Freeze-fracture characteristics of the ocellar retina of the honeybee,Apis mellifica carnica pollm. (Hymenoptera : Apidae)

Freeze-fracture electron microscopy has been utilized to investigate membrane specializations in the dark-adapted retina of the ocellus of worker honeybee, Apis mellifica (Hymenoptera : Apidae).The distal segment of the photoreceptor cell is unilaterally composed of short microvilli, termed rhabdomeres. All microvilli are oriented in the same direction. Transversally fractured rhabdomerss show the typical hexagonal arrangement of the microvillar profiles. The plasma membrane limiting each microvillus, is rich in particles (4000/μm² on the protoplasmic (P) face). Two kinds of P-face particles are seen: one type is about 4nm in diameter and the other has diameters of 8 – 10nm. Because of their size and density, these particles may mainly correspond to photopigments. Plaques of exoplasmic (E) face particles present in the glial cells sheathing the photoreceptor cells, are thought to represent gap junctions. Special differentiations of the smooth endoplasmic reticulum (ER) are found in the perirhabdomeric cytoplasm closely associated with microvilli. The membranes of the ER are wound up to form large concentric bodies. These bodies may play an important role in formation of new microvilli. Furthermore, conspisuous bodies with stomata are found in the perirhabdomeric cytoplasm.     

A helical,polymeric extracellular protein associated with the luminal surface of Haemonchus contortus intestinal cells

The structure of the intestinal cells of the parasitic nematode Haemonchus contortus is described. The cells have numerous microvilli about 0.09 μ in diameter; most being 5.5–7.5 μ in length. The microvillar (plasma) membrane is coated with a layer of amorphous material (glycocalyx) about 60 Å thick which is electron dense in sectioned preparations. Associated with the surface of this material, and filling the spaces between the microvilli, are filaments in the form of helices about 400 Å in diameter and of variable pitch. The helices appear to be flexible but they are aligned approximately with the long axes of the microvilli. There are up to ten helices per microvillus; they extend beyond the tips of the microvilli and are up to 10 μ long. The material has been obtained nearly pure in small amounts. It is primarily protein and it is proposed that it should be called contortin. The monomeric form (of molecular weight about 60,000) has been identified with a Y-shaped structure with arms about 45 Å long and 25 Å wide seen in negatively stained preparations. The helical filament appears to be formed by lateral polymerization of pairs of these units.     

Integument of the tapeworm scolex. 1. Freeze-fracture of the syncytial layer, microvilli and discoid bodies

The tegument of cestodes is the most important and structurally complex metabolic interface between these parasites and the hostile environment in which they reside. In spite of the complex metabolic, regulatory and immunological properties of this layer of syncytial cytoplasm, which are relatively well known, the detailed fine structural anatomy of the cestode tegument remains equivocal. The present study therefore reports the freeze-fracture morphology of the tapeworm (Hymenolepis diminuta) tegument. The most important features revealed by analysis of platinum replicas of freeze-fractured tapeworm scolex-neck tegument include: (a) presence of highly ordered linear and/or circumferentially-orientated rows of intramembrane particles situated on the PF fracture face of microvillar plasma membrane, which may participate in movements of the microvilli, (b) presence of apparent 'pores' (11 nm in diameter) at the tips of the tegumentary microvilli, which could serve as regulated gates through which extramicrovillar surface coating materials can be extruded, and (c) the alignment of cytoplasmic discoid bodies into positions at the bases of the surface microvilli such that they could move into the core of each microvillus and thereby release their contents for extrusion (via the pores) onto the outer surface of the microvilli. Concomitantly, the limiting membrane of the discoid bodies could be added to the tegument plasma membrane and thereby contribute to the rapid turnover of the tegumentary surface. This study provides the first detailed account of the ultrastructural anatomy of the tapeworm tegument and is intended to serve as a point of reference for future investigations of tapeworm tegumentary functions.     

An ordered membrane-cytoskeleton network in squid photoreceptor microvilli

To study the organization of microvilli in the photoreceptor cells of an invertebrate. X-ray diffraction patterns were obtained from aldehyde-fixed squid retinas to a resolution of (40 Å)^?1 and correlated with results from electron microscopy and sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Squid photoreceptor microvilli are packed in extensive hexagonal arrays; in addition each microvillus has a hexagonal substructure. Image reconstruction from thin section electron micrographs shows that the microvilli are linked together with specialized membrane junctions at their neighbour contacts, and phosphotungstic acid-stained sections show a central cytoskeleton connected to the membrane by side-arms.The X-ray patterns also reveal two axial periodicities in the microvilli. A weak and diffuse (50 Å)^?1 band is tentatively assigned to rhodopsin molecules ordered in the plane of the membrane. In addition, an arc at (85 Å)^?1 is attributed to a cytoplasmic or extracellular structure.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the isolated microvilli shows that the major component, rhodopsin, comprises about 50% of the total protein. There are two major detergent-insoluble polypeptides with molecular weights of 145,000 and 42,000. The 42,000 component is identified as actin by papain digestion fragment mapping.Cephalopod photoreceptors are highly sensitive to the polarization vector of linearly polarized light. In consequence, the linear rhodopsin chromophores must be aligned relative to the microvillar axes. The membrane junctions and cytoskeleton described here may provide a mechanism for maintaining this rhodopsin alignment.     

Rapid synthesis of photoreceptor membrane and assembly of new microvilli in a crab at dusk

Summary Large areas of photoreceptor membrane are synthesized in the retinula cells of the crab Leptograpsus variegatus at dusk. Initially, new membrane differentiates from rough endoplasmic reticulum (ER) as large tubules of smooth ER. These tubules transform to concentric ellipsoids of closely apposed pairs of membranes (doublet ER), sometimes passing through an intervening crenate form. The new membrane is transported through bridges of cytoplasm that cross the palisade to the rhabdom region, from which the remains of the rhabdomeres that were built during the previous dusk have been dissolved. The degradation of the old microvilli of one rhabdomere is accomplished without affecting neighbouring rhabdomeres of other cells. New microvilli are assembled in situ from sheets of doublet ER, which are converted to tubules oriented in the same direction as the future microvilli. The cytoplasmic face of the ER remains the cytoplasmic face of the tubules, which become progressively narrower, partly by further longitudinal division, until the final diameter of the microvillus is reached. A central core is often seen in transverse sections of mature microvilli. It may be involved in the final consolidation, but rhabdomeric microvilli are not formed in the same manner as those of intestinal brush border cells. There is no evidence that new membrane passes through the Golgi compartment before incorporation into the rhabdom, as is the case for rod outer segment membrane in vertebrate photoreceptors.     

Ultrastructural localization of actin in muscle, epithelial and secretory cells by applying the protein A-gold immunocytochemical technique

Actin-immunoreactive sites have been localized at the electron microscope level by the protein A-gold technique in striated and smooth muscle cells as well as in epithelial and secretory cells. The combination of the highly sensitive protein A-gold technique with the good ultrastructural preservation and retention of antigenicity obtained using low-temperature embedding conditions has allowed a very precise identification of the labelled structures with high resolution. In striated muscle cells the labelling was obtained over the myofilaments and the Z-band, mainly at its periphery. Labelling was also observed at the edge of the intercalated discs of the cardiac muscle cells. In smooth muscle cells the labelling was present over the myofilaments; the dense plaques associated with the plasma membrane were labelled at their periphery where actin filaments have been reported to anchor. In epithelial cells of the duodenum and the renal convoluted proximal tubule, the labelling occurred over the filamentous core of the microvilli and over the cell web. Gold particles were often present over, or closely associated with, the cell membrane at the tip of the microvilli or of invaginations and vesicular structures. At the level of the junctional complexes the gold particles were aligned at the edge of the dense zones. In pancreatic endocrine and exocrine secretory cells, actin-immunoreactive sites were revealed over the Golgi apparatus, mainly at the level of the inner cisternae in the maturing face over or closely associated with the membranes of the condensing vacuoles and secretory granules, and also over the plasma membrane. Microvilli and cell web were also labelled. Finally, in fibroblasts, gold particles were associated with the membrane of vesicular structures. The consistent finding of actin-immunoreactive sites closely associated with membranes of secretory granules and vesicular structures brings support to the proposal that contractile proteins might play an important role in transcellular transport and protein secretion.     

Chromatin structure visualization by immunoelectron microscopy

Antibodies elicited in rabbits by chromatin and by purified histone H2B have been used to study the structure of chromatin by immunoelectron microscopy. Chromatin spread on grids reveals a structure of closely packed spherical particles with an average diameter of 104 Å, arranged either in clusters or in linear arrays of beads, some of which have a supercoil-like arrangement. No DNA strings connecting the beads could be observed. Upon antibody binding, the diameter of the particles increases up to 300 Å. This size is compatible with a model where one layer of gamma globulin molecules 110 Å long encircles a sphere of chromatin 100 Å in diameter. The presence of rabbit gamma globulins on the enlarged beads has been verified by the addition of ferritin-labeled goat anti-rabbit gamma globulins. Anti-chromatin sera which react with nonhistone proteins but not with free histones or DNA react with more than 95% of the beads; this suggests that most of the beads contain nonhistone proteins. Since the number of nonhistone proteins is large, it is improbable that each sphere contains a full complement of these proteins. We therefore suggest that the various chromatin spheres contain different types of nonhistone proteins. About 90% of the chromatin spheres reacted with antibodies to histone H2B, suggesting that most of the chromatin beads contain this type of histone.     

A new putative cellulose-synthesizing complex ofColeochaete scutata

Summary Cells of the charophycean alga,Coleochaete scutata active in cell wall formation were freeze fractured in the search for cellulose synthesizing complexes (TCs) since this alga is considered to be among the most advanced and a progenitor to land plant evolution. We have found a new TC which consists of two geometrically distinctive particle complexes complementary to one another in the plasma membrane and occasionally associated with microfibril impressions. In the E-fracture face is found a cluster of 8–50 closely packed particles, each with a diameter of 5–17 nm. Most of these particles are confined within an 80 nm circle. In the P-fracture face is found an 8-fold symmetrical arrangement of 10 nm particles circumferentially arranged around a 28 nm central particle. The TCs ofC. scutata are quite distinctive from the rosette/globule TCs of land plants. The 5.5×3.1 nm microfibril inC. scutata is also distinctive from the 3.5×3.5 nm microfibril typical of land plants. The phylogenetic implications of this unique TC in land plant evolution are discussed.     

Glucose transport in isolated brush-border and lateral-basal plasma-membrane vesicles from intestinal epithelila cells

Free-flow electrophoresis was used to separate microvilli from the lateral basal plasma membrane of the epithelial cells from rat small intestine. The activities of the marker enzyme for the microvillus membrane, i.e. alkaline phosphatase (EC 3.1.31), was clearly separated from the marker for the lateral-basal plasma membrane, i.e. the (Na⁺, K⁺)-ATPase (EC 3.6.1.3). A microvillus membrane fraction was obtained with a high specific activity of alkaline phosphatase (an 8-fold enrichement over the starting homogenate). The lateral-basal plasma membrane fraction contained (Na⁺, K⁺)-ATPase (5-fold over homogenate) with some alkaline phosphatase (2-fold over homogenate).Glucose transport was studied in both membrane fractions. The uptake of d-glucose was much faster than that of l-glucose in either plasma membrane, d-Glucose uptake could be accounted for completely by its transport into an osmotically active space. Interestingly, the characteristics of the glucose transport of the microvillus membrane were different from those of the lateral-basal plasma membrane. In particular: Na⁺ stimulated the d-glucose transport by the microvillus membrane, but not by the lateral-basal plasma membrane. In addition, the glucose transport of the microvillus membrane was much more sensitive to phlorizin inhibition than that of the lateral-basal plasma membrane.These experiments thus provide evidence not only for an asymmetrical distribution of the enzymes, but also for differences in the transport properties with respect to glucose between the two types of plasma membrane of the intestinal epithelial cell.     

Regulation of intestinal brush border microvillus length during development by the G- to F-actin ratio

We examined ultrastructural changes in developing chicken intestinal microvilli and correlated these with changes in the G- to F-actin ratio and the amount of actin per milligram cell protein. Three discrete morphological and temporal changes occur during late microvillus morphogenesis: an increase in microvillus number associated with microvilli becoming hexagonally packed on the cell surface; an increase in core actin filament number; and an increase in the length of microvilli. Dramatic rises in the amount of cell actin occur at the time of the first two morphological changes. Changes in the G- to F-actin ratio suggest that increases in the level of monomeric actin drive the elongation phase of microvillus growth since immediately prior to growth the G- to F-actin ratio shifts from its embryonic and adult 3:7 ratio to a 1:1. Our results also indicate, but do not prove, that an increase in the amount of G-actin precedes the rise in level of F-actin and growth of microvilli by 1 day, implying that an increase in the content of G-actin stimulates actin polymerization. Our findings also suggest that the G- to F-actin ratio and their absolute amounts, perhaps in combination with cytoskeletal protein turnover and/or the pool size of actin binding proteins, plays a role in restricting the mature constant length of microvilli.     

A Freeze-Fracture Study of the Cortex of Xenopus laevis Eggs

The organization of the cortex of Xenopus laevis eggs was investigated by freeze-fracture electron microscopy. The cortical endoplasmic reticulum (CER) formed a network surrounding and interconnecting the cortical granules. It formed junctions with the plasma membrane and was confluent with the ER in subcortical regions. Intramembranous particles (IMP₁) were only present in the P face of the CER, the E face being apparently devoid of pits and particles. Arrays of densely packed IMP₁, having a mean diameter of 17 nm, were restricted to the microvillar region of the plasma membrane. The cortical granule membrane also contained IMP₁ (mean diameter, 21 nm) that were sparsely and randomly distributed. Several types of cortical granule seemed to exist based on an analysis of the distribution of the different IMP sizes.     

Ultrastructural alterations induced by Amphotericin B in the plasma membrane of Epidermophyton floccossum as revealed by freeze-etch electron microscopy

Freeze-etch electron microscopic observations demonstrated that Amphotericin B, a potential antifungal polyene induced profound ultrastructural alterations in the plasma membrane of cells of a human pathogenic fungus, Epidermophyton floccosum; aggregation of membrane-associated particles (85-Å) and formation of depressions (or craters) on the inner fracture face.Thin-sectioning electron microscopy showed “vesiculation” of plasma membranes of Amphotericin B-treated Epidemophyton cells.It can be suggested from the preliminary results that the fungal cells which contain ergosterol as the major sterol are highly sensitive to Amphotericin B.     

Surface morphology and functions of pharyngeal structures in the larval lamprey Petromyzon marinus

To gain insight into the multiple functions of a complex biological structure, the morphology of the pharynx of the larva (ammocoete) of the lamprey Petromyzon marinus was investigated with scanning electron microscopy and histochemistry (PAS and Alcian blue). Features studied include the gills, the parabranchial chambers external to the gills, intrapharyngeal ciliary tracts, the ridged pharyngeal roof, the floor, and the intrapharyngeal taste buds. Significant findings are: (1) All (nonciliated) cells lining these structures are covered with microvilli or microridges. The pattern and packing density of these membrane features vary among different pharyngeal structures. The lumenar membranes of pharyngeal lining cells overlie a mucous prosecretion in the apical cytoplasm, suggesting that the microvilli/ridges on these membranes function to anchor mucus. (2) Patterns of microvilli/ridges on the gill respiratory lamellae differ among ammocoetes of different species. (3) Pharyngeal osmo-regulatory cells (“chloride cells”) could not be identified on the basis of the microvillus/ridge pattern. (4) Two types of ciliary tracts are present within the pharynx. One has tall (x = 13 μm) and densely packed cilia, whereas the other has shorter (x = 7 μm) and less densely packed ones. Because mucus covers both types of tracts their function appears to involve the transport of mucus. (5) Food particles were found on the lateral surfaces of the gill filaments and on the surfaces of the parabranchial chambers. It appears that goblet cells in the epithelia of these regions secrete mucus in which the particles are trapped.     

Effects of tunicamycin on thin-section and freeze-fracture images of microvilli of the duodenal epithelial cells of the mouse

Summary The effect of tunicamycin, which is known to inhibit the synthesis of N-linked glycoprotein, on the duodenal absorptive epithelial cell of the mouse was studied in thin-section as well as freeze-fracture images. In tunicamycin-treated animals, the apical part of the epithelial cell was almost negative to the PAS reaction. Moreover, microvilli of the epithelial cell became shorter, larger in diameter, and fewer in number in tunicamycin-treated mice. In addition, freeze-fracture images revealed that the population density of membrane particles of the microvillus membrane was lowered by tunicamycin treatment. These results may indicate that the inhibition of synthesis of N-linked glycoprotein causes a decrease of membrane supply from the Golgi apparatus to the apical plasma membrane.This study was supported by grants from the Ministry of Education, Science and Culture.     

Annexins in rat enterocyte and hepatocyte: an immunogold electron-microscope study

In the present study, immunogold labeling of ultrathin sections of rat small intestine and liver has been used to obtain insights into the ultrastructural localization and possible functions of annexins. In enterocytes, annexins II, IV, and VI are found at the periphery of the core of each microvillus and of the rootlets, but are absent from the interrootlet space. Annexins II, IV, and VI are also observed close to the interdigitated plasma membrane. In hepatocytes, only annexin VI is found to be concentrated within the microvilli in the bile canaliculi, on the inner face of the sinusoidal cell surface, particularly in the space of Disse, and all along the plasma membrane. Annexin VI is also detected in mitochondria of enterocytes and hepatocytes. These localizations are in agreement with the concept of a close calcium-dependent association of annexins with membranes and cytoskeletal proteins, particularly with actin. Moreover, they support the hypothesis of an involvement of annexins in exocytotic and endocytotic processes, which take place in epithelial cells.     

ULTRASTRUCTURAL ORGANIZATION OF CHLOROPLAST THYLAKOIDS OF THE GREEN ALGA "OOCYSTIS MARSSONII"

Intact cells of "Oocystis marssonii" were thin sectioned and freeze-etched, using conventional and double-recovery techniques. Thylakoids extend the length of the single chloroplast and occur in stacks of three to five. The peripheral thylakoids in a stack often alternate between adjacent stacks. Interpretation of double-recovery results suggests that membranes in unstacked regions are asymmetrical, with one face smooth and the matching face covered with closely packed 85–90 Å diameter particles. Adjacent membranes in stacked regions evidently share 170 Å diameter particles, and either membrane in a stacked region may fracture. The two fracture planes thus made possible may expose nearly entire 170 Å particles or only the upper portion of such particles, creating in the latter case images of 125–135 Å diameter particles. Fracture planes in all cases appear to occur through the interior of the membrane, in the plane between the hydrophobic ends of the lipid bilayer proposed in numerous membrane models.     

Freeze-fracture and etching studies on membrane damage on human erythrocytes caused by formation of intracellular ice

The present study examined the damaging effect of intracellular ice on plasma membranes of human erythrocytes. Ice crystals of 0.2–2.0 μm in diameter were formed within the cells as the result of rapid freezing of erythrocytes at the cooling rates around 8000 °C/min. Freeze-fracture and etching studies revealed the ultrastructural alterations of membranes caused by the formation of intracellular ice.In the membrane regions which were in direct contact with intracellular ice, depressions resembling “worm-eaten spots” ranging from 400 to 3000 Å in diameter were observed both on the etched protoplasmic fracture faces (PF) and the exoplasmic surfaces (ES); no perforations were detected in the worm-eaten spots as visualized by slight etching, but artificial destructions occurred on these worm-eaten spots following the increase of etching. The most important phenomenon concerning membrane damage was that in the worm-eaten spots the fracture did not occur along the inner hydrophobic plane of membrane.It was suggested that the formation of intracellular ice in direct contact with a membrane brought about molecular disorganization of bilayer membrane. The presence of these altered membrane regions seems to be responsible for the postthawed hemolysis of the intracellularly frozen erythrocytes.     

Freeze fracture studies on the annelid septate junction

Summary Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80–120 Å, the distance between two particles (centre to centre) is 150–250 Å. Additionally striking rows of large particles (long diameter 150–200 Å). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100–150 Å, centre to centre distance approx. 250 Å) whereas the particles of the extracellular face (EF, mean diameter 200–250 Å) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.     

Ultrastructural pathology of the thyroid folliculo-papillary and follicular carcinoma

Twenty cases of thyroid carcinoma (10 follicular and 10 folliculo-papillary) were ultrastructurally studied. In the follicular carcinoma the most striking features were: microfollicular cavities with microvilli from the apical surface of the tumorous cells, intracellular microlumens, swollen mitochondria sometimes containing electrondense bodies and tightly packed filaments. In the solid sheaths light and dark cells were present. Golgi complexes were disposed in small dense cristae. The nuclei were large, round, oval or with a folded appearance. In the folliculo-papillary carcinoma were found nuclei with an irregular shape containing stage I and stage II inclusions, dilated endoplasmic sacks, closely packed, sometimes dystrophic mitochondria, dense bodies or tightly packed parallel filaments and numerous phagolysosomes. The peroxidase activity wa present as black precipitates in the nuclear envelope or around colloid droplets. The acid phosphatase activity was found as unhomogeneous precipitates inside the lysosomes. From this study it could be concluded that the follicular and folliculo-papillary carcinomas have some common ultrastructural features; the ultrastructural and cytoenzymological patterns suggest marked alteration of the synthesis, storage and secretion of thyroid hormones.     

Visual behaviour and the structure of dark and light-adapted larval and adult eyes of the New Zealand glowworm Arachnocampa luminosa (Mycetophilidae: Diptera)

Both larval and adult New Zealand cave glowworms exhibit reactions to light; their photoreceptors must, therefore, be regarded as functional. The two principal stemmata of the larva possess large biconvex lenses and voluminous rhabdoms. Approximately 12 retinula cells are present. In light-adapted larvae the diameter of the rhabdom is 8 μm and that of an individual microvillus is 49.5 nm. Dark-adapted eyes have rhabdoms that measure 14 μm in cross section and microvilli with an average diameter of 54 nm. The compound eye of the adult comprises approximately 750 ommatidia, each with a facet diameter of 27–28 μm. A facet is surrounded by 1–6 interommatidial hairs which are up to 30 μm long. The interommatidial angle is 5.5°. Cones, consisting of 4 crystalline cone cells, are of the ‘acone’ type. Pigment granules in the primary pigment cells are twice as large as those of the retinula cells which measure 0.6–0.75 μm in diameter. The rhabdom is basically of the dipteran type, i.e. six open peripheral rhabdomeres surround 2 central rhabdomers arranged in a tandem position. The microvilli of cells 1–6 and cell 8 have diameters ranging from 68 to 73 nm, but those of the distally-located central rhabdomere 7 are 20% larger. This is irrespective of whether the eye is dark or light-adapted. In the latter the cones are long and narrow, the screening pigment granules closely surround the rhabdomeres, and the rhabdom is less voluminous than that of the dark-adapted eye.