Seawater at the nanoscale: marine gel imaged by atomic force microscopy

The present study introduces atomic force microscopy (AFM) as a tool for characterization of marine gel network and marine biopolymers self-assembly, not accessible by other techniques. AFM imaging of marine gel samples collected in summers 2003 and 2004 in the northern Adriatic Sea provided insight into molecular organization of gel network and associations between polysaccharide fibrils in the network. Initial stages of biopolymers self-assembly were visualized by AFM in a phytoplankton bloom experiment performed in the same aquatorium. Based on AFM imaging and differential scanning calorimetry, the marine gel is characterized as a thermoreversible physical gel and the dominant mode of gelation as crosslinking of polysaccharide fibrils by hydrogen bonding which results in helical structures and their associations. Direct deposition of whole seawater on freshly cleaved mica followed by rinsing was the procedure that caused the least impact on the original structures of biopolymer assemblies in seawater.     

Atomic force microscopy characterization of silver nanoparticles interactions with marine diatom cells and extracellular polymeric substance

This study highlights the capacity of atomic force microscopy (AFM) for investigating nanoparticle (NP) algal cell interaction with a subnanometer resolution. We designed a set of AFM experiments to characterize NP size, shape, and structure to visualize changes in the cell morphology induced by NPs and to characterize NP interaction with the extracellular polymeric substance (EPS). Samples for AFM imaging were prepared using the same protocol-drop deposition on mica and imaged in air. Here we address the interactions of Ag NPs with ubiquitous, lightly silicified marine diatoms Cylindrotheca fusiformis and Cylindrotheca closterium and their EPS. In natural seawater used throughout this study, the single Ag NPs adopted truncated tetrahedron morphology with particle heights of 10, 20, 30, and 40 nm. This size class Ag NPs penetrates the cell wall through the valve region built of silica NPs embedded in organic matrix. The Ag NPs cause a local damage inside the cell without disintegration of the cell wall. The EPS production has been shown to increase as a feedback response to Ag NP exposure and may contribute to detoxification mechanisms. Imaging EPS at high resolution revealed the incorporation of Ag NPs and their aggregates into the EPS-gel matrix, proving their detoxifying capacity.     

Polysaccharide synthesis in relation to nodulation behavior of Rhizobium leguminosarum.

In this study, we characterized four Tn5 mutants derived from Rhizobium leguminosarum RBL5515 with respect to synthesis and secretion of cellulose fibrils, extracellular polysaccharides (EPS), capsular polysaccharides, and cyclic beta-(1,2)-glucans. One mutant, strain RBL5515 exo-344::Tn5, synthesizes residual amounts of EPS, the repeating unit of which lacks the terminal galactose molecule and the substituents attached to it. On basis of the polysaccharide production pattern of strain RBL5515 exo-344::Tn5, the structural features of the polysaccharides synthesized, and the results of an analysis of the enzyme activities involved, we hypothesize that this strain is affected in a galactose transferase involved in the synthesis of EPS only. All four mutants failed to nodulate plants belonging to the pea cross-inoculation group; on Vicia sativa they induced root hair deformation and rare abortive infection threads. All of the mutants appeared to be pleiotropic, since in addition to defects in the synthesis of EPS, lipopolysaccharide, and/or capsular polysaccharides significant increases in the synthesis and secretion of cyclic beta-(1,2)-glucans were observed. We concluded that it is impossible to correlate a defect in the synthesis of a particular polysaccharide with nodulation characteristics.     

Fibrillar beta-lactoglobulin gels: Part 1. Fibril formation and structure

As a prelude to experimental and theoretical work on the mechanical properties of fibrillar beta-lactoglobulin gels, this paper reports the structural characterization of beta-lactoglobulin fibrils by electron and atomic force microscopy (AFM), infrared and Raman spectroscopy, and powder X-ray diffraction. Aggregates formed by incubation of beta-lactoglobulin in various alcohol-water mixtures at pH 2, and in water-trifluoroethanol (TFE) at pH 7, were found to be wormlike (approximately 7 nm in width and <500 nm in length), with a "string-of-beads" appearance. Longer (approximately 7 nm in width, and >1 microm in length), smoother, and seemingly stiffer fibrils formed on heating aqueous beta-lactoglobulin solutions at pH 2 and low ionic strength, although there was little evidence for the higher-order structures common in most amyloid-forming systems. Time-lapse AFM also revealed differences in the formation of these two fibril types: thermally induced aggregation occurring more cooperatively, in keeping with a nucleation and growth process. Only short stiff-rods (<20 nm in length) formed on heating beta-lactoglobulin at pH 7, and only complex three-dimensional "amorphous"aggregates in alcohols other than TFE at this pH. Studies of all of the pH 2 fibrils from beta-lactoglobulin, by Raman and infrared spectroscopy confirmed beta-sheet as mediating the aggregation process. Interestingly, however, some evidence for de novo helix formation for the solvent-induced systems was obtained, although it remains to be seen whether this is actually incorporated into the fibril-structure. In contrast to other amyloid systems, X-ray powder diffraction provided no evidence for extensive repeating "crystalline" structures for any of the pH 2 beta-lactoglobulin fibrils. In relation to amyloid, the lactoglobulin fibrils bear more resemblance to protofilaments than to higher-order fibril structures, these latter appearing more convincingly for thermally induced insulin fibrils (pH 2) also included in the AFM study.     

PRODUCTION OF MUCILAGE BY THE ADRIATIC EPIPELIC DIATOM CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE) UNDER NUTRIENT LIMITATION

The carbon partitioning of the epipelic diatom Cylindrotheca closterium (Ehrenberg) Reiman and Lewin isolated from the Adriatic Sea was studied in the laboratory under varying scenarios of nutrient limitation. Total number of cells, photosynthesis measured at 695 μmol photons·m ^{− 2}·s ^{− 1} irradiance (P_{695- μ mol}), chlorophyll ( a + c ) content, respiration, extracellular polymeric substances (EPS), total particulate carbohydrate (TPC), and dissolved carbohydrate were evaluated under nitrogen and phosphorus deficiencies in culture. The highest total number of cells was found in the control, whereas the nitrogen-limited treatment showed the lowest value. During the transition phase of growth, photosynthesis in the nitrogen-limited treatment was 3-fold lower than in the phosphorus-limited treatment and 4-fold lower than in the control. Differences in respiration rates and chlorophyll ( a + c ) content were even more marked. Dissolved carbohydrate remained the same in all the treatments, whereas during the transition and stationary phase, EPS presented the highest values under phosphorus limitation and the lowest in the control treatment. The production of EPS was closely linked to the periods of carbon assimilation (transition phase) in the nutrient depleted treatments, especially in the phosphorus-limited treatment. These results point out the relevance of the nutrient imbalance (nitrogen or phosphorus) in the production of EPS by the benthic or resuspended diatoms and suggest that these diatoms play an important role in nutrient-unbalanced systems like sediments or marine snow.     

Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.     

Time of Flight-Secondary Ion Mass Spectrometry on isolated extracellular fractions and intact biofilms of three species of benthic diatoms

Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS) was used to study compositional characteristics of Extracellular Polymeric Substances (EPS) and compared these to characteristics of the EPS-matrix of intact diatom biofilms. Three benthic diatoms species were investigated, Cylindrotheca closterium, Navicula mutica and Nitzschia cf. brevissima. Comparison of the ToF-SIMS spectra of sequentially extracted EPS-fractions by cluster analysis and multidimensional scaling analysis (MDS) indicated that soluble and bound EPS were not distinguishable based on their ion spectra. On the contrary the water insoluble bicarbonate soluble (WIBS)-EPS-fraction formed a distinct cluster showing that this material was compositionally different from the other EPS-fractions. Ion spectra of the EPS-fractions were dissimilar to results obtained from intact biofilms. This suggested that during the extraction procedure, the structure of the EPS irreversibly changed, which alters the fragmentation patterns of the extracellular surface layer. Furthermore, from the examination of the positive ion spectra it was shown that the overall composition of EPS in the intact biofilms was different between diatom species. In spite of these differences, several common peak patterns were shared between different species. This suggests the presence of common structural components in the EPS of these diatoms that may play a role in building the surface EPS-layer.     

Structural Identification of Individual Helical Amyloid Filaments by Integration of Cryo-Electron Microscopy-Derived Maps in Comparative Morphometric Atomic Force Microscopy Image Analysis

The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297–391), termed ‘dGAE’. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are structurally closely related to the paired helical filaments (PHFs) isolated from Alzheimer’s disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.     

Isolation and biochemical characterization of extracellular polymeric secretions (EPS) from modern soft marine stromatolites (Bahamas) and its inhibitory effect on CaCO3 precipitation

Bahamian soft marine stromatolites consist of cyanobacterial biofilms and carbonate sand grains (ooids) embedded in their extracellular polymeric secretions (EPS). EPS were isolated from natural marine stromatolites and the laboratory cultured stromatolite forming cyanobacterium isolate Schizothix sp. Laboratory investigations were conducted to examine biochemical characteristics and the role of EPS in the inhibition of CaCO3 precipitation. EPS consisted of acid polysaccharides and proteins. SDS-PAGE and amino acid analysis suggested that EPS from both soft marine stromatolite and Schizothrix sp. mat contained small proteins (38 kD and 45 kD) enriched in aspartic acid and glutamic acid. Also, immuno blotting suggested that natural EPS contain high molecular weight acid polysaccharide (500 k) which may represent cross-linked products of laboratory cultured Schizothrix sp. acid polysaccharide (300 k). EPS from both soft marine stromatolite and laboratory cultured Schizothrix sp. inhibited CaCO3 precipitation in vitro, as determined using pH drift assays examining pH decrease which occur in response to CaCO3 precipitation. PH drift assays of enzymatically and chemically modified EPS isolated from soft marine stromatolite and laboratory cultured Schizothrix sp. indicated that both uronic acids and protein fractions may be involved in the inhibition of CaCO3 precipitation.     

The nanometer-scale structure of amyloid-Β visualized by atomic force microscopy

Amyloid-Β (AΒ) is the major protein component of neuritic plaques found in Alzheimer's disease. Evidence suggests that the physical aggregation state of AΒ directly influences neurotoxicity and specific cellular biochemical events. Atomic force microscopy (AFM) is used to investigate the three-dimensional structure of aggregated AΒ and characterize aggregate/fibril size, structure, and distribution. Aggregates are characterized by fibril length and packing densities. The packing densities correspond to the differential thickness of fiber aggregates along az axis (fiber height above thex-y imaging surface). Densely packed aggregates (≥100 nm thick) were observed. At the edges of these densely packed regions and in dispersed regions, three types of AΒ fibrils were observed. These were classified by fibril thickness into three size ranges: 2–3 nm thick, 4–6 nm thick, and 8–12 nm thick. Some of the two thicker classes of fibrils exhibited pronounced axial periodicity. Substructural features observed included fibril branching or annealing and a height periodicity which varied with fibril thickness. When identical samples were visualized with AFM and electron microscopy (EM) the thicker fibrils (4–6 nm and 8–12 nm thick) had similar morphology. In comparison, the densely packed regions of ～≥100 nm thickness observed by AFM were difficult to resolve by EM. The small, 2- to 3-nm-thick, fibrils were not observed by EM even though they were routinely imaged by AFM. These studies demonstrate that AFM imaging of AΒ fibrils can, for the first time, resolve nanometer-scale,z-axis, surface-height (thickness) fibril features. Concurrentx-y surface scans of fibrils reveal the surface submicrometer structure and organization of aggregated AΒ. Thus, when AFM imaging of AΒ is combined with, and correlated to, careful studies of cellular AΒ toxicity it may be possible to relate certain AΒ structural features to cellular neurotoxicity.     

Impact of sludge retention time on MBR fouling: role of extracellular polymeric substances determined through membrane autopsy

The impact of sludge retention time (SRT) on the biofouling of a membrane bioreactor (MBR) by extracellular polymeric substances (EPS) was investigated. The MBR was operated at 60 and 20 d SRT. The gel layer (recovered through optimized membrane autopsy methods) and the cake layer were analyzed for their content and profile of EPS proteins and polysaccharides. The change to a shorter SRT led to decreased membrane filterability, concomitant with a higher expression of EPS proteins in the cake layer, which were identified as being mainly related with biosynthesis and stress functions. The gel layer was more substantial in internal fibers, with polysaccharides being the major component in this layer. With the decrease in SRT (and filterability decrease), the overall polysaccharide content and sugar variety increased. In conclusion, SRT impacted not only on the quantity but also the composition of EPS molecules, and both were shown to be important in biofouling.     

Subcellular location and composition of the wall and secreted extracellular sulphated polysaccharides/proteoglycans of the diatomStauroneis amphioxys Gregory

Summary Extracellular polysaccharide/proteoglycan (EPS) mucilages play a crucial role in maintaining the structure of the extensive algal sheets that appear along the undersurface of nearshore Antarctic sea ice during the austral spring. In this study we have determined the composition and ultrastructural location of a family of novel sulphated polysaccharides/proteoglycans from the pennate ice diatomStauroneis amphioxys Gregory. They occur as soluble EPS in the culture supernatant, as an intercellular mucilage sheet, and as components of a distinct organic layer (diatotepum) underlying the silicious cell wall. The ultrastructural location and quantitative extraction of the mucilage EPS and the major diatotepum polysaccharides with hot water and alkali, respectively, was monitored by light and electron microscopy. The EPS and wall components were purified by Ultrafiltration, anion exchange and gel filtration chromatographies, and their monosaccharide composition was determined by gas-chro-matography mass spectrometry. The soluble and mucilage EPS, and major diatotepum polysaccharides/proteoglycans had an apparent molecular mass greater than 2 × 10⁶ Da on gel. They contained a similar complex monosaccharide composition that includes glucuronic acid and galactose as the major sugars and significant levels of rhamnose, fucose, arabinose, xylose, mannose, glucose and the mono-O-methylated monosaccharides 3-O-methylrhamnose, 3-O-methylfucose, 3-O- and 4-O-methylxylose. The ratios of Gal to GlcA, which together account for 45% of the monosaccharides, varied from 0.8 (in the soluble EPS) to 2.3 (in diatotepum polysaccharides). The level of sulphation also varied from 5–15% (w/w), with the mucilage EPS being the most highly sulphated. The soluble EPS also contains a small amount of protein (ca. 5%, v/w) which cochromatographs with the polysaccharide during gel filtration and anion exchange chromatographies suggesting that it may be a sulphated proteoglycan. They are clearly distinct from a sulphated glucuronomannan that remained in the alkali-insoluble fraction and may be tightly associated with the silica wall components. The amount of mucilage EPS increased during logarithmic growth but decreased during stationary phase, when most of the EPS was found in the soluble pool. These changes correlate with the breakdown of the mucilage sheet and dispersal of diatom colonies during stationary growth. Interestingly, the soluble EPS from stationary-growth cultures was indistinguishable from the mucilage EPS of logarithmic- or stationary-phase cells, suggesting that the dissolution of the intercellular mucilage was not due to a change in EPS composition. The possibility that cell motility may be required for mucilage formation and the significance of these polysaccharides in the under-ice community is discussed.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

High-resolution atomic force microscopy of soluble Abeta42 oligomers

Soluble oligomers and protofibrils are widely thought to be the toxic forms of the Abeta42 peptide associated with Alzheimer's disease. We have investigated the structure and formation of these assemblies using a new approach in atomic force microscopy (AFM) that yields high-resolution images of hydrated proteins and allows the structure of the smallest molecular weight (MW) oligomers to be observed and characterized. AFM images of monomers, dimers and other low MW oligomers at early incubation times (< 1h) are consistent with a hairpin structure for the monomeric Abeta42 peptide. The low MW oligomers are relatively compact and have significant order. The most constant dimension of these oligomers is their height (approximately 1-3 nm) above the mica surface; their lateral dimensions (width and length) vary between 5 nm and 10nm. Flat nascent protofibrils with lengths of over 40 nm are observed at short incubation times (< or = 3h); their lateral dimensions of 6-8 nm are consistent with a mass-per-length of 9 kDa/nm previously predicted for the elementary fibril subunit. High MW oligomers with lateral dimensions of 15-25 nm and heights ranging from 2-8 nm are common at high concentrations of Abeta. We show that an inhibitor designed to block the sheet-to-sheet packing in Abeta fibrils is able to cap the heights of these oligomers at approximately 4 nm. The observation of fine structure in the high MW oligomers suggests that they are able to nucleate fibril formation. AFM images obtained as a function of incubation time reveal a sequence of assembly from monomers to soluble oligomers and protofibrils.     

Assembly of alpha-synuclein fibrils in nanoscale studied by peptide truncation and AFM

α-Synuclein (α-Syn) fibrils are the major component of Lewy bodies that are closely associated with the pathogenesis of Parkinson’s disease, but the mechanism for the fibril assembly remains poorly understood. Here we report using a combination of peptide truncation and atomic force microscopy (AFM) to elucidate the self-assembly and morphology of the α-Syn fibrils. The results show that protease K significantly slims the fibrils from the mean height of ∼6.6 to ∼4.7 nm, whereas chaotropic denaturant urea completely breaks down the fibrils into small particles. The in situ enzymatic digestion also results in thinning of the fibrils, giving rise to some nicks on the fibrils. Moreover, N- or C-terminally truncated α-Syn fragments assemble into thinner filaments with the heights depending on the peptide lengths. A nine-residue peptide corresponding to the homologous GAV-motif sequence can form very thin (∼2.2 nm) but long (>1 μm) filaments. Thus, the central sequence of α-Syn forms a fibrillar core by cross-β-structure that is flanked by two flexible termini, and the orientation of the fibril growth is perpendicular to the β-sheet structures.     

Myxococcus xanthus chemotaxis homologs DifD and DifG negatively regulate fibril polysaccharide production

The extracellular matrix fibrils of Myxococcus xanthus are essential for the social lifestyle of this unusual bacterium. These fibrils form networks linking or encasing cells and are tightly correlated with cellular cohesion, development, and social (S) gliding motility. Previous studies identified a set of bacterial chemotaxis homologs encoded by the dif locus. It was determined that difA, difC, and difE, encoding respective homologs of a methyl-accepting chemotaxis protein, CheW, and CheA, are required for fibril production and therefore S motility and development. Here we report the studies of three additional genes residing at the dif locus, difB, difD, and difG. difD and difG encode homologs of chemotaxis proteins CheY and CheC, respectively. difB encodes a positively charged protein with limited homology at its N terminus to conserved bacterial proteins with unknown functions. Unlike the previously characterized dif genes, none of these three newly studied dif genes are essential for fibril production, S motility, or development. The difB mutant showed no obvious defects in any of the processes examined. In contrast, the difD and the difG mutants were observed to overproduce fibril polysaccharides in comparison with production by the wild type. The observation that DifD and DifG negatively regulate fibril polysaccharide production strengthens our hypothesis that the M. xanthus dif genes define a chemotaxis-like signal transduction pathway which regulates fibril biogenesis. To our knowledge, this is the first report of functional studies of a CheC homolog in proteobacteria. In addition, during this study, we slightly modified previously developed assays to easily quantify fibril polysaccharide production in M. xanthus.     

Short and long range order of the morphology of silk from Latrodectus hesperus (Black Widow) as characterized by atomic force microscopy.

The surfaces of both stretched and unstretched silk threads from the cobweb weaver, Latrodectus hesperus (Black Widow) have been examined by atomic force microscopy (AFM). AFM images of cobweb scaffolding threads show both unordered and highly ordered regions. Two types of fibers within the threads were observed: thicker (approximately 300 nm in diameter) fibers oriented parallel to the thread axis and thinner (10-100 nm) fibrils oriented across the thread axis. While regions which lacked parallel fibers or fibrils were observed on threads at all strain values, the probability of observing fibers and/or fibrils increased with strain. High-resolution AFM images show that with increasing strain, both mean fiber and fibril diameters decrease and that fibrils align themselves more closely with the thread axis. The observation of fibers and fibrils within the cobweb threads has implications for current models of the secondary and tertiary structure and organization of spider silk.     

The formation and development of cellulose-synthesizing linear terminal complexes (TCs) in the plasma membrane of the marine red algaErythrocladia subintegra Rosenv.

Summary The formation and development of linear terminal complexes (TCs), the putative cellulose synthesizing units of the red algaErythrocladia subintegra Rosenv., were investigated by a freeze etching technique using both rotary and unidirectional shadowing. The ribbon-like cellulose fibrils ofE. subintegra are 27.6 ± 0.8 nm wide and only 1–1.5 nm thick. They are synthesized by TCs which are composed of repeating transverse rows formed of four particles, the TC subunits. About 50.4 ± 1.7 subunits constitute a TC. They are apparently more strongly interconnected in transverse than in longitudinal directions. Some TC subunits can be resolved as doublets by Fourier analysis. Large globular particles (globules) seem to function as precursor units in the assembly and maturation of the TCs. They are composed of a central hole (the core) with small subunits forming a peripheral ridge and seem to represent zymogenic precursors. TC assembly is initiated after two or three gobules come into close contact with each other, swell and unfold to a nucleation unit resembling the first 2–3 transverse rows of a TC. Longitudinal elongation of the TC occurs by the unfolding of globules attached to both ends of the TC nucleation unit until the TC is completed. The typical intramembranous particles observed inErythrocladia (unidirectional shadowing) are 9.15 ± 0.13 nm in diameter, whereas those of a TC have an average diameter of 8.77 ± 0.11 nm. During cell wall synthesis membranes of vesicles originating from the Golgi apparatus and which seem to fuse with the plasma membrane contain large globules, 15–22 nm in diameter, as well as tetrads with a particle diameter of about 8 nm. The latter are assumed to be involved in the synthesis of the amorphous extracellular matrix cell wall polysaccharides. The following working model for cellulose fibril assembly inE. subintegra is suggested: (1) the ribbon-like cellulose fibril is synthesized by a single linear TC; (2) the number of glucan chains per microfibril correlates with the number of TC subunits; (3) a single subunit synthesizes 3 glucan chains which appear to stack along the 0.6 nm lattice plane; (4) lateral aggregation of the 3-mer stacks leads to the crystalline microfibril.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement