X-ray and dissolution studies of paramylon storage granules fromEuglena

Summary Paramylon is the -1,3 glucan storage carbohydrate in the euglenoid algae. Mature paramylon granules are highly crystalline, fibrillar, and have a complex substructure. X-ray diffraction was used to demonstrate that mature paramylon granules are much more crystalline than immature granules. Freeze-etch electron microscopy showed that in mature granules, the microfibrils are organized in highly ordered arrays while the microfibrils of immature granules are less organized. The data suggest that the high crystallinity of paramylon is due to higher-order aggregates of microfibrils and the interaction of water with the microfibrils. The dissolution of paramylon was recorded by darkfield videomicroscopy. In a 0.5 N NaOH solution, paramylon dissociates in a regular manner into its constituent 4 nm microfibrils, and the central region of the granule is the last remaining refractile area during the dissolution process.Abbreviation LN liquid nitrogen     

Fabrication of doughnut-shaped particles from spheroidal paramylon granules of Euglena gracilis using acetylation reaction

An anhydrous type of paramylon, the micro-sized granular storage carbohydrate (β-1,3-glucan) of Euglena, was transformed from a spheroidal to a doughnut-like shape by acetylation. Fourier transform infrared spectroscopic measurements suggested that the doughnut formation is due to removal of accessible regions of paramylon particles by acetylation of glucans. A time-course observation of the paramylon granules during acetylation by using field emission scanning electron microscopy revealed that the doughnut-making process begins with the removal of an outer membrane of the granule and that the central region of the granules is preferentially removed with the survival of a thick rim part to give the doughnut-like particles.     

THE INTRAMEMBRANOUS PARTICLE PROFILE OF THE PARAMYLON MEMBRANE DURING PARAMYLON SYNTHESIS IN EUGLENA (EUGLENOPHYCEAE)1

Paramylon is the β-1, 3-glucan storage product in euglenoid algae. It is a fibrous crystal that occurs as membrane-bound granules in the cytosol. The role of the surrounding membrane in paramylon synthesis was investigated by the use of freeze-etch electron microscopy. When Euglena gracilis Klebs strain Z (Pringsheim) cells were frozen in supercooled liquid nitrogen, the fracture plane primarily was throuh the paramylon membrane. A large intramembranous particle (IMP, mean diam range 5.6-6.5 nm) and a small IMP (mean diam range 9.6-10.3 nm) were predominant in both PF (protoplasmic fracture) and EF (exoplasmic fracture) faces of the paramylon membrane. During paramylon synthesis induction, the ratio of small to large IMPs increased in both fracture faces. The IMP density decreased in both fracture faces concomitant to paramylon synthesis increase. These changes in IMP profile and density suggest that the paramylon membrane is involved in the synthesis of paramylon.     

Degradation of Paramylon by Euglena gracilis

SYNOPSIS. Protozoa of the order Euglenida contain a polysaccharide storage product, paramylon, composed of 1, 3-linked glucose molecules arranged into an extremely resistant granule. An enzyme was purified from the soluble phase of Euglena gracilis which would degrade this polysaccharide to single glucose residues, providing the integrity of the paramylon granule was 1st disrupted by dilute base. This enzyme, a β-1, 3 glucanase, had optimal activity at pH 5.0 and 60 C and bound tightly to base-disrupted paramylon substrate tho not to the intact granules. The specific activity of the enzyme was doubled when cell cultures reached stationary phase, the phase where net carbohydrate utilization began. An ATP-dependent hexokinase reaction was also present in Euglena homogenate. No phosphorylase activity has been found in Euglena. It is suggested, therefore, that Euglena do utilize their paramylon as a carbohydrate reserve and the mechanism of this utilization is by exo-hydrolytic cleavage to free glucose followed by phosphorylation and glycolysis.     

A Comparative Study of the Storage Carbohydrate Granules from Euglena (Euglenida) and Pavlova (Prymnesiida)1,2

ABSTRACT The storage carbohydrate granules from Euglena and Pavlova were compared by light and electron microscopy. Freezeetch studies demonstrated that while both types of granules are crystalline, they have different structures. The elemental microfibril of the euglenoid granule measures 4 nm, and the elemental striation of the granule from Pavlova is 22 nm. The granules each have a unique X-ray diffraction pattern. The storage carbohydrate granules from Pavlova are not the same as paramyton, and the term “paramylon” should be reserved for the euglenoid storage carbohydrate.     

Structure of Acetobacter cellulose composites in the hydrated state

The structure of composites produced by the bacterium Acetobacter xylinus have been studied in their natural, hydrated, state. Small-angle X-ray diffraction and environmental scanning electron microscopy has shown that the ribbons have a width of 500 A and contain smaller semi-crystalline cellulose microfibrils with an essentially rectangular cross-section of approximately 10 x 160 A(2). Incubation of Acetobacter in xyloglucan or pectin results in no changes in the size of either the microfibrils or the ribbons. Changes in the cellulose crystals are seen upon dehydration of the material, resulting in either a reduction in crystal size or an increase in crystal disorder.     

Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.     

The three-dimensional morphology of aggregates of native cotton cellulose microfibrils

The three-dimensional morphology of native bacterial cellulose is confirmed by scanning electron microscopy. In addition, it is shown by scanning electron microscopy, and transmission electron microscopy with positive staining by phosphotungstic acid ions that aggregates of microfibrils of native cotton cellulose have a similar structure. The results are consistent with previous reports on microfibrils of algal cellulose. These observations exclude a simple spinneret process as a mechanism of formation of the microfibrils of these sources of cellulose.     

Structure and association of wall fibrils produced by regenerating tobacco protoplasts

A study has been made of the wall fibrils produced by tobacco protoplasts, using scanning electron microscopy in conjunction with negative staining. It has been shown that the fibres seen in scanning electron microscopy correspond to aggregates of microfibrils. These aggregates are only visible where they are lifted clear of the protoplast surface. Negative staining of fixed protoplasts shows that the aggregation of microfibrils into the fibres visible in scanning electron microscopy is probably produced by air-drying. Gentle disruption of microfibrils produces both random broken fragments and bundles of short pieces of fibrillar material about 60 nm in length. This material is present in undisrupted young walls, but not in undisrupted older walls. The microfibrils in young walls seem much more fragile and liable to breakage than those in older walls. These results are discussed in terms of the interpretation of scanning electron microscope images and the mechanism of cellulose microfibril formation by higher plants.Abbreviations SEM Scanning electron microscopy     

Architecture of the cell wall of a green alga,Oocystis apiculata

Summary The cell wall of a green alga,Oocystis apiculata, was visualized by electron microscopy after preparation of samples by rapid-freezing and deep-etching techniques. The extracellular spaces clearly showed a random network of dense fibrils of approximately 6.4 nm in diameter. The cell wall was composed of three distinct layers: an outer layer with a smooth appearance and many protuberances on its outermost surface; a middle layer with criss-crossed cellulose microfibrils of approximately 15–17 nm in diameter; and an inner layer with many pores between anastomosing fibers of 8–10 nm in diameter. Both the outer and the inner layer seemed to be composed of amorphous material. Cross-bridges of approximately 4.2 nm in diameter were visualized between adjacent microfibrils by the same techniques. The cross-bridges were easily distinguished from cellulose microfibrils by differences in their dimensions.     

The microfibrils of connective tissue: II. Immunohistochemical detection of the amyloid P component

Immunohistochemical methods were used for the detection of the amyloid P component in the microfibrils of two regions: the zonule of the eye and the connective tissue of the foot pad in 20- to 50-gm mice. Following fixation by immersion in 4% formaldehyde, the eyes and foot pads were embedded in paraffin, and sections were immunostained for light microscopy by using antiamyloid P component antiserum followed by peroxidase-antiperoxidase procedure. For electron microscopy, formaldehyde-fixed tissues were immunostained for the amyloid P component with protein A-gold by using either thin Lowicryl sections or frozen sections which were then embedded in Epon for thin sectioning. In the zonule of the eye, the light microscope showed that zonular fibers were strongly immunostained for the amyloid P component; there was also weak staining of the nonpigmented ciliary epithelium at the distal end of the fibers and of the zonular lamella at their proximal end. The electron microscope revealed clear-cut immunolabeling of the microfibrils making up zonular fibers as well as of individual microfibrils. In the foot pad, the light microscope detected a weak diffuse staining of connective tissue, whereas the electron microscope showed immunolabeling restricted to microfibrils. It was concluded that the amyloid P component was present in, or associated with, microfibrils. Purified amyloid P component was prepared and examined in the electron microscope after either negative staining or routine processing. After negative staining, it appeared as flat pentagonal units, frequently associated into columns. After routine processing, the units looked like cross sections of microfibrillar tubules. The dimensions of the units matched those of the hypothetical segments of the tubules. It was concluded that this tubule consisted of a column of amyloid P units. The cohesion of the units within the column was likely to be reinforced by the bands present at the surface of microfibrils.     

RESTING CYST FORMATION OF EUTREPTIELLA GYMNASTICA (EUGLENOPHYCEAE) IN THE NORTHERN COASTAL BALTIC SEA1

Resting cyst formation of Eutreptiella gymnastica Throndsen was observed during a mesocosm experiment, where nutrient enrichment had induced almost a unialgal bloom. Cells and resting cysts of E. gymnastica were examined in scanning (SEM) and transmission electron microscopy (TEM) and light microscopy. Mature cysts were spherical, with a smooth thick mucilaginous cover that appeared layered when observed with the TEM. Intermediate forms were spherical and lacking flagella and a mucilaginous cover; the euglenoid pellicular striation and canal opening were easily visible. The volume of these intermediate spherical cells and mature cysts was estimated to have increased threefold compared to flagellated cells and contained many paramylon grains. When the cells were grazed by zooplankton, the paramylon grains passed the gut intact and were packed into fecal pellets. Intact undigested paramylon grains were observed in SEM after the breaking up of the pellets.     

Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

The ultrastructure of the renal corpuscle, the neck segment, the proximal tubule and the intermediate segment of the kidney of a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) was examined by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and freeze-fracture technique. The glomerular filter apparatus consists of the podocyte epithelium, a distinct basement membrane, a subendothelial space and the capillary endothelium. Emanating from the podocyte cell body, several long primary processes encircle neighboring capillaries. The short slender foot processes originating from the primary processes interdigitate with those from other primary processes, thereby forming the meandering filtration slit. Thick bundles of microfilaments are found in the primary processes, but absent in the foot processes. The basement membrane consists of a lamina rara externa and a rather thin lamina densa (50 nm thickness). The wide subendothelial space contains abundant microfibrils, a few collagen fibrils and many thin processes of mesangial cells. The endothelium is flat and fenestrated (compared to mammals displaying relatively few fenestrations); some of the fenestrations are bridged by a diaphragm. The glomerular mesangium is made up of the mesangial cells and a prominent mesangial matrix containing microfibrils and collagen fibrils. The cells of the neck and intermediate segments display numerous cilia with their microtubules arranged in the typical 9 + 2 pattern. The basal bodies of the cilia are attached to thick filaments with a clear crossbanding pattern of 65 nm periodicity. The proximal tubule is composed of cells typical for this segment (PT cells) and light cells lacking a brush border (bald-headed cells). The PT cells measure 10-25 micron in height and 15-30 micron in width and do not interdigitate at their lateral borders with each other. Their basolateral cell membrane is amplified by many folds projecting into lateral intercellular spaces and into basal recesses. The brush border is scarce and composed of loosely arranged short microvilli.     

Cell wall extension results in the coordinate separation of parallel microfibrils: evidence from scanning electron microscopy and atomic force microscopy

Enlargement of the cell wall requires separation of cellulose microfibrils, mediated by proteins such as expansin; according to the multi-net growth hypothesis, enlargement passively reorients microfibrils. However, at the molecular scale, little is known about the specific movement of microfibrils. To find out, we examined directly changes in microfibril orientation when walls were extended slowly in vitro under constant load (creep). Frozen-thawed cucumber hypocotyl segments were strained by 20-30% by incubation in pH 4.5 buffer or by incubation of heat-inactivated segments in alpha-expansin or a fungal endoglucanase (Cel12A). Subsequently, the innermost layer of the cell wall was imaged, with neither extraction nor homogenization, by field-emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). AFM images revealed that sample preparation for FESEM did not appreciably alter cell wall ultrastructure. In both FESEM and AFM, images from extended and non-extended samples appeared indistinguishable. To quantify orientational order, we used a novel algorithm to characterize the fast Fourier transform of the image as a function of spatial frequency. For both FESEM and AFM images, the transforms of non-extended samples were indistinguishable from those of samples extended by alpha-expansin or Cel12A, as were AFM images of samples extended by acidic buffer. We conclude that cell walls in vitro can extend slowly by a creep mechanism without passive reorientation of innermost microfibrils, implying that wall loosening agents act selectively on the cross-linking polymers between parallel microfibrils, rather than more generally on the wall matrix.     

Lattice images from ultrathin sections of cellulose microfibrils in the cell wall of Valonia macrophysa Kütz.

The crystalline ultrastructure and orientation of cellulose microfibrils in the cell wall of Valonia macrophysa were investigated by means of high-resolution electron microscopy of ultrathin (approx. 28 nm) sections. With careful selection of imaging conditions, ultrastructural aspects of the cell wall that had remained unresolved in previous studies were worked out by direct imaging of crystal lattice of cellulose microfibrils. It was confirmed that each microfibril is a single crystal having a lateral dimension of 20·20 nm², because lattice images of 0.39 nm resolution were clearly recorded with no major disruption in the whole area of the cross section of the microfibril. There was no evidence for the existence of 3.5-nm elementary fibrils which have been considered to be basic crystallographic and morphological units of cellulose in general. It was also confirmed that the axial directions (crystallographic fiber direction) of adjacent microfibrils in each single lamella of the cell wall are opposite to each other.     

Ultrastructural properties of ciliary zonule microfibrils

Conventional electron microscopy and rotary shadowing techniques have provided conflicting interpretations of microfibril ultrastructure. To address this issue, we have used quick-freeze deep-etch (QFDE) microscopy to obtain 3-dimensional surface views of microfibrils that have not been fixed, dehydrated, or stained with heavy metals. By this approach, microfibrils appear as tightly packed rows of bead-like subunits that do not display the interbead filamentous links seen by other methods. At regular 50-nm intervals along the microfibril length, a larger bead is often recognized which tends to be aligned with those from adjacent microfibrils when the microfibrils are in bundles. This evidence of organized lateral associations of microfibrils is supported by the observation of small filaments that span between the adjacent microfibrils. When QFDE microscopy was used to examine microfibrils exposed to sonication, partially dissociated microfibrils with the more typical "beads on a string" appearance were observed. Beads are also seen alone, as monomers, often with an array of small thread-like filaments extending from the bead in a "crab-like" manner. Our results suggest that the beads on a string appearance of sonicated microfibrils may result from a partial loss of protein components from the interbead domains, thus leading to exposure of a filamentous substructure. It is possible, therefore, that this phenomenon might also contribute to the beads on a string appearance of microfibrils seen using other electron microscopy techniques.     

The microfibrils of connective tissue: I. Ultrastructure

The ultrastructure of connective tissue microfibrils was examined in two sites: the ciliary zonule of the eye and the foot pad, in 20-day-old mice perfused with glutaraldehyde. The microfibrils were classified into two categories, referred to as typical and atypical. Typical microfibrils predominate in both sites; they are unbranched, straight or gently curving, tubular structures of indefinite length with an overall diameter of 12.8 +/- 1.7 nm in the zonule and 13.8 +/- 2.8 nm in the foot pad. They are composed of two parts: tubule proper and surface band. The tubule is 7- to 10-nm wide and characterized in cross section by an approximately pentagonal wall and an electron-lucent lumen containing a 1- to 2-nm bead referred to as a spherule. When longitudinal sections of microfibrils are examined at high magnification, the wall of the tubule does not appear as a continuous line but as a series of successive dots. The interpretation of these findings is that the tubule is composed of successive annular segments with an approximately pentagonal outline. The surface band is a 3-nm-wide, ribbon-like structure wrapped around the tubule. The band has dense borders called tracks. Along the tracks, densely stained, 4.6-nm-long "spikes" are attached at 4.0-nm intervals. The wrapping of the bands is somewhat irregular. They may be in a transverse position across single or several microfibrils, in which case each band might constitute a distinct belt; more frequently, the bands are oblique and appear to form a continuous helix. It is proposed that surface bands play a role in holding together the juxtaposed segments making up a tubule. A model has been constructed to represent the association of tubule and band into a typical microfibril. Atypical microfibrils, which are more common in foot pad than in ciliary zonule, appear wavy, lack a definite tubule, and are characterized by distorted, irregular surface bands. They are attributed to proteolysis of typical microfibrils.     

On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electronmicroscopic, X-ray diffraction and chemical study.

The nets produced by protoplasts of Saccharomyces cerevisiae in liquid culture media consisted of microfibrils about 20 nm wide, forming flat, fairly straight bundles of variable width and length, up to about 500 nm wide and 4 mum long. Ends of microfibrils were seldom found. They were not attacked by chitinase or dilute acids, but the net structure disappeared in 3% (w/v) NaOH, leaving about 60% dry wt of the nets as partly microfibrillar clusters. The X-ray powder pattern from the nets, in contrast to that from normal walls, exhibited a set of well-defined rings which identified two micro-crystalline constituents: chitin and unbranched chains of beta-(1 leads to 3)-linked D-glucose residues. These latter were the alkali-soluble fraction. The X-ray diagram of the glucan, corresponding to that of paramylon, indicated an in vivo crystal modification. Up to 15% dry wt was chitin which was found de novo by the protoplasts. A fine net structure of microfibrils about 7-5 to 10 nm thick with meshes about 20 to 60 nm wide was demonstrated in normal walls, forming the entire inner layer and consisting mainly of yeast glucan. This glucan and chitin were only slightly crystalline in these walls. The features of the glucan and chitin of the protoplast nets indicate that enzymes active in normal wall formation were differentially removed or inactivated by the liquid medium.     

Cell wall architecture of the elongating maize coleoptile

The primary walls of grasses are composed of cellulose microfibrils, glucuronoarabinoxylans (GAXs), and mixed-linkage beta-glucans, together with smaller amounts of xyloglucans, glucomannans, pectins, and a network of polyphenolic substances. Chemical imaging by Fourier transform infrared microspectroscopy revealed large differences in the distributions of many chemical species between different tissues of the maize (Zea mays) coleoptile. This was confirmed by chemical analyses of isolated outer epidermal tissues compared with mesophyll-enriched preparations. Glucomannans and esterified uronic acids were more abundant in the epidermis, whereas beta-glucans were more abundant in the mesophyll cells. The localization of beta-glucan was confirmed by immunocytochemistry in the electron microscope and quantitative biochemical assays. We used field emission scanning electron microscopy, infrared microspectroscopy, and biochemical characterization of sequentially extracted polymers to further characterize the cell wall architecture of the epidermis. Oxidation of the phenolic network followed by dilute NaOH extraction widened the pores of the wall substantially and permitted observation by scanning electron microscopy of up to six distinct microfibrillar lamellae. Sequential chemical extraction of specific polysaccharides together with enzymic digestion of beta-glucans allowed us to distinguish two distinct domains in the grass primary wall. First, a beta-glucan-enriched domain, coextensive with GAXs of low degrees of arabinosyl substitution and glucomannans, is tightly associated around microfibrils. Second, a GAX that is more highly substituted with arabinosyl residues and additional glucomannan provides an interstitial domain that interconnects the beta-glucan-coated microfibrils. Implications for current models that attempt to explain the biochemical and biophysical mechanism of wall loosening during cell growth are discussed.     

Penetration of chemicals into the Malus leaf cuticle

The adaxial leaf cuticle of Malus pumila was examined by electron microscopy to determine possible avenues for transcuticular movement of foliarly applied chemicals. Cutin-embedded polysaccharide microfibrils originated at the outer epidermal cell wall and occasionally extended to the cuticle surface. Lamellae, ca. 4 nm wide, usually were oriented parallel to the cuticle surface. When oriented perpendicular to the surface, they extended nearly to the subjacent wall layer from the surface. Aqueous solutions of uranyl acetate, silver nitrate and phenyl mercuric acetate applied to the cuticle surface of leaf segments floated on solutions of phosphate salts or thiocarbohydrazide (TCH) reacted within the cuticle to form insoluble electron-opaque deposits indicative of their avenues of transcuticular movement. Uranyl phosphate deposits were observed only in the polysaccharide microfibrils of chloroform: methanolextracted leaves. Silver-TCH deposits were observed in the microfibrils of both extracted and nonextracted leaf cuticles. Phenyl mercuric acetate-TCH deposits were randomly dispersed throughout the extracted cuticle and not associated with the polysaccharide microfibrils.Abbreviations TCH thiocarbohydrazide - PMA phenyl mercuric acetate