Polyanion-mediated mineralization — assembly and reorganization of acidic polysaccharides in the Golgi system of a coccolithophorid alga during mineral deposition

Summary Immunolocalization of two highly acidic polysaccharides (PS-1 and PS-2) in a calcifying algaPleurochrysis carterae is described throughout the mineralization process, from before crystal nucleation through the cessation of crystal growth. This unicellular coccolithophorid alga is a useful model for mineralization because it produces calcified scales known as coccoliths in homogeneous cell culture. PS-1 and PS-2 were localized in the crystal coats of mature coccoliths and in electron dense Golgi particles. The polyanions are synthesized in medial Golgi cisternae and co-aggregate with calcium ions into discrete 25 nm particles. Particle-laden vesicles bud from cisternal margins and fuse with a coccolith-forming saccule containing an organic oval-shaped scale which forms the base of the future coccolith. The particles are localized on the base before the onset of mineral deposition and are present in the coccolith saccule throughout the period of crystal (CaCO₃) nucleation and growth. During the final phase of coccolith formation, the particles disappear, and the mature crystals acquire an amorphous coat containing PS-1 and PS-2 polysaccharides which remain with the mineral phase after the coccoliths are extruded from the cell. Postulated mechanisms of polyanion-mediated mineralization are reviewed and their relevance to the calcification of coccoliths is addressed.Abbreviations PS-1 polysaccharide one - PS-2 polysaccharide two - BSA bovine serum albumin - SDS sodium dodecyl sulfate - MES 2-(N-morpholino)-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - DHA 3-deoxy-lyxo-2-heptulosaric acid - TCA trichloroacetic acid     

THE FINE STRUCTURE OF HYMENOMONAS (CRICOSPHAERA) CARTERAE. II. OBSERVATIONS ON SCALE AND COCCOLITH PRODUCTION

The fine structure of the marine coccolithophorid Hymenomonas (Cricosphaera) carterae (Braarud & Fagerland) Manton & Peterfi is reported. Details of the formation of the circular organic body scales are presented. Their formation is shown to be closely linked with the presence of tubules found within the Golgi cisternae. The details of coccolith production are also discussed. The formation of the organic matrix scale appears to be associated with a densely staining organelle, the intracellular coccolith pre-cursor. The precise mechanisms involved in the mineralization of the organic matrix scale is not known but 2 possibilities are discussed. The production of coccoliths in H. carterae is compared with coccolith production in other coccolithophorids that have been investigated finestructurally.     

Involvement of Acidic Polysaccharide Ph-PS-2 and Protein in Initiation of Coccolith Mineralization,as Demonstrated by In Vitro Calcification on the Base Plate

Coccolithophorids, unicellular marine microalgae, have calcified scales with elaborate structures, called coccoliths, on the cell surface. Coccoliths generally comprise a base plate, CaCO₃, and a crystal coat consisting of acidic polysaccharides. In this study, the in vitro calcification conditions on the base plate of Pleurochrysis haptonemofera were examined to determine the functions of the base plate and acidic polysaccharides (Ph-PS-1, -2, and -3). When EDTA-treated coccoliths (acidic polysaccharide-free base plates) or low pH-treated coccoliths (whole acidic polysaccharide-containing base plates) were used, mineralization was not detected on the base plate. In contrast, in the case of coccoliths which were decalcified by lowering of the pH and then treated with urea (Ph-PS-2-containing base plates), distinct aggregates, probably containing CaCO₃, were observed only on the rim of the base plates. Energy dispersive X-ray spectroscopy (EDS) confirmed that the aggregates contained Ca and O, although X-ray diffraction analysis did not reveal any evidence of crystalline materials. Also, in vitro mineralization experiments performed on EDTA-treated coccoliths using isolated acidic polysaccharides demonstrated that the Ca-containing aggregates were markedly formed only in the presence of Ph-PS-2. Furthermore, in vitro mineralization experiments conducted on protein-extracted base plates suggested that the coccolith-associated protein(s) are involved in the Ca deposition. These findings suggest that Ph-PS-2 associated with the protein(s) on the base plate rim initiates Ca²⁺ binding at the beginning of coccolith formation, and some other factors are required for subsequent calcite formation.     

Polysaccharide localization, coccolith formation, and Golgi dynamics in the coccolithophorid Hymenomonas carterae

The periodic acid-thiocarbohydrazide-silver proteinate staining technique according to Thiéry (1967) was employed for visualization of the ultrastructural localization of polysaccharides in the coccolithophorid alga Hymenomonas carterae . Preferential staining was observed in the Golgi apparatus, including constituents and precursors of "scales" and " coccoliths " (scales with a rim of elaborate CaCO3 crystals), which are extruded and become part of the cell wall. Cells fixed in the presence of the polyanion-precipitating agent cetylpyridiniumchloride showed a voluminous coat over the crystalline matter of the coccolith giving the extracellular coccoliths the appearance of being glued together. Soluble acid polysaccharides are thought to occur in the coat. Evidence is presented that the coat and the crystalline matter are produced simultaneously. The excellent stainability of the Golgi apparatus allowed study of its morphology in considerable detail, and permitted a tentative reconstruction of the formation of coccoliths and scales and of the Golgi dynamics in general. The question of whether Golgi cisternae are mobile or static entities is dealt with.     

Formation and function of the “Xestoleberis‐spot” in Xestoleberis hanaii (Crustacea: Ostracoda)

The crescent sculpture of the so‐called “Xestoleberis‐spot” develops inside the calcified valve of the family Xestoleberididae. Electron microscopic observations on both, intermoult and postmoult stages of Xestoleberis species reveal that the “Xestoleberis‐spot” system consists of three elements; two calcified chambers, a vesicle of electron‐dense material and an uncalcified procuticle. The formation and function of the “Xestoleberis‐spot” system are discussed. In conclusion, the “Xestoleberis‐spot” system functions as the muscle attachment site for several antennal muscles, and provides the material for chitinous fibers in the exocuticle of outer lamella. The unique cuticular structures of the family Xestoleberididae are due to the “Xestoleberis‐spot” system.     

Ultrastructure and taxonomy ofJomonlithus littoralis gen. et sp. nov. (Class prymnesiophyceae), a coccolithophorid from the Northwest Pacific

A new coccolithophorid genusJomonlithus withJ. littoralis as the type species, is described based on specimens isolated from a sand sample collected at the mouth of Nakagawa river, Ibaraki, Japan. This genus is characterized by the coccolith which is composed of an organic base-plate scale and calcified rim elements made up of two different subelements.J. littoralis has been found in several localities along the coast of Japan. Culture and ultrastructure studies gave special attention to the cell cycle, coccolith and scale morphology and the ultrastructure of the cellular organelles. It was confirmed thatJomonlithus is similar toWigwamma Mantonet al., Papposphaera Tangen andPappomonas Manton et Oates on the basis of coccolith morphology and toCricosphaera Braarud andHymenomonas Stein in cellular structure.     

Three Types of Acidic Polysaccharides Associated with Coccolith of Pleurochrysis haptonemofera: Comparison with Pleurochrysis carterae and Analysis Using Fluorescein-Isothiocyanate-Labeled Lectins

In the coccolithophorid microalgae acidic polysaccharides are considered to be involved in the formation of the calcified scale, coccolith. Characteristics of the acidic polysaccharides extracted from the cell surface of the coccolithophorid Pleurochrysis haptonemofera were analyzed. The acidic polysaccharides on the cell surface can be detected by measuring fluorescence of cells after fluorescein-isothiocyanate-labeled lectin staining by flow cytometry. Flow cytometric analyses revealed that the acidic polysaccharides remained on the cell surface even after CaCO₃ in the coccolith was dissolved by lowering pH, but they were extracted by subsequent EDTA or EGTA treatment, suggesting that they are bound not into the CaCO₃ crystals of the coccolith, but onto the surface via Ca²⁺. Analyses of the acidic polysaccharides by anion exchange chromatography, colloidal precipitation with divalent cations, and polyacrylamide gel electrophoresis (PAGE) revealed that P. haptonemofera has 3 types of acidic polysaccharides (Ph-PS-l, -2, and -3). The PAGE patterns suggested that Ph-PS-2 has a repeated structure with a broad range of molecular weight, as in Pleurochrysis carterae, while Ph-PS-1 and -3 contain several minor components in addition to a major component, respectively. The minor components in Ph-PS-1 and -3 that have not been found in P. carterae might be characteristic of P. haptonemofera. Analyses of both the cell surface treated by various concentrations of EDTA and EGTA and the extracts suggested that Ph-PS-2, which is distinguishable by a higher affinity to concanavalin A, is bound onto the coccolith surface more intensely than the other two types of acidic polysaccharides.     

THE FINE STRUCTURE AND SCALE FORMATION OF CHRYSOLEPIDOMONAS DENDROLEPIDOTA GEN. ET. SP. NOV.(CHRYSOLEPIDOMONADACEAE FAM. NOV., CHRYSOPHYCEAE)1

Chrysolepidomonas gen. nov. is described for single-celled monads with two flagella, a single chloroplast, and distinctive canistrate and dendritic scales. The type species, Chrysolepidomonas dendrolepidota sp. nov., is described for the first time. The canistrate scales bear eight “bumps” on the top surface, and the dendriticscales have a tapered base with a quatrifid tip. These organic scales are formed in the Golgi apparatus and storred in a scale reservoir. The scale reservoir is bounded on two sides by the R₁ and R₂ in microtubular roots of the basal apparatus. The cyst (=stomatocyst, statospore) forms endogenously by means of a silica deposition vesicle. The outer cyst surface is smooth, and the pore region is unornamented. Two other organisms bearing canistrate and dendritic scales, previously assigned to the genus Sphaleromants, are transferred to the genus Chrysolepidomonas. They are C.angalica sp. nov. and C. marine(Pienaar) comb. nov. The distinguishing features of Chrysolepidomonas and Sphaleromantis are discussed. A new family, Chrysolepidomonadceae fam. noc., is described for flagellates covered with organic scales.     

Coccolith crystals ofPleurochrysis carterae: Crystallographic faces, organization, and development

Summary The crystallographic and morphological configuration of the mineral ring associated with the coccoliths ofPleurochrysis carterae was determined by transmission electron microscopy and electron diffraction. Mature Pleurochrysis coccoliths consist of an oval organic base plate, a distal rim of interlocking calcite crystals, and a narrow ribbon of organic material which tethers the mineral ring to the base plate. Crystals of two distinct forms (R and V units) alternate about the rim in a quasi regular manner; their crystallographicc-axes are aligned parallel to and inclined about 63° to the coccolith plane, respectively. The mineral ring has four platelike elements: the distal-shield and outer-tube elements which form the V unit, and the proximal-shield and inner-tube elements which form the R units. The platy surfaces of both tube elements correspond to the common (10 4) rhombohedral faces of calcite, and the plates of the proximal-shield element are prismatic (2 0) faces. The plates of the distal-shield element are rather curved and their orientation does not correspond to a favorable calcite face; however, for convenience they are described as approximately ( 108) faces, faces which rarely, if ever, develop in inorganic sources of calcite. During coccolith development the earliest habits observed for both V and R units correspond to rectangular parallelepipeds. Outgrowth from the initial V unit begins by expansion of (10 4) faces which form the platy surfaces of the outer-tube element. Throughout this period of development the mineral ring is flexible, at least in an isolated state. Subsequent outgrowth of the inner-tube and proximal-shield elements from the initial R unit produces a rigid interlocking ring. The unusual ( 108) faces of the distal-shield element develop after the crystals are locked in place. Organic structures in intimate association with the mineral phase during its nucleation and growth include the coccolith ribbon, the calcium-polyanion particles, and the membrane of the coccolith vesicle. These structures are described in reference to their putative functions in regulating the development of V and R units.Abbreviation PS polysaccharide     

The body wall of cheilostome Bryozoa. I. The ectocyst of Watersipora nigra (Canu and Bassler)

The cuticle of Watersipora nigra is at first translucent, but it later becomes black and differentiates into two layers. It is composed, at least in part, of a protein-polysaccharide complex. Calcified parts are three-layered: (1) an outer, cuticular layer, (2) a calcium carbonate skeleton deposited on a matrix of acid mucopolysaccharide, and (3) a “skeletal membrane.” The relationships of these layers indicate that the skeleton is intracuticular. A layer of cuticular material, the “intercalary cuticle” is present in lateral walls, but not transverse walls; it may become calcified in some species. The cuticles of calcified and uncalcified parts of cheilostomes are not necessarily homologous.     

Further observations on the genus Pappomonas Manton et Oates with special reference to P. virgulosa sp. nov. from West Greenland

Electron microscopy of dried preparations of Arctic nanoplankton has permitted a second species of the recently erected genus Pappomonas Manton et Oates to be described and named. P. virgulosa sp. nov. is characterised by coccolith appendages composed of a central shaft ending in a cluster of four finger-like rods. There are also calcified plates of characteristic construction, without appendages. Detailed observations on the coccoliths, especially on the shapes and arrangement of component calcite crystallites and the presence of unmineralised components, have permitted further comparisons to be made with equivalent structures in Papposphaera lepida Tangen, recently investigated in the southern hemisphere, as well as with Pappomonas flabellifera Manton et Oates. As a working hypothesis, an interpretation of coccolith morphology in all three taxa has been formulated in terms of known structural components of uncalcified scales in Chrysochromulina spp. The need for fuller information on certain details, notably the coccolith bases in Pappomonas spp. is stressed.     

Coccolith production inHymenomonas carterae

Summary The formation of coccoliths inHymenomonas carterae was studied. It was found that the Golgi body was directly involved in the production of the baseplate scale, the organic matrix membrane and the deposition of calcium carbonate to produce the coccolith. The ICP body is no longer regarded as being involved in coccolith production but more with the getting rid of excess coccoliths within the cell. Because the ICP body contains acid hydrolases it is suggested that this organelle now be referred to as a residual body.Financial assistance from the Council for Scientific and Industrial Research is gratefully acknowledged.     

CALCIFICATION BY COCCOLITHOPHORIDS: EFFECTS OF pH AND Sr1

Cells of Coccolithus huxleyi which fail to deposit CaCO₃ and form coccoliths often occur as unwanted components in cultures used for studies of calcification. Non-calcified cells generally cannot be made to recalcify, but they can be removed from cultures by treatment at elevated pH or by a method based on faster sinking of calcified cells. Lowering the concentrations of nitrate, phosphate, or trace metals in the medium did not restore calcifying ability of non-calcified cells. However, addition of strontium did promote recalcification of decalcified Cricosphaera carterae grown under calcium limitation. Strontium seemed to promote coccolith attachment to cells rather than to affect calcium uptake or coccolith formation itself.     

Ultrastructure of Mucocysts in Peranema trichophorum (Euglenophyceae)1

The “mucigenic” or “muciferous” bodies of Peranema trichophorum are further characterized here as unique extrusive organelles, the mucocysts. Intracellular and ejected mucocysts have characteristic shapes that may represent different developmental stages. Mucocysts found near the Golgi apparatus are membrane-bounded, elongate, tubular structures with amorphous contents of low electron density. Subpellicular mucocysts are often aligned with pellicular striae and have dense contents, which are separated by an electron-lucent zone from granular material at the tips. Ejected mucocysts are uniform in structure and consist of an inner tube with helical striations, an outer tube with a diamond-shaped pattern, and a dense middle band. Fine fibrils, visible only after mucocyst discharge, emanate from the tips. Mucocysts may also protrude through the pellicle and discharge mucilaginous materials into the medium. Acid phosphatase activity is localized within the subpellicular mucocysts, suggesting that they may be involved in release of hydrolytic enzymes into the medium.     

Lepidotrachelophyllum fornicis,n. g., n. sp., a Ciliate with an External Layer of Organic Scales (Ciliophora,Litostomatea, Haptoria)1

Lepidotrachelophyllum fornicis n. g., n. sp. was discovered in White Lake, Ontario, Canada, under winter ice. The genus is Trachelophyllum-like, being highly flattened, elongate, and very extensible. The major feature that separates it from other genera in the family Trachelophyllidae is the presence of a dense layer of organic scales which covers the exterior of the cell and through which the cilia emerge. The scales are composed of filamentous material which is organized as an ovoid structure. The “rim” of the baseplate is formed of interwoven filaments. The baseplate is broken by circular or polygonal apertures. The same filaments form an arched superstructure broken by even larger, less regular apertures.     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.     

Paraphysomonas faveolata sp. nov. (Chrysophyceae), a fourth marine species with meshwork body-scales

Paraphysomonas faveolata sp. nov., the eighth described species of this genus of colourless chrysophycean flagellates, has heterokont flagellation, parabasal nucleus and silicified body-scales. It is the fourth known species with meshwork scales, these being of two types: flat “cobweb” scales and scales with a planar “honeycombed” extension arising from a “cobweb” base. The organism is compared with the other species of the genus and the case considered for placing forms with meshwork scales in a new genus, separate from forms with spined scales. This step is not taken and the taxonomic status of the genus is discussed in relation to the Ochromonadaceae and other genera of the Synuraceae.     

Coccolith biomineralisation studied with atomic force microscopy

Biomineralisation can only be understood as an interplay between organic and mineral phases. With this objective, we conducted an investigation of coccoliths using atomic force microscopy (AFM), an ultra-high resolution technique that requires no surface coating and can be used in air or under solution at ambient conditions of temperature and pressure. The detailed morphology, crystal structure, organic scales and organic coating of the coccolith species Coccolithus pelagicus , Helicosphaera carteri and Oolithotus fragilis were investigated. The fine structure of coccoliths is very complex, with the calcite either being smooth, dominated by steps or tuberculate; organic cover can be either granular or fibrous. Behaviour of coccolith surfaces during dissolution is influenced both by mineral and organic material and different surface types show variable resistance to dissolution. The organic coating protects element faces against etching. Through atomic resolution AFM, it is possible to establish the crystallographic structure of the distal shields of C. pelagicus and O. fragilis . Though elements of both species are dominated by stable crystal faces, there are important differences between them, with the external edge of elements being parallel to a cleavage direction in C. pelagicus but parallel to the atomic rows in O. fragilis . Thus, there is evidence that the biomineralisation of each species, and also of select areas of coccoliths of the same species, is markedly different.     

Further Observations on the Fine Structure of Acanthamoeba palestinensis (Reich, 1933). The Golgi Complex,Microbodies, and Mitochondria1

The fine structure of the trophozoite of Acanthamoeba palestinensis with a special emphasis on the Golgi complex, microbodies, and mitochondria has been examined. Golgi complexes are distributed throughout the cytoplasm but are most abundant in the perinuclear region. Usually two Goigi complexes are found in the same plane on opposite sides of the nucleus. One of them appears to be in an intimate association with the nuclear membrane. The region of contact contains compact cisternae, vesicles of various sizes, as well as granular and amorphous electron-dense material. Structural changes in the nuclear envelope are also observed in this area. A structure consisting of a Golgi complex and electron-dense microtubule organizing center, comparable to the centrosphere of other Acanthamoeba species, has been observed. Microbodies, surrounded by a single unit membrane and containing a granular matrix and tubular inclusions, are scattered throughout the cytoplasm. These organelles, circular (～1 μm in diameter) or ovoidal (～1 μm in length and ～0.5 μm in width) in section, have often an irregular outline. These microbodies are probably the morphological equivalent of peroxisomes and glyoxysomes. Most mitochondria show a typical structure including tubular cristae and intracristal inclusions. Occasionally mitochondria with two apposed double membranes running through the midline are found. Such atypical cristae have never been reported in small amoebae before.     

The Ultrastructural Feature and Distribution of Microbodies in Mature Embryo Cells of Pinus bungeana

The material of pine seeds used in this investigation was collected in 1982 from Peking. The microbodies of mature embryo ceils are very well developed and their diameter averages about 2–3 μm, even up to 4.3 μm. The appearance is usually ovoid or elliptic. The microbodies are essentially glyoxysomes. The microbody matrix is composed of two types of substances, one type is of a finely granular material in a densely arrangement (Plate Ⅲ Fig. 6); the other is of coarsely granular or flocculant in appearance and the elements of the matrix are loosely distributed. These matrices usually contain an amorphous inclusion or crystalline arrays in regular arrangement. The inclusion sometimes occupies a small portion of the microbody matrix (Plate Ⅲ, Figs, 5, 6) and sometimes the inclusion occupies nearly the entire glyoxysome (Plate Ⅱ, Fig. 3). It is interesting that the “pockets” frequently appear in the microbodies of mature embryo cells, and those are actually as a result of invagination in microbodies (Plate Ⅱ, Fig. 4). In addition, an electron-transparent “oil body-like space” occurs occasionally in microbody (Plate Ⅰ, Fig. 1). The periphery of “space” is a constitutive part of matrix or continuing with the matrix. This “space” may be due to the degradation in a part of the matrix. While the periphery of the pocket is membranaceous and an electron-opaque cytoplasmic groundplasm was found within the pocket. The microbodies of mature embryo cells in Pinus are mainly distributed in pericolumn cells of the root cap and cortical cells of the hypocotyl. Besides the dominant organelles of lipid bodies in the cells of above mentioned tissues, there are also microbodies, amyloplasts, mitochondria, plastids, endoplasm reticulum and Golgi apparatus, of which the microbodies are the most aboundant organelles. In contrast, the microbodies and other organelle are rare in the parenchyma of the cotyledons in Pinus. Their common and outstanding characteristics in various tissues of mature embryo is that the entire cytoplasm of the cells is almost full of the lipid bodies, and each organelle is directly surrounded by a number of lipid bodies (Plate Ⅰ—Ⅲ, Figs. 1–6). Because of the other organelles are rare in parenchyma of the cotyledons, the lipid bodies are so appressed with each other that the inlaid periphery of lipid bodies frequently occurs in some degree. To sum up, based upon 'the state of distribution of microbodies in mature embryo tissues, cotyledons of Pinus could be considered as the main storage organ of nutrient substances, while the root cap and hypocotyl are the important sites of glyoxysome metabolism. The function of glyoxysomes is to convert lipid into the carbohydrates and to transfer the latter to embryos for growth.