Freeze-fracture Technique Research Articles

41] Purification of yeast vacuolar membrane H+-ATPase and enzymological discrimination of three ATP-driven proton pumps in Saccharomyces cerevisiae

Publisher Summary This chapter describes the purification and properties of vacuolar membrane H + -ATPase, and its enzymological discrimination from other ATP-driven proton pumps, F 1 F 0 -ATPase from mitochondria, and H + translocating ATPase from the plasma membrane of Saccharomyces cerevisiae . In the yeast Saccharomyces cerevisiae , vacuoles are the largest organelles, occupying about 25% of the cell volume, and they are postulated to function as lysosomes and as a storage compartment in which the bulk of basic amino acids, S-adenosylmethionine, and polyphosphates are localized. These observations have suggested the existence of some specific transport systems in the vacuolar membranes. Vacuoles are the largest organelles in yeast cells, but they contain only 0.2% of total cell protein. To analyze the functions of vacuoles and vacuolar membrane ATPase, it is crucial to obtain pure materials in hand. The orientation and size of vacuolar membrane vesicles are studied by electron microscope using a freeze-fracturing technique. Many small particles are seen on the concave face of the vesicles, while the convex face is fairly smooth.

Arthropod immune system. III. Septate junctions in the hemocytic capsule of the german cockroach, Blattella germanica

Granulocytes (GRs) and/or plasmatocytes (PLs), the two major immunocytes in arthropods, participate in cellular encapsulation of foreign tissue. Although gap and desmosome junctions have been reported in insect capsules, smooth septate junctions are being reported for the first time by both thin section and freeze-fracture techniques in Blattella germanica. In 7-day-old capsules, the septa are 23 nm thick, faintly ‘scalloped’ and slightly curved in appearance; the interseptal space has a periodicity of about 5 nm. In freeze-fractured capsules, the septa are associated on both sides with the corresponding intramembranous structures, belonging to the plasma membranes of the two junction-forming GRs. The intercellular space is 27 nm wide. There are 36–40 septa/1 μm junctional length. The junctions show furrows on the extracellular fracture face (E) and the complementary regular rows of intramembranous particles on the cytoplasmic face (P). The septate junctions often occur in the region of the capsule that also shows the presence of gap junctions, but only rarely that of desmosomes. The septate junctions are in close proximity with mitochondria. It is suggested that the function of these junctions is to produce compact capsules.

Freeze-fracture studies of myofiber plasma membrane in X chromosome-linked muscular dystrophy (mdx) mice.

The structure of the muscle plasma membrane of extensor digitorum longus muscles of X chromosome-linked muscular dystrophy (mdx) mice was studied by freeze-fracture technique at several time points after birth. The common denominator of the abnormalities was the decreased density of orthogonal arrays throughout all the time points examined. The results demonstrated that the ultrastructural features of the muscle plasma membrane alterations in mdx mice were similar to those in Duchenne dystrophy.

Distribution of filipin-sterol complexes in Japanese abalone sperm during the acrosome reaction.

The distribution of membrane sterols in the acrosome reaction of Japanese abalone sperm was studied by freeze-fracture techniques using a polyene antibiotic, filipin, as a probe for membrane sterols. In the unreacted sperm, the plasma membrane covering the acrosome showed a random distribu-tion of filipin-sterol complexes, while in the outer acrosomal membrane, filipin-sterol complexes were rarely observed. In the initial stage of the acrosome reac-tion, filipin-sterol complexes were found densely distributed in the plasma mem-brane throughout the acrosomal region. On the subjacent outer acrosomal mem-brane, filipin-sterol complexes were densely packed in the apical area, cor-responding to the truncated cone region. This apical area consistently showed such regionalization of filipin-sterol complexes during the acrosome reaction. As the acrosomal process elongated, the intramembrane particle (IMP)-free areas which appeared in the acrosomal process membrane were found to be displaced by densely distributed filipin-sterol complexes. These results indicate a possible regionalization of membrane sterols related to the acrosome reaction.

Junctional specializations in the seminiferous epithelium of an insect ( Triatoma infestans): A freeze fracture and lanthanum tracer study

Intercellular junctions in the seminiferous epithelium of the testis of Triatoma infestans were examined by conventional electron microscopy, tannic acid fixation, electronopaque tracers, and freeze-fracture techniques. Distinctive aspects of the intercellular junctions are described in different regions of the testis follicles. In the basal region, close to the basal lamina, smooth septate junctions intermingled with gap junctions were observed between sustentacular cells. In the parabasal regions of the seminiferous epithelium, plated septate junctions, ‘molluscous and arthropod’ type (according to the classification of Green, 1981), were observed. Over the above junctions, in the central regions, also located between sustentacular cell membranes, parallel rows of intramembrane particles form successive belts of widely spaced septate junctions in an atypical configuration. Invertebrate gap junctions were also observed between adjacent spermatocyte membranes.

Effect of proteases and other treatments on the proliferative assembly of tight junction strands in the rat prostate tissue

As already observed with the freeze-fracture technique by Kachar and Pinto da Silva (1981) incubation of excised rat ventral prostate tissue at 37 degrees C results in proliferation of tight junction strands over the lateral membranes of the epithelial cells, an effect not occurring after incubation at 4 degrees C. Our investigations have shown that a pretreatment with protease inhibitor Contrykal at 4 degrees C suppresses the proliferation of tight junction strands at 37 degrees C. On the other hand the added proteases trypsin and pronase induce the proliferation already at 4 degrees C. The effect was lowered by higher concentrations of pronase. Incubation with solutions of the non-ionic detergent Triton X-100 at 4 degrees C could also induce tight junction proliferation, whereas this effect was nearly absent after an incubation at 4 degrees C with a hyperosmotic solution of arabinose and with 50% acetone. No relation was found between proliferation of tight junction strands and cluster formation of intramembraneous particles. In conclusion of the results a small protein or a peptide resembling fusions proteins is assumed as trigger for formation of tight junction strands, acting by destabilization of the bilayer arrangement of membrane lipids.

Formula omitted]: structural preferences in pure and mixed model membranes

The structural preferences of the pH-sensitive phospholipid, N- succinyldioleoylphosphatidylethanolamine ( N- succinyl-DOPE ), have been examined alone and in mixtures with DOPE by 31P-NMR, fluorescence energy transfer, and freeze-fracture techniques. The basic polymorphic behavior of pure N- succinyl-DOPE and DOPE/N- succinyl-DOPE lipid systems and the influence of calcium and pH were investigated. It is shown that, similar to other negatively charged acidic phospholipids, N- succinyl-DOPE adopts the bilayer organization upon hydration. This structure is maintained at both pH 7.4 and 4.0 in the presence or absence of calcium. In the mixed lipid system, N- succinyl-DOPE can stabilize the non-bilayer lipid, DOPE, into a bilayer structure at both pH 7.4 and 4.0 at more than 10 mol% N- succinyl-DOPE , although a narrow 31P-NMR lineshape is observed at acidic pH values. This corresponds to the presence of smaller vesicles as shown by quasi-elastic light scattering measurements. Addition of equimolar calcium (with respect to N- succinyl-DOPE ) to the DOPE/N- succinyl-DOPE systems induces the hexagonal H II phase at both pH values. In unilamellar systems with similar lipid composition the addition of Ca 2+ results in membrane fusion as indicated by fluorescence energy-transfer experiments. These findings are discussed with regard to the molecular mechanism of the bilayer to hexagonal H II phase transition and membrane fusion and the utility of N- succinyl-DOPE containing pH-sensitive vesicles as drug-delivery vehicles.

Structural relationships in the human odontoblast layer, as demonstrated by freeze-fracture electron microscopy

The structural relationships in the odontoblastic cell layer of fully developed permanent human teeth were studied with the freeze-fracture technique. A good three-dimensional comprehension of the spatial arrangement of the cells was obtained. The odontoblasts displayed three types of membrane specializations. At the apical part, spot desmosomes (macula adherens) were abundant. Numerous gap junctions (nexuses) were distributed all over the cells. Tight junctions (zonula occludens) were found apically on the odontoblasts in some teeth. Between the tightly packed odontoblasts, fibers with a diameter of 0.1 to 1.0 μm were seen running through the cell layer. The true nature of these cell processes could not be established, but one group of them had features (shape and diameter) in common with unmyelinated nerve fibers previously described. Such fine caliber fibers regularly formed morphological contacts (gap junctions) with the odontoblasts. Fibrillar structures of the so-called von Korff type between odontoblasts were not observed. The odontoblasts were also seen to be in close contact with the subodontoblastic fibroblasts via gap junctions and desmosomes.

Freeze-fracture studies of reactive myelinated nerve fibres after diffuse axonal injury.

We have studied the axonal and myelin sheath response in diffuse axonal injury after angular acceleration using the freeze-fracture and thin section techniques. It was found that the glial-axonal junction was intact until 1 h after injury. But upon loss of the nodal axolemma specialisations, after 3 to 4 h, the dimeric particles of the glial-axonal junction (GAJ) were lost and, by 6 h, the myelin lamellae became separated from the axonal remnant. There was a correlated loss of glial membrane specialisations of the GAJ during this separation. In the internodal region a suggestion of membrane damage occurred after 20 min but discrete myelin dislocations (particle-free areas) were not found until 1-h survival and were extensive by 6 h. Areas of loosely organised myelin occurred between intact axons at 7-28 days after injury. No evidence for growth cone formation was obtained.

Characterization of dodecylphosphocholine/myelin basic protein complexes

The stoichiometry of myelin basic protein (MBP)/dodecylphosphocholine (DPC) complexes and the location of protein segments in the micelle have been investigated by electron paramagnetic resonance (EPR), ultracentrifugation, photon correlation light scattering, 31P, 13C, and 1H nuclear magnetic resonance (NMR), and electron microscopy. Ultracentrifugation measurements indicate that MBP forms stoichiometrically well-defined complexes consisting of 1 protein molecule and approximately 140 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into DPC/MBP aggregates. EPR spectral parameters and 13C and 1H NMR relaxation times indicate that the addition of MBP does not affect the environment and location of the labels or the organization of the micelles except for a slight increase in size. Previous results indicating that the protein lies primarily near the surface of the micelle have been confirmed by comparing 13C NMR spectra of the detergent with and without protein with spectra of protein/detergent aggregates containing spin-labels. Electron micrographs of the complexes taken by using the freeze-fracture technique confirm the estimated size obtained by light-scattering measurements. Overall, these results indicate that mixtures of MBP and DPC can form highly porous particles with well-defined protein and lipid stoichiometry. The structural integrity of these particles appears to be based on protein-lipid interactions. In addition, electron micrographs of aqueous DPC/MBP suspensions show the formation of a small amount of material consisting of large arrays of detergent micelles, suggesting that MBP is capable of inducing large changes in the overall organization of the detergent.

Lysophosphatidyl choline-induced demyelination. A freeze-fracture study.

Focal demyelination was produced in the rat sciatic nerve by microinjection of lysophosphatidyl choline (LPC). The demyelinating lesion was examined over the following 48 h using the freeze-fracture technique to examine myelin, Schwann cell and axonal membranes. Myelin lamellae were replaced by myriad spherical or oval membranous vesicles. The axonal and Schwann cell plasma membranes remained intact and the latter showed a large increase in caveolae-associated pores in some nerve fibres. The lysis of myelin lamellae and membranous vesicle formation are related to the known action of LPC on myelin and its membrane fusogenic properties. The importance of calcium ion influx and membrane protein aggregation and depletion in vesiculation are discussed.

Plasmodium falciparum: Freeze-fracture of the gametocyte pellicular complex

Freeze-fracturing has been used to study the architecture of the pellicular complex of the gametocytes of Plasmodium falciparum. The gametocyte is surrounded by three membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes is split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on their surfaces. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. In gametocytes, unlike in sporozoites, the intramembranous particles are always distributed randomly and lack any definite pattern or orientations. A unique feature of gametocytes revealed by the freeze-fracturing technique is the presence of several transverse sutures on the middle membrane that encircle the gametocyte and give it a segmented appearance.

Leishmania chagasi: In vitro differentiation of promastigotes monitored by flow cytometry

A sequential development from a less infective to an infective stage of Leishmania promastigotes growing in culture has been previously reported. The aim of this work was to investigate whether freeze-fracture electron microscopy and flow cytometry would be able to provide some reliable morphological markers of in vitro differentiation of Leishmania chagasi promastigotes. The flow cytometry technique discriminates between the L. chagasi promastigotes from the different stages of their in vitro differentiation. The “forward scatter” intensity of the parasite, very high 15 hr after seeding when the parasites were very condensed and with a high DNA content per particle, strongly decreased during the culture course. Parallel experiments have shown a striking correlation between forward scatter intensity, growth curves, and infectivity of promastigote populations. By contrast, freeze-fracture techniques showed that in either less infective or infective promastigote plasma membranes, the intramembrane particles density in protoplasmic fracture faces (about 2800/μm 2) and in exoplasmic fracture faces (about 1000/μm 2) was independent of the time of cultivation. The amount of filipin lesions, which reflects the cholesterol content within the plasma membrane, was also constant throughout the culture course. Both data suggest that the architecture of the plasma membrane is an intrinsic characteristic of the promastigote stage. This study shows that whereas freezefracture electron microscopy does not provide markers for the differentiation of Leishmania promastigotes, flow cytometry may on the other hand be of value as a screening test for promastigote populations allowing the characterization of their developmental stages in in vitro Cultures.

Small vesicle bouton synapses on the distal half of the lateral dendrite of the goldfish Mauthner cell: freeze-fracture and thin section study.

To understand principles of synaptic integration, it is necessary to define the types of synapses on a particular neuron and their distribution. Thin sectioning and double replica freeze-fracture techniques were employed to characterize the small vesicle bouton (SVB) synapses on the distal half of the Mauthner (M) cell lateral dendrite, which probably mediate a remote dendritic inhibition. Three morphologically distinct SVB synapses, types A, B, and C, were found. These three SVB synapses form roughly 90% of the synapses on the distal half of the lateral dendrite, with types A and B being most common. The SVB A synapse is characterized by mostly oval and round synaptic vesicles, a discrete presynaptic active zone with a highly variable shape, and a postsynaptic active zone with no apparent particle aggregate in either the E or P face. At the SVB B synapse, most of the synaptic vesicles are flat. A very high particle density is present throughout the presynaptic P face, and vesicle attachment sites are dispersed over much of the presynaptic membrane. Postsynaptic P face particle aggregates are subjacent to the presynaptic vesicle attachment sites, and are often large and anastomosing. The SVB C synapse is characterized by synaptic vesicle profiles that vary from flattened to round. The SVB C cytoplasm was unclouded by the flocculent material that characterized SVBs A and B. The presynaptic active zones at the SVB C synapse are discrete, and macular or oblong. No particle aggregates are apparent in the postsynaptic active zone. Small, macular particle aggregates were found in nonactive zone regions of the postsynaptic E face of all three types of SVBs. Small subsurface cisterns were also observed underlying the M cell membrane at all three types of SVB synapses. Neither the postsynaptic E face aggregates nor the subsurface cisterns were ever observed directly subjacent to presynaptic active zones, but were often seen adjacent to active zones. Short, straight rows of particles and short cylinders were often seen in both pre- and postsynaptic surrounding zone regions of SVB A and C synapses. These structures are thought to represent tight junctions.

Estrogen-induced synaptic remodelling in adult rat brain is accompanied by the reorganization of neuronal membranes

Adult cycling female rats were injected with estradiol valerate (2 mg/100 g body wt.), a treatment which has previously been shown to result in synaptic remodelling in the arcuate nucleus and constant vaginal estrus. During the 32 weeks following estrogen treatment, arcuate nucleus neuronal plasma membranes were quantitatively assessed for intermembrane (IMP) number and size using freeze-fracture techniques. Neuronal membranes from untreated cycling females, females injected with oil and untreated males were also studied. Untreated rats had dimorphic sexual phenotypes in membrane organization; female rats had more IMP than males, mainly due to greater numbers of small ( < 10 nm) particles. These sex differences were observed in perikarya and dendritic shafts, but not in dendritic spines. Following estrogen treatment the density of IMP in membranes from females decreased. The IMP changes were found only in neuronal perikarya and dendritic shafts, not in dendritic spines, and were mainly due to a massive decrease in the number of small ( < 10 nm) IMP which was only partially offset by an increase in the number of large ( ⪖ 10 nm) IMP. Thus, by 32 weeks after estradiol valerate treatment, the number and size of IMP in neuronal membranes from females were not different from those seen in normal males. These results strengthen the idea that estradiol may affect the turnover of certain neuronal membrane components in sex-steroid sensitive areas of the brain.

Junctions between lens cells in differentiating cultures: Structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated “lentoids” are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eightfold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.

Freeze-fracture study of the lateral-line canal organ of the Japanese sea eel, Lincozymba nystromi.

Specializations of apical surfaces of hair cells, supporting cells and marginal cells in the lateral-line canal organ of Japanese sea eel, Lincozymba nystromi, were examined with a freeze-fracture technique. Apical surfaces of hair cells have a lower density of intramembrane particles (IMP) than those of the surrounding supporting cells. Density of IMP on the stereocilia is almost the same as that on the apical surface of hair cells. Junctions between hair and supporting cells were tighter than those between two supporting cells; those between supporting and marginal cells were tighter than those between hair and supporting cells, and those between two marginal cells were the tightest in the lateral-line canal organ.

Effects of ethanol and prostaglandin on rat gastric mucosal tight junctions

The effect of 16,16 dimethyl prostaglandin E 2 (PG) upon tight junctions (TJs) of adjacent surface mucous cells (SMCs) as a possible mechanism by which PGs mediate their protective effects was studied using transmission electron microscopy (TEM) and freeze fracture (FF) techniques. Fasted rats were subcutaneously injected with 10 μg/kg of PG or an equal volume of saline, followed 30 min later by 1 ml of oral 100% ethanol or saline. Ten or sixty minutes later, animals were sacrificed and stomach blocks were prepared for TEM or FF using standard techniques. Electron micrographs (×60,000) were obtained and the distance between SMC inner membrane leaflets was measured with a micrometer and expressed as TJ width. Stomach blocks for FF were stored at 4°C, cryoprotected, freeze fractured, and photographed by TEM (×30,000). At 0.5-μm intervals, measurements of TJ strand number and depth were made. No statistical differences were found in TJ width or strand number of SMCs among the various experimental groups when compared with controls at each sacrifice time. At the 60 but not 10 min sacrifice time, TJ depth was greatly increased in cells treated with 10 μg/kg PG prior to ethanol exposure ( P < 0.025) in contrast to control mucosae. We conclude that 16,16-dimethyl PGE 2 induces no changes in the structural composition of TJs as a possible explanation for its protective properties. The increase in TJ depth observed in ethanol exposed mucosa following PG pretreatment at the 60 min sacrifice time is most likely related to the architectural restructuring that occurs during reconstitution of damaged surface epithelium.

Postreplication labeling of E‐leaflet molecules: Membrane immunoglobulins localized in sectioned, labeled replicas examined by TEM and HVEM

Conventional freeze-fracture techniques were combined with immunogold labeling and with plastic embedding and sectioning to analyze the distribution of membrane immunoglobulins (mIgs) and their associated intramembrane particles (IMPs) in E-face replicas of murine B-lymphocyte plasma membranes. Immunogold labels were applied to cells after the process of freeze-fracture and replication. Conventional stereoscopic transmission electron microscopic examination of sectioned, labeled replicas (SLRs) revealed that the gold-labeled mIgs were bound to and localized on the outer leaflets of split and replicated membranes. The gold labels were attached to the external determinants of the mIg molecules, which were retained beneath and contiguous with the replicated E-faces. The mIgs were also localized on the external surface of unreplicated microvilli. In addition, thick sections examined by high-voltage transmission electron microscopy (HVEM) revealed large expanses of replica with well-resolved IMPs. mIgs colocalized with small-diameter (less than 60 A) IMPs in E-face replicas of B-lymphocytes whose mIgs were patched by anti-immunoglobulin. Thus, postreplication E-surface labeling of split and replicated membranes is a high-resolution technique that is suitable for the study of membrane protein distribution in E-face replicas and contiguous nonreplicated tissue.

A freeze fracture technique for examining human hair medulla with Scanning and Transmission Electron Microscopy

Examination of hair medulla by transmission electron microscopy (TEM) is difficult because of the keratinous composition of hair and because of sectioning problems that result from insufficient infiltration and nonmiscibility of hair with embedding resins, even those of low viscosity. Although longitudinally cutting or tearing fibers will expose the medulla for embedment or direct viewing, considerable disruption occurs in its structure. Less disruption results from the use of freeze fracture techniques for either transmission or scanning electron microscopy (SEM).Freshly plucked human scalp and beard hairs were submersed in liquid nitrogen for a minimum of three minutes, held at proximal and distal ends with Dumont #10 tweezers, and slowly bent to an arc until the specimens broke at the apex. Customarily, clean bevelled fractures occurred along the tips of the arcs and exposed not only the medulla but also the cortex and cuticle. The fractured specimens were then removed from liquid nitrogen.