Organisation of wing cuticle in Locusta migratoria linnaeus,Tropidacris cristata linnaeus and Romalea microptera beauvais (Orthoptera : Acrididae)

The composite fibrous architectures of the wing cuticles of Locusta migratoria, Tropidacris (= Eutropidacris) cristata and Romalea microptera (Orthoptera : Acrididae) have been established. The wing cuticle in all the 3 species consists of: (i) an exocuticle, which is either pigmented or birefringent, and which under an electron microscope shows constantly helicoidal architecture of chitin microfibrils; (ii) endocuticle, which shows alternately birefringent and isotropic layers when sectioned transversely across the wing veins; these layers show helicoidal and unidirectional architecture, respectively of chitin microfibrils under the electron microscope. In transverse section, the chitin microfibrils appear as clear rods (2.8 nm in diameter) in a darkly stained matrix. However, in the hinge called the “claval furrow”, these microfibrils are considerably larger, being 25 nm in diameter. This presumably gives sufficient hardness to the claval hinge, which is the most vulnerable area for wear and tear during flight. The pore canals follow the parabolic pattern of microfibrils in the helicoidal layer, but remain straight in the unidirectional layers. The thickness of wing cuticle increases up to about 10–12 days, the time at which the acridids most probably attain the optimum flight ability. It is suggested that changes in the wing cuticle are related to increased wing beat frequency and speed of flight with age, and may help in resisting the simultaneous increase in the bending and twisting forces on the wing.     

Two separate zones of helicoidally orientated microfibrils are present in the walls ofNitella internodes during growth

Summary The dynamic changes in microfibril architecture in the internode cell walls of the giant unicellular algaNitella translucens were studied during cell expansion. Thin section electron microscopy in conjunction with mild matrix polysaccharide extraction techniques revealed three distinct architectural zones in the walls of fully grown cells. These zones were related to distinct phases of growth by monitoring changes in cell wall architecture of internodes during active cell expansion. The initial microfibril deposition before the onset of active cell growth is helicoidal. A helicoid is a structurally complex but ordered arrangement of microfibrils that has been detected increasingly often in higher plant cell walls. During active cell elongation microfibrils are deposited transversely to the direction of cell elongation as shown in earlier studies by birefringence measurements in the polarizing microscope. The gradual decline in cell elongation corresponds with a final helicoidal deposition which continues after cell expansion ceases entirely.The continual presence of the initial helicoidal zone in the outer wall region during the whole growth process suggests that these microfibrils do not experience strain reorientation and are continually reorganized, or maintained, in a well ordered helicoidal arrangement.     

Surface replicas of pulmonary endothelial cells in culture.

Pulmonary endothelial cells possess a variety of enzymes on their surface. However, the topographical distribution of these enzymes is not known. In this report we describe a simple technique for the preparation of surface replicas of pulmonary endothelial cells using an unmodified critical point drying apparatus and a high vacuum freeze-etch unit. The technique should be applicable for use with all cells in monolayer culture. Endothelial cells grown on glass slides or coverslips were washed thoroughly, fixed and dehydrated. The monolayers were then critical point dried. Surface replicas were prepared in a Balzer's freeze-etch unit. The advantages of surface replicas are that they can be examined with the resolving power of the transmission electron microscope and can be used to examine whole cells. The potential for the surface replica technique may lie in mapping specific enzymes, receptors and cell surface factors. Therefore, to provide a basis for such studies, we have described the appearance of pulmonary endothelial cells in surface replicas.     

An Electron Microscope Examination of Acetobacter xylinum Showing the Ultrastructure of the Cells and the Association of Cellulose Microfibrils

A surface-pellicle-forming strain of A. xylinum was examinedby electron microscopy. Both thin-sections and freeze-etch replicasshowed the ultrastructure of the cells to be typically Gram-negative,the envelope consisting of two membranes with the pcptidoglycanlayer between. The cells did not possess a capsule. Thin-sectionsof cells stained with ruthenium-red showed specific depositslocalized within the periplasm. Cellulose microfibrils wereseen to arise directly from the cell surface, but each cellformed only a limited number of microfibrils. Frecze-etch replicassuggested that the microfibrils arose from particles locatedon or within the outer membrane.     

The structure of cellulose-producing bacteria, Acetobacter xylinum and Acetobacter acetigenus.

The structure of the pellicles and cells of the cellulose-producing bacteria, Acetobacter xylinum and Acetobacter acetigenus, was studied by transmission electron microscopy of thin sections and freeze-etch replicas of glucose-stimulated cell suspensions, quiescent cell suspensions, and discrete pellicles. These bacteria have a relatively thin cell wall in section, with several irregular features superimposed on an otherwise simple, Gram-negative morphology. There are no flagella or pili. Unfixed, unextracted cells, viewed as whole mounts, show spherical or ellipsoidal bodies of undetermined composition which disappear after extraction with water or ethanol and propylene oxide. For both species, there are several kinds of cell surface irregularities, some of which are localized protrusions of the cell envelope. A variety of irregularities is seen frequently on cells in the first minutes of glucose incubation, on cells in a discrete pellicle, on quiescent cells, and on starved cells. Immediately after the addition of glucose to cellulose-free cells in suspension culture, fine fibrils appear on and (or) near the cell envelope. The fine fibrils are frequently as small as 3 nm in diameter in both freeze-etch and thin-section preparations and are frequently associated with freshly synthesized cellulose fibrils. Starved cells in suspensions free of (classical) microfibrils sometimes reveal stubs of an extracellular structure whose morphology resembles that of a nascent cellulose fibril.     

Biomineralization of limpet teeth: a cryo-TEM study of the organic matrix and the onset of mineral deposition

The continuously growing limpet radula contains teeth at various stages of maturity and thus provides an excellent opportunity for studying the processes and mechanisms of their mineralization. We report here on our structural investigations of the pre-formed chitin matrix and the initial deposition and growth of goethite (α-FeOOH) crystals within the matrix. By using cryo-techniques, in which unstained sections of the teeth are examined in a frozen-hydrated state in a transmission electron microscope (TEM), we were able to characterize the process without introducing artifacts normally associated with the staining, dehydration, and embedding required for conventional TEM. The unmineralized matrix consists of relatively well ordered, densely packed arrays of chitin fibers, with only a few nanometers between adjacent fibers. There are clearly no pre-formed compartments that control goethite crystal size and shape; rather, crystals must push aside or engulf the fibers as they grow. By examining teeth nearly row-by-row around the onset of mineralization, we were able to image the first-formed mineral within the chitin matrix. These linear deposits of goethite appear to nucleate on the chitin fibers, which thus control the orientation of the crystals. Crystal growth, on the other hand, is apparently not influenced by the matrix, in contrast to many other biomineralization systems.     

Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which beta(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis.     

cuticleDB: a relational database of Arthropod cuticular proteins

Background  

The insect exoskeleton or cuticle is a bi-partite composite of proteins and chitin that provides protective, skeletal and structural functions. Little information is available about the molecular structure of this important complex that exhibits a helicoidal architecture. Scores of sequences of cuticular proteins have been obtained from direct protein sequencing, from cDNAs, and from genomic analyses.     

The production of microfibrils at the surface of isolated tomato-fruit protoplasts

Summary Two stages occur in the development of a wall by certain isolated tomato fruit protoplasts. A primary multi-lamellar system develops, beneath which other substances are layed down. The deep-etching modification of the freeze-etch technique clearly reveals that a major component of the second stage consists of cellulose microfibrils which are layed down in close association with the outer face of the plasmalemma.     

A proposed solution to a fine-structural puzzle: The organization of gill cuticle in a crayfish (Panulirus)

Crayfish gill cuticle is approximately 2 μm thick and comprises an epicuticle and an endocuticle, which is subdivided into outer and inner layers. Sections demonstrate indistinct lamellae in the outer endocuticle and vertically striated lamellae in the inner endocuticle. Microfibrils cannot be seen in sections. Difficulties in interpretation of the fibrous architecture of the cuticle from thin sections have been overcome by examining tilted series of micrographs of sections and also by making freeze-fracture replicas of the cuticle, which reveal the microfibrils clearly. A model for the endocuticle based on a helicoidal configuration of microfibrillar laminae is proposed and the vertically striated structures seen in sections of the outer layer are accounted for by including regular rows of particles oriented perpendicular to microfibrils. The model is compared with cuticles and coverings reported from other invertebrates.     

On the structure of the cuticle of Mecoptera

The chitin architecture of Mecoptera cuticle is of two kinds: helicoidal and helicoidal preferred with the preferred layers being cross-plied. Comparison of both systems of terminology currently in use to differentiate the subtypes of cuticle indicates that neither provides much information about the arrangement of chitin within cuticle and that both give information only about the extent of hardening in cuticle. All of the specimens of solid cuticle broken in tension exhibited a similar fracture behaviour in which the exocuticle failed in a brittle manner and the endocuticle failed plastically. The mode of endocuticular failure is dependent upon the arrangement of chitin microfibres within this region. The ultrastructural patterns of chitin microfibres determined by electron microscopy cannot be related to current notions about the phylogenetic interrelationships among Mecoptera and the usefulness of chitin fibre arrangement as a phylogenetic tool remains an open question.     

Demonstration of rotational symmetries in alpha-keratin microfibrils of porcupine quill tip

Selected electron micrographs of transversely, obliquely and longitudinally sectioned microfibrils of transversely sectioned porcupine quill tip are shown to possess 2-fold radial, 3-fold radial and 5-fold polygonal rotational symmetries. These symmetries are verified with a rotation technique, and are similar to edge, corner and face projections of a pentagonal dodecahedron. The a-keratin microfibril is therefore suggested to be composed of a linear arrangement of morphologically identical microfibrillar subunits which approximate the shape of a pentagonal dodecahedron. The various line patterns present in electron micrographs of microfibrils are explained by different orientations of this three-dimensional shape within the thickness of the section. Previous electron microscopic models for the structure of the microfibrils are incompatible with these results. The image averaging methods used to arrive at these earlier models are discussed, and are thought to yield results which must differ from the demonstrated rotational symmetries.     

Development of elastic fibers of nuchal ligament, aorta, and lung of fetal and postnatal sheep: an ultrastructural and electron microscopic immunohistochemical study

The morphogenesis of elastic fibers of the nuchal ligament, aorta, and lung of sheep was studied by light microscopy, transmission electron microscopy, and immunohistochemical methods for the detection of elastin. The degree of maturation of the amorphous materials of elastic fibers was assessed morphologically in preparations stained by the tannic acid and periodic acid methenamine-silver methods. With both of these methods, the amorphous components of mature fibers stained less intensely than did those of immature fibers. Elastic fibers in early stages of development consisted of many microfibrils and few, small, branching masses of immature amorphous material. Thicker fibers were formed by the coalescence of growing masses of amorphous materials. In late stages of formation of elastic fibers, the mature amorphous materials were associated with few microfibrils; and they were partially surrounded by immature amorphous materials associated with many microfibrils. Antielastin antibody reacted evenly with amorphous materials in very early stages of elastic-fiber development, but reacted only with the other zones of amorphous materials in later stages; it also reacted with the microfibrils in all stages. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin on their surfaces. This conclusion is in agreement with ultrastructural observations showing 1) that development of microfibrils precedes that of the amorphous material and 2) that the microfibrils adjacent to the immature amorphous materials are covered with small amounts of tannic acid-positive amorphous materials. These observations suggest that microfibrils serve as sites for elastin deposition, both in early elastogenesis and in subsequent growth of elastic fibers. However, the nature of the interaction between elastin and microfibrils remains unknown.     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes

Freeze-fracture-etch replicas of concentrated DNA solutions which appeared, by polarized light microscopy, to be in a cholesteric-like liquid crystalline state were examined by high resolution transmission electron microscopy (TEM). Individual DNA molecules were resolvable, and the microscopic morphologies observed for such replicas confirmed the cholesteric organization of DNA molecules in this liquid crystalline state. Furthermore, replica morphologies were strikingly similar to TEM images of dinoflagellate chromosomes in both thin section and freeze-etch replicas, providing strong support for the cholesteric DNA packing model proposed for the organization of DNA in these chromosomes by Bouligand and Livolant.     

Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs

Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine.     

Ultrastructure and chemical composition of the ballistospore wall ofConidiobolus obscurus

The ballistospores of the entomopathogenConidiobolus obscurus are spheroidal cells with a papilla corresponding to the zone of attachment on the sporophore. They are covered by a mucus responsible for the attachment of the spore to the insect. Chemical and cytochemical investigations of the nature of the wall components have been undertaken to better understand fungus-insect interactions in entomopathology. β(1→3)-Glucans and chitin together represented the main components of the wall which did not contain chitosan and uronic acids. Transmission electron microscopy revealed that the spore wall was composed of a thick electron-lucent inner layer and a thin outer electron-dense layer, the latter being absent at the papilla region. The spore is covered by a mucilaginous layer that upon spore impact on a substratum, is dispersed and forms a halo around the spore. Shadow replicas of the discharged spores showed that they are covered by rodlets except on the papilla which displayed a chitinous, microfibrillar structure. The ontogeny of the rodlets deposited on the surface of young spores was characterized by a progressive organization of separate rodlets and then a clustering of the rodlets in fascicles. Shadow replicas and chemical and enzymatic investigations of the halo surrounding discharged spores showed that the mucus was composed of long β(1→3)-glucan microfibrils embedded in amorphous proteins partly covered by rodlets discharged from the spore surface.     

Ultrastructure and localization of wall polymers during regeneration and reversion of protoplasts ofSchizophyllum commune

Summary Cell-wall regeneration and reversion of protoplasts ofSchizophyllum commune were investigated using electron microscopic methods and X-ray diffraction.After 3 hours of regeneration protoplasts have formed a loosely organized wall which does not react with Thiéry's stain for periodic acid sensitive carbohydrates. This wall largely consists of chitin microfibrils which are adpressed to the plasmalemma and which are covered by loose aggregates of alkali-soluble S-glucan (-1,3-glucan). Both components are microcrystalline, at least partly. Walls formed in the presence of polyoxin D only consist of thick loose fibers of S-glucan.From 3 hours onward the inner chitin microfibrils of the wall of the primary cells become embedded in alkali-insoluble material that stains heavily with the Thiéry reagent and probably is similar to the R-glucan of the mature wall (i.e., -1,3--1,6-glucan). The outer chitin microfibrils remain free of this matrix and are covered by S-glucan only.Bud-like structures that arise have the same wall architecture as the primary cells,i.e., only the inner chitin microfibrils are embedded in R-glucan and the S-glucan forms a fluffy coat. The walls of hyphal tubes that arise are distinct, however, in that all chitin microfibrils are embedded in R-glucan and the S-glucan forms a compact coat.Cytoplasmic vesicles are sparse in primary cells except at the sites of emergence of budlike structures and hyphae. They continue to be present in the apex of growing hyphae.     

Fine mapping of tropoelastin-derived components in the aorta of developing chick embryo

Summary Affinity-purified antitropoelastin antibodies have been used to localize tropoelastin-derived components in aortas from chick embryos of different age by immunoelectron microscopy. Staining in the matrix is first noted at day 3 associated with irregular bundles of filaments resembling microfibrils, in the absence of amorphous elastin deposits. Amorphous material, which rapidly accumulates at later stages, is heavily labelled, while surrounding microfibrils are only poorly labelled. By contrast, a more intense staining of microfibrils persists in regions in which amorphous material is not morphologically evident. These observations indicate that the initial accumulation of elastin requires microfibrils, while the two components are not in close association in the subsequent growth of the amorphous core of the fibre. Intracellular staining is evident in the secretory apparatus of the cell and in peripheral large vesicles. Differentiated cells also show regions of close contact with elastic fibres in which immunological staining for elastin is very close to the cell membrane.