junctions and desmosomes. The proximal tight junction was fascia occludens, whereas the distal tight junction was zonula occludens. Between adjacent ameloblasts, mature gap junctions were frequent. The stratum-intermedium cells were connected to each other and to the stellate-reticulum cells and ameloblasts by well-developed desmosomes, gap junctions and fascia or macula-type tight junctions. Stellate-reticulum cells were inter-connected by many extensive cytoplasmic processes, in which well-developed desmosomes, small gap junctions and occasional macula-type tight junctions appeared. Thus fascia or macula-type tight junctions as well as many desmosomes seem to serve in mechanical, cell-to-cell adhesion during tooth formation. Frequent and large gap junctions between adjacent stratum-intermedium cells and between the stratum intermedium and the base of the ameloblast suggest that, in relation to enamel formation, these two cell layers form a functional unit.

Cryomicroscopy of liquid and semiliquid specimens: Direct imaging versus replication

Direct imaging techniques are compared to freeze-fracture replication as applied to electron microscopic specimens of frozen liquid and semiliquid systems. The processes of cryofixation and sample preparation are discussed as are the problems of contrast and resolution. The pros and cons of the two techniques indicate that direct imaging and freeze fracturing are complementary rather than alternative techniques.

Glial membrane specializations and the compartmentalization of the lamina ganglionaris of the housefly compound eye.

Membrane specializations in the lamina ganglionaris of the housefly are investigated using conventional thin-section EM, freeze-fracture replication and the diffusion of colloidal lanthanum. All glial cells in the lamina are coupled by gap junctions. Desmosomes also link all glia except the epithelial glia. Extensive glia-glial and glia-neuronal septate junctions are present in the pseudocartridge zone and nuclear layer. Septate junctions in the nuclear layer intermingle with bands of interglial and glia-neuronal tight junctions. Tight junctions are also found between satellite and epithelial glia at the border of the nuclear and plexiform layers, between adjacent epithelial glial cells in the plexiform layer, between epithelial and marginal glia at the proximal boundary of the optic neuropil, between marginal glial cells, and between marginal glia and axons. Colloidal lanthanum, introduced through an incision in the cornea, penetrates the retina but is occluded from the neuropil by septate junctions in the pseudocartridge zone. The disposition of tight and septate junctions is described in relation to the compartmentalization of the lamina. Two major compartments are delineated. The first represents the nuclear layer and contains the cell bodies of second-order visual neurons (monopolar neurons). The second compartment constitutes the plexiform layer of the lamina. Within the plexiform layer, each optic cartridge is partitioned into a separate subcompartment. Also, tracheoles and axons of long visual fibres are isolated from the optic cartridges by glial tight junctions. Morphological evidence for compartmentalization is correlated with previously established electrical properties of the insect lamina ganglionaris.

Connection of lamellar body to intracellular membrane system in type II alveolar cells of human lung.

We examined the type II alveolar cells of the human lung with an electron microscope, using the freeze-fracture replication technique, and found connections of some lamellar bodies to barlike structures, which seemed to be a kind of endoplasmic reticulum system. This morphologic finding suggested that the human lung has an additional pathway for surfactant synthesis in type II alveolar cells.

Thin-section, tracer, and freeze-fracture study of the smooth-ended maturation ameloblasts in rat incisors.

The morphology and functional roles of the smooth-ended maturation ameloblasts (SAs) of rat incisors were examined by means of routine thin sections, tracer experiments, and freeze-fracture replication. SAs possessed two sets of junctional complexes consisting of tight junctions (fasiae occludentes) and gap junctions at the proximal and distal ends. Neither the proximal nor the distal junctional complex formed a complete barrier around the cell; intravenously injected horseradish peroxidase (HRP) reached the developing enamel surface through SAs extracellular spaces. SA supranuclear cytoplasm included such various cytoplasmic vesicles as the multivesicular body (MVB), large and vacuoles, dense body, and coated vesicles. The HRP that reached the enamel surface was incorporated into some coated vesicles and small vacuoles via coated pits and membrane invaginations of the distal cell surface of SA. Then, in the process of time, it migrated into the MVB and large endocytic vacuoles. These results indicate that the SA layer forms an extracellular transfer route for metabolites between papillary layer and the enamel surface and that SAs absorb exogenous protein.