Macromolecular recognition directs calcium ions to coccolith mineralization sites

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Science  05 Aug 2016:
Vol. 353, Issue 6299, pp. 590-593
DOI: 10.1126/science.aaf7889


Many organisms form elaborate mineralized structures, constituted of highly organized arrangements of crystals and organic macromolecules. The localization of crystals within these structures is presumably determined by the interaction of nucleating macromolecules with the mineral phase. Here we show that, preceding nucleation, a specific interaction between soluble organic molecules and an organic backbone structure directs mineral components to specific sites. This strategy underlies the formation of coccoliths, which are highly ordered arrangements of calcite crystals produced by marine microalgae. On combining the insoluble organic coccolith scaffold with coccolith-associated soluble macromolecules in vitro, we found a massive accretion of calcium ions at the sites where the crystals form in vivo. The in vitro process exhibits profound similarities to the initial stages of coccolith biogenesis in vivo.

Mineralized structures formed by organisms are hybrid materials, characterized by the intimate association of organic macromolecules within and/or around the inorganic phase (13). The hierarchical assembly of the organic and inorganic components is accountable for the superior materials properties that biominerals exhibit (4). Soluble organic macromolecules control mineralization by interacting with the developing mineral. Such interactions can affect the morphology of the growing crystal, stabilize a transient amorphous precursor phase, or inhibit precipitation in solution (57). The insoluble organic components of biominerals, usually forming scaffold structures, also have been shown to influence crystal nucleation and growth (810). These observations have led to the general view that the localization of crystallization is determined by direct interactions between nucleating macromolecules and the developing mineral.

One of the prominent examples demonstrating high degree of control over crystallization are the calcitic scales produced by coccolithophores (11, 12). These ubiquitous marine microalgae, which are the main calcifying phytoplankton, produce complex arrays of calcite crystals, termed coccoliths (13). Each coccolith is formed inside a specialized vesicle and upon completion, it is extruded to the cell surface to form an extracellular shell (Fig. 1, A and B) (11). Coccolith biogenesis starts with formation of an organic scale, called the base plate, inside the coccolith vesicle (14, 15). Calcite crystals nucleate on the periphery of the base plate with their crystallographic orientation being precisely controlled (13). Ultrastructural studies on coccoliths have led to the hypothesis that crystal nucleation is mediated by specific chemical moieties at the base-plate periphery (13, 16). The initial simple crystals grow and develop genus-specific, complex morphologies (15, 17). Acidic polysaccharides, which become tightly associated with the mineral phase during its formation, further affect crystal nucleation and growth (7, 1820) (supplementary text).

Fig. 1 Coccolith-associated organic components.

(A) Scanning electron microscopy (SEM) image of a P. carterae coccolith shell. (B and C) SEM images of isolated coccoliths consisting of calcite crystals and a base plate (*). In (C), the coccolith is oriented upside down, displaying its bottom side. The base plate covers only one crystal type [inner circle of crystals indicated with white arrowheads, also known as “V-type” crystals (13, 16)], whereas the second type of crystals are only in contact with the base-plate circumference (outer circle of crystals indicated with black arrowheads, also known as “R-type” crystals). The inset shows an AFM phase-contrast image of a similar sample with the base-plate fibers covering only the “V-type” crystals. (D) AFM phase-contrast image of the fibrous bottom side of a demineralized base plate on polylysine-functionalized mica. (E) AFM height-image showing the topography of the top side of a demineralized base plate. (F) AFM height-image of the top side of a demineralized base plate imaged in solution. Some swelling of the inner area is observed.

We tested the proposed functions of the organic building blocks of coccoliths individually and in a holistic context in vitro. For this, we isolated coccoliths from live Pleurochrysis carterae cells using a mild harvesting procedure, preserving the coccolith-associated organic material as close as possible to its native state. P. carterae coccoliths consist of two types of morphologically distinct calcite crystals that are placed in an alternating order along the base-plate periphery. Previous characterization of the organic constituents of Pleurochrysis coccoliths have shown that the organic base plate is composed of cellulosic fibers and proteins, and that the soluble fraction is dominated by acidic polysaccharides (7, 17, 2125) (supplementary text).

Atomic force microscopy (AFM) of isolated and dried base plates on a negatively charged mica surface showed a radial array of fibers, characteristic for the bottom side of the base plate (Fig. 1, C and D) (15, 17). When the mica surface was functionalized with positively charged polylysine, however, the base plates adsorbed preferentially with their mineral-associated side facing up (Fig. 1E). This observation suggests distinct surface-charge properties of the two sides of the base plate. The most prominent feature of the mineral-associated side (top side) is a thickening at the periphery of the base plate, raising 2 to 3 nm above the ~10-nm-thick inner area. This topological feature, previously recognized as a potential crystal nucleation site (16), was also prominent when the base plates were imaged in solution (Fig. 1F). Amino acid and monosaccharide analyses of isolated base plates showed them to be composed of a glucose-rich polymer, which could be cellulose, additional polysaccharides, and yet unidentified proteins (table S1 and fig. S1), as previously reported (22, 23). The soluble organic fraction of the coccoliths contained the three known acidic polysaccharides (21) as well as unidentified proteins (figs. S1 and S2).

We tested the ability of the isolated base plates to nucleate calcite in vitro using the ammonium carbonate diffusion method (26). A CaCl2 solution, with or without the base plates, was placed in a closed desiccator. CO2 and ammonia diffused into the solution via evaporation of the solid ammonium carbonate salt, thus slowly raising the saturation degree of the solution relative to calcium carbonate. In the absence as well as in the presence of base plates, rhombohedral calcite crystals grew (Fig. 2, A and B). Several experimental modifications, such as varying calcium and/or base-plate concentrations, varying diffusion rates, and adding inorganic additives, yielded similar results (see supplementary text). However, the addition of the coccolith-associated soluble macromolecules changed the mineralization products entirely. When the soluble macromolecules were mixed with the base plates (or if the crude organic extract was used without separating the insoluble and soluble components), no calcite precipitation was observed. Instead, each base plate was decorated by a prominent ring of aggregated particles (Fig. 2, C and D). These ~20-nm particles attach exclusively to the periphery of the base plate. Aggregation of the nanoparticles is restricted to the edge of the top side of the base plate (i.e., the side that nucleates the crystals in vivo), with a layer of particles projecting inward and a second, narrower layer projecting outward (Figs. 2, D to F, and 3A). The aggregated nanoparticles were observed at calcium concentrations >0.1 mM, while at calcium concentrations >10 mM, unspecific precipitation of particles dominated (fig. S3). Aggregate formation was pH dependent, with acidic conditions (pH <4) inhibiting the reaction (fig. S4). We tested the elemental specificity of the aggregation behavior using other cations—Sr, Mg, and Na. Only Sr ions yielded particle aggregates at the base-plate periphery but always accompanied by unspecific precipitation in the surrounding of the base plate. Mg ions showed only unspecific precipitation, and Na ions caused no precipitation (fig. S5).

Fig. 2 Calcium-mediated formation of particle aggregates at the base-plate periphery.

(A) SEM image of a calcite crystal growing in the absence of organic components. (B) SEM image showing the outcome of a mineralization experiment with purified base plates as organic additive. The inset shows base plates lying on top of each other. (C) SEM image of a base plate after a mineralization experiment, including also the soluble coccolith-associated macromolecules. Inset shows lower magnification. (D) AFM height-image of the aggregated particles on the base-plate periphery. Arrowheads indicate the boundary between the narrow outer layer and inner ring. (E) SEM image of two base plates after a mineralization experiment. The base plate in the upper part of the image is facing up, whereas the one in the lower part is facing down. Owing to the upside down orientation, the inner part of the aggregate ring is shaded by the base plate, whereas the uncovered outer ring (arrowheads) produces bright contrast. (F) A scheme showing the specific locations on the base plate where the particle aggregates (red balls) form during a mineralization experiment in the presence of the soluble macromolecules (black knots).

Fig. 3 Compositional analysis of the aggregates on the base-plate periphery.

(A) HAADF-STEM image of base plates recovered from a mineralization experiment. Inset shows a higher-magnification image of the base-plate periphery with two separate rings of particle aggregates. (B) Bright-field TEM image of base plates from the same mineralization experiment, and (C) the corresponding EELS map showing calcium-rich locations with bright pixels. (D) Carbon K-edge EELS spectra of three different locations in the sample. (E) ATR-FTIR spectra of base plates and soluble macromolecule (SM) mixtures before (blue) and after (red) a mineralization experiment. The differences between the spectra are due to residual ammonium vibrations (~1400 cm−1) and possibly altered conformations of the macromolecules. None of the characteristics carbonate peaks, as shown in a spectrum of amorphous calcium carbonate (ACC), are detected.

High-angle annular dark-field scanning–transmission electron microscopy (HAADF-STEM) images showed that the electron-dense particles are assembled into two rings separated by a ~5-nm gap (Fig. 3A). Electron energy-loss spectroscopy (EELS) mapping showed that the aggregate particles are indeed calcium-rich (Fig. 3, B and C). When we analyzed the EELS spectrum of the aggregates at the carbon K-edge, expecting features characteristic for carbonate, the spectrum was identical to that of organic carbon from the base plate and the supporting film (Fig. 3D). We acquired attenuated total reflectance Fourier-transformed infrared (ATR-FTIR) spectra of bulk precipitates from a mineralization experiment and again found no evidence for carbonate (Fig. 3E). The lack of involvement of carbonate ions in the precipitation of the calcium-rich aggregates on the base-plate periphery was corroborated by observing identical aggregates that formed in the absence of ammonium carbonate vapor (fig. S6).

To follow the reaction in situ, we took advantage of carboxylic residues in the soluble macromolecules (21) and conjugated a hydroxylamine-functionalized fluorophore to a fraction of them. The fluorescently labeled macromolecules showed a uniform fluorescence pattern when mixed with base plates or with calcium (Fig. 4, A and B). However, when base plates, fluorescently labeled macromolecules, and calcium were mixed, oval-shaped fluorescent rings with dimensions similar to those of the base plates were observed (time from mixing to image acquisition was 3 min) (Fig. 4C). After 30 min, the background fluorescence from unbound macromolecules diminished whereas the ring fluorescence did not change in intensity, suggesting the aggregation process to be completed within 3 min (Fig. 4, D to F). Colocalization of fluorescence signals from the soluble macromolecules and calcium ions was observed when calcein, a fluorescent chelator of calcium ions, was added to the reaction (fig S7). These observations show that the aggregation reaction is almost instantaneous when calcium is present and that the soluble macromolecules are a substantial component of the aggregates.

Fig. 4 In situ monitoring of the aggregation reaction with fluorescently labeled soluble macromolecules.

(A and D) Fluorescence images of a mixture containing the base plates and the labeled soluble macromolecules (SM), 3 and 30 min after mixing. (B and E) Images of a mixture containing the soluble macromolecules and calcium ions. After 30 min, scattered, weakly labeled aggregates emerge, corresponding to unspecific aggregation. (C and F) Images of a mixture with all three components. The inset shows a higher magnification of a single base plate. Acquisition time was identical for all images.

Our results suggest that templating the nucleation of calcite from a supersaturated solution is not an intrinsic feature of the P. carterae base plate. However, the calcium-mediated recognition between soluble macromolecules and the base-plate periphery is a highly specific process that drives large amounts of complexed calcium ions to the site where crystals ultimately nucleate. Such an additional step to the biomineralization process—i.e., localizing concentrated calcium pools by ion-binding before crystal nucleation—can provide an additional level of control over crystal nucleation. On the base plates, this process generates a geometric segregation of the concentrated calcium pool into two distinct rings. Such partitioning could facilitate the nucleation of two distinct crystal orientations, as occurs in vivo.

The in vitro observations presented here exhibit similarities to coccolith biogenesis as observed inside of cells. First, the base plate and the soluble macromolecules were shown to colocalize inside the coccolith vesicle, where the macromolecules are associated with a concentrated, as yet unidentified, calcium phase in the form of ~20-nm particles (16, 17). Second, these calcium-bearing nanoparticles have been shown to aggregate on the base-plate periphery before the onset of crystallization (16). Third, calcium has a longer residence time in the cell relative to carbon (27), supporting a distinct calcium accumulation process, which is followed by carbonate incorporation, presumably by active transport into the coccolith vesicle.

Dynamic light scattering showed that in the presence of calcium, the soluble macromolecules undergo a concentration-dependent aggregation process (fig. S8). The calcium concentration needed to saturate the aggregation potential of the macromolecules was higher (>1 mM) than the concentration needed to induce the aggregation on the base plate (<1 mM). This suggests that the higher calcium-binding affinity of the base-plate periphery facilitates the dominance of the specific aggregation pattern over nonspecific precipitation. When the polysaccharide alginate, which is rich in carboxylic acid residues, was tested as a substitute for the soluble macromolecules, no particle aggregates formed on the base-plate periphery (fig. S9). In addition, enzymatic degradation of the soluble proteins did not hinder the aggregation reaction (table S2 and fig S2). These observations suggest that neither the presence of carboxylate residues alone nor the presence of the soluble proteins can account for the macromolecular recognition reaction. On the base-plate side, we can rule out simple geometric affinity to the edges of the scale, because the very similar cellular structures called “body scales” (15) do not calcify in vivo and also do not show affinity for the calcium-loaded macromolecules in vitro (fig. S10). Supporting the notion of site-specific chemical interactions, AFM phase-contrast images in solution demonstrated that the surface of the base-plate periphery is chemically distinct from that of the base-plate interior (fig. S11).

The cooperative role of the organic template and soluble macromolecules in directing a calcium reservoir to the site of mineralization represents a sophisticated strategy for biological control over crystallization. It extends the role of biomolecules, which not only establish a controlled chemical environment for mineralization, but are also involved in specifying the localization of the process via specific macromolecular interactions. The paradigm of mineralization control through macromolecular recognition may be of relevance to many other biominerals, where organic templates and soluble macromolecules are well studied but the interactions between them are unclear.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S11

Tables S1 and S2

References (2836)

References and Notes

  1. Acknowledgments: We are grateful to A. Skeffington, N. Kröger, and U. Armbruster for critically reading the manuscript; M. Brzezinka for help with SDS–polyacrylamide gel electrophoresis; and A. Erban for gas chromatography–mass spectrometry analysis. This research was supported by the Max-Planck Society, Deutsche Forschungsgemeinschaft (DFG) grants Sche1637/3-1 to A.S., and FA835/9-1 to D.F. A.G. is supported by an Alexander von Humboldt postdoctoral fellowship. D.F. acknowledges financial support from the European Research Council (Starting Grant MB2 No.256915). P.F. acknowledges financial support from the Gottfried Wilhelm Leibniz Preis of the DFG. All data used to support the conclusions in this manuscript are provided in the supplementary materials.
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