Diatom adhesive mucilage contains distinct supramolecular assemblies of a single modular protein.

A previous study used atomic force microscopy saw-tooth retraction curves to characterize the adhesive mucilage pads of the diatom Toxarium undulatum. The major mucilage component consisted of adhesive nanofibers (ANFs) made up of modular proteins arranged into cohesive units, each containing a set number of modular proteins aligned in parallel. This study shows that T. undulatum adhesive mucilage is a biocomposite containing four additional adhesive components, including single modular proteins that are likely to be the structural units from which the ANFs are assembled. Two further distinct supramolecular assemblies were observed to coexist with ANFs (ANFs II and III), along with a continuum of single modular proteins through oligomers made up of varying numbers of modular proteins arranged in parallel. All components of the adhesive biocomposite produce a characteristic force spectrum with the same interpeak distance (35.3 +/- 0.3 (mean +/- SE) nm), suggesting they are derived from discrete supramolecular assemblies of the same modular protein, but they are distinguishable from one another based on the rupture force, persistence length, and interpeak force measured from their saw-tooth curves.

Single adhesive nanofibers from a live diatom have the signature fingerprint of modular proteins.

The adhesive and mechanical properties of a cell-substratum adhesive secreted by live diatom cells were examined in situ using atomic force microscopy. The resulting force curves have a regular saw-tooth pattern, the characteristic fingerprint of modular proteins, and when bridged between tip and surface can repeatedly be stretched and relaxed resulting in precisely overlaying saw-tooth curves (up to approximately 600 successive cycles). The average rupture force of the peaks is 0.794 +/- 0.007 (mean +/- SE) nN at a loading rate of 0.8 microm/s and the average persistence length is 0.026 +/- <0.001 (mean +/- SE) nm (fit using the worm-like chain model). We propose that we are pulling on single adhesive nanofibers, each a cohesive unit composed of a set number of modular proteins aligned in register. Furthermore, we can observe and differentiate when up to three adhesive nanofibers are pulled based upon multimodal distributions of force and persistence length. The high force required for bond rupture, high extensibility (approximately 1.2 microm), and the accurate and rapid refolding upon relaxation, together provide strong and flexible properties ideally suited for the cell-substratum adhesion of this fouling diatom and allow us to understand the mechanism responsible for the strength of adhesion.

Adhesion and motility of fouling diatoms on a silicone elastomer.

Recent demands for non-toxic antifouling technologies have led to increased interest in coatings based on silicone elastomers that 'release' macrofouling organisms when hydrodynamic conditions are sufficiently robust. However, these types of coatings accumulate diatom slimes, which are not released even from vessels operating at high speeds (>30 knots). In this study, adhesion strength and motility of three common fouling diatoms (Amphora coffeaeformis var. perpusilla (Grunow) Cleve, Craspedostauros australis Cox and Navicula perminuta Grunow) were measured on a poly-dimethylsiloxane elastomer (PDMSE) and acid-washed glass. Adhesion of the three species was stronger to PDMSE than to glass but the adhesion strengths varied. The wall shear stress required to remove 50% of cells from PDMSE was 17 Pa for Craspedostauros, 24 Pa for Amphora and >>53 Pa for Navicula; the corresponding values for glass were 3, 10 and 25 Pa. In contrast, the motility of the three species showed little or no correlation between the two surfaces. Craspedostauros moved equally well on glass and PDMSE, Amphora moved more on glass initially before movement ceased and Navicula moved more on PDMSE before movement ceased. The results show that fouling diatoms adhere more strongly to a hydrophobic PDMSE surface, and this feature may contribute to their successful colonization of low surface energy, foul-release coatings. The results also indicate that diatom motility is not related to adhesion strength, and motility does not appear to be a useful indicator of surface preference by diatoms.

Brefeldin A affects adhesion of zoospores of the green alga Enteromorpha.

Primary adhesion of zoospores of the green macroalga Enteromorpha to substrata involves a massive release of adhesive glycoproteins from Golgi-derived, membrane-bounded vesicles in the anterior region of the spore, followed by rapid curing. This process is sensitive to low concentrations (5-10 microg x ml(-1)) of the secretion-inhibiting antibiotic, brefeldin A (BFA). The proportion of cells that settled in BFA was reduced by approximately 50%, but the effect was fully reversed by washing in seawater to remove the BFA. Ultrastructural observations showed that BFA caused the breakdown of Golgi stacks in the majority of cells examined. When settled cells were subjected to shear stress, a greater proportion of those settled in the presence of BFA were detached, compared with controls, indicating reduced adhesion strength in the presence of the antibiotic. The most likely reason for this is that strong adhesion to substrata either requires the synthesis of extra adhesive materials beyond those present in the swimming spore, or the secretion of an additional component required for adhesive curing. The novel use of atomic force microscopy in force modulation mode demonstrated that the adhesive secreted by most spores in the presence of BFA did not undergo the rapid curing process typical of control spores. However, some variation between zoospores was observed, with some cells showing no ultrastructural changes and normal adhesive curing. These results are discussed in relation to variations observed in the propensity and competence of spores to settle, which may be reflected in differential requirements for de novo synthesis and secretion of materials needed for full adhesion.

Identification of a 41 kDa protein embedded in the biosilica of scales and bristles isolated from Mallomonas splendens (Synurophyceae, Ochrophyta).

Cells of the photosynthetic protist Mallomonas splendens (Synurophyceae, Ochrophyta) are encased within a highly patterned wall or scale case that consists of silicified scales and bristles. In an effort to understand the mechanisms that unicellular protists utilize to produce elaborate, mineralized structures of great complexity and hierarchical structure, we identified and characterized a 41 kDa protein from purified scales/bristles isolated from M. splendens (SP41 for Scale Protein of 41 kDa). A cDNA encoding this protein was isolated and sequence analysis indicated that it is a novel protein. Polyclonal antibodies were generated against bacterially expressed SP41 and used to localize the protein throughout scale and bristle morphogenesis. Immunoelectron microscopy confirmed the biochemical data that SP41 is a component of mature scales and bristles, the protein localizing to silicified components of the purified extracellular matrix. During scale and bristle biogenesis within the cell, SP41 is deposited into a specialized Silica Deposition Vesicle (SDV) concomitant with silica deposition, a highly regulated event during scale and bristle formation. These results argue for SP41 playing a role in morphogenesis and/or silicification within the SDV during scale and bristle biogenesis.

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha.

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7-10 microm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 microm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 +/- 1.7 mN m(-1) (mean +/- SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m(-1). The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 x 10(6) +/- 0.05 x 10(6) N m-2 (mean +/- SE; n=30). Within minutes of release the adhesive underwent a progressive 'curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives.

Pleuralins are involved in theca differentiation in the diatom Cylindrotheca fusiformis.

Diatom cells are encased within a silica-based cell wall (frustule) that serves as armour-like protection for the enclosed protoplast. Maintaining the integrity of the frustule requires a precise coupling between the biogenesis of new frustule components and the cell cycle. Thus far, the molecular mechanisms by which this coupling is achieved are unknown. This study demonstrates that pleuralins (formerly HEPs), a previously characterized family of diatom cell wall proteins, are involved in cell cycle-dependent frustule development. The frustule is made up of two, overlapping half-shells termed the epitheca and hypotheca. Both thecae are morphologically identical, yet immunolocalisation with anti-pleuralin antibodies demonstrates that their protein composition is clearly different. During interphase, pleuralins are associated only with the epitheca, where they are confined to the inner surface of the terminal elements (pleural bands) in the region of overlap with the hypotheca. At cell division, pleuralins also become associated with the newly formed pleural bands of the hypotheca. Remarkably, this process is concomitant with the functional conversion of the parental hypotheca into the epitheca of one of the progeny cells. These results indicate that developmentally controlled association of pleuralins with the frustule is involved in hypotheca-epitheca differentiation, which is a crucial process to ensure proper frustule development.

Diatom gliding is the result of an actin-myosin motility system.

Diatoms are a group of unicellular microalgae that are encased in a highly ornamented siliceous cell wall, or frustule. Pennate diatoms have bilateral symmetry and many genera possess an elongated slit in the frustule called the raphe, a feature synonymous with their ability to adhere and glide over a substratum, a process little understood. We have used cytoskeleton-disrupting drugs to investigate the roles of actin, myosin, and microtubules in diatom gliding or motility. No effect on diatom gliding was observed using the cytochalasins, known actin inhibitors, or the microtubule-inhibitors oryzalin and nocodazole. The latrunculins are a new group of anti-actin drugs, and we show here that they are potent inhibitors of diatom gliding, resulting in the complete disassociation of the raphe-associated actin cables. The recovery of actin staining and motility following latrunculin treatment was extremely fast. Cells exposed to latrunculin for 12 h recovered full function and actin staining within 5 sec of the drug being removed, demonstrating that the molecular components required for this motility system are immediately available. Butanedione monoxime (BDM), a known myosin inhibitor, also reversibly inhibited diatom gliding in a manner similar to the latrunculins. This work provides evidence that diatom gliding is based on an actin/myosin motility system.

Extracellular matrix assembly in diatoms (Bacillariophyceae). Iii. Organization Of fucoglucuronogalactans within the adhesive stalks of achnanthes longipes

Achnanthes longipes is a marine, biofouling diatom that adheres to surfaces via adhesive polymers extruded during motility or organized into structures called stalks that contain three distinct regions: the pad, shaft, and collar. Four monoclonal antibodies (AL.C1-AL.C4) and antibodies from two uncloned hybridomas (AL.E1 and AL.E2) were raised against the extracellular adhesives of A. longipes. Antibodies were screened against a hot-water-insoluble/hot-bicarbonate-soluble-fraction. The hot-water-insoluble/hot-bicarbonate-soluble fraction was fractionated to yield polymers in three size ranges: F1, >/= 20,000, 000 Mr; F2, congruent with100,000 Mr; and F3, <10,000 Mr relative to dextran standards. The congruent with100,000-Mr fraction consisted of highly sulfated (approximately 11%) fucoglucuronogalactans (FGGs) and low-sulfate (approximately 2%) FGGs, whereas F1 was composed of O-linked FGG (F2)-polypeptide (F3) complexes. AL.C1, AL.C2, AL.C4, AL.E1, and AL.E2 recognized carbohydrate complementary regions on FGGs, with antigenicity dependent on fucosyl-containing side chains. AL.C3 was unique in that it had a lower affinity for FGGs and did not label any portion of the shaft. Enzyme-linked immunosorbent assay and immunocytochemistry indicated that low-sulfate FGGs are expelled from pores surrounding the raphe terminus, creating the cylindrical outer layers of the shaft, and that highly sulfated FGGs are extruded from the raphe, forming the central core. Antibody-labeling patterns and other evidence indicated that the shaft central-core region is related to material exuded from the raphe during cell motility.

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans.

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and > 200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion.

A scale associated protein of Apedinella radians (Pedinellophyceae) and its possible role in the adhesion of surface components.

Monoclonal antibodies have been generated against cell surface components of the unicellular phytoflagellate Apedinella radians (Pedinellophyceae). One monoclonal antibody, designated Arg 1E5/1B1, labels a scale associated protein (SAP) of 145 kDa. Immunofluorescence microscopy of whole cells as well as immunoelectron microscopy of whole cell mounts and thin sections using Arg 1E5/1B1 have shown that the SAP is located on the proximal surface of body scales and spine-scales. Its specific location suggests that the SAP may play a role in the adhesion of these surface components to the cell membrane and/or to one another. The potential of monoclonal antibody Arg 1E5/1B1 as a tool to study cell surface morphogenesis and the role of the endomembrane system in A. radians is discussed.

"Transfer connections": specialized pathways for nutrient translocation in a red alga?

"Transfer connections" are morphologically and developmentally distinct pit connections in Polysiphonia (Ceramiales). They are intracellular rather than extracellular and have been observed between all cells of the diploid carposporophyte plus those specialized cells of the gametophyte suspected of providing nutritive materials to it.

Fine-structural studies of the gametes and embryo of Fucus vesiculosus L. (Phaeophyta). III. Cytokinesis and the multicellular embryo.

Condensation of the chromosomes during the first cell division following fertilization of the brown alga Fucus vesiculosus L. is accompanied by the almost complete disappearance of the nuclear envelope. Golgi vesicles and other small vesicles appear within the spindle, which has paired centrioles at each end. A large amount of rough endoplasmic reticulum is in the surrounding cytoplasm during mitosis, and many vesicles at the spindle margin are encircled by stacks of endoplasmic reticulum. Annulate lamellae are observed during mitosis. The envelope which initially reforms around the chromatin in telophase has unevenly spaced nuclear pores. Cytokinesis results primarily by vesicle addition to a centripetal furrow. Mitochondria and chloroplasts concentrate around the partition site, possibly in association with microfilaments. Fibrillar material is added rapidly to the space between the daughter cells from vesicle discharge of both cells and seems to spread into the older cell wall surrounding the embryo. The rhizoid daughter cell contains numerous mitochondria and hypertrophied Golgi bodies whose vesicles increasingly pack the cell. The thallus daughter cell is packed with a variety of vesicles, and the nucleus is surrounded by many dilated cisternae of rough endoplasmic reticulum. By the four-cell stage, chloroplasts of the rhizoid cells have weakly staining lamellae, while chloroplasts of the thallus cells are actively dividing with deeply staining lamellae.

Fine-structural studies of the gametes and embryo of Fucus vesiculosus L. (Phaeophyta). II. The cytoplasm of the egg and young zygote.

Following fertilization, there are rapid changes in the appearance of the Fucus egg. Large electron-translucent vesicles (V1) accumulate fibrillar material, and following pronuclear fusion, they are largely electron-opaque. These vesicles (V1) are formed originally in unfertilized eggs by smooth endoplasmic reticulum (SER) after release of the eggs from the oogonium. Golgi complex hypertrophy follows fertilization, and this increased activity continues throughout early embryogenesis. Wall formation begins after penetration of the egg by the sperm. Vesicles (V2) of unknown origin, which have homogeneously fibrillar contents, and Golgi vesicles (V3) merge with SER-derived vesicles (V1) after wall formation begins. Osmiophilic bodies are a prominent feature of the egg and embryo. They are penetrated by SER, and subsequently there is a loss of electron-opaque material. Alternatively, they discharge concentrically whorled material into the cytoplasm. The nuclear surface of the egg is convoluted in the period close to fertilization, and electron-opaque material is segregated in the cytoplasmic matrix lying within the nuclear invaginations.

Fine-structural studies of the gametes and embryo of Fucus vesiculosus L. (Phaeophyta). I. Fertilization and pronuclear fusion.

In the marine brown alga, Fucus vesiculosus L., the sperm pronucleus is delimited by an envelope following penetration of the eff by the sperm. This envelope disintegrates as the pronucleus begins its migration through the cytoplasm of the egg. The highly condensed chromatin of the sperm pronucleus disperses slightly following disintegration of the envelope. Microtubules of unknown origin are associated with the sperm pronucleus during its migration. The flagellar microtubules remain in the peripheral cytoplasm but lose their tight 9 + 2 configuration. The sperm eyespot and mitochondria follow the pronucleus through the cytoplasm toward the egg pronucleus. The mitochondria of the sperm are distinguished from those of the egg by their longitudinally oriented cristae and by electron-opaque material in the intracristal space. The pronucleus of the egg becomes convoluted along the surface nearest to the advancing sperm pronucleus. Immediately prior to pronuclear fusion, many egg mitochondria aggregate in the vicinity of the sperm pronucleus. At this time, only the portion of the sperm pronucleus facing the egg pronucleus is surrounded by an envelope. The egg mitochondria disperse rapidly after pronuclear fusion. The sperm mitochondria and eyespot are still in the perinuclear region in 16-h-old embryos. At this time, the osmiophilia of the sperm eyespot has increased, and the sperm mitochondrial membranes are less distinct than in earlier stages. The fine-structural features of fertilization in Fucus are discussed in relation to the fertilization patterns in other cryptogams and marine invertebrates and to polar axis determination in the Fucaceae.