Characterization of an Endoplasmic Reticulum-associated Silaffin Kinase from the Diatom Thalassiosira pseudonana

The formation of SiO₂-based cell walls by diatoms (a large group of unicellular microalgae) is a well established model system for the study of molecular mechanisms of biological mineral morphogenesis (biomineralization). Diatom biomineralization involves highly phosphorylated proteins (silaffins and silacidins), analogous to other biomineralization systems, which also depend on diverse sets of phosphoproteins (e.g. mammalian teeth and bone, mollusk shells, and sponge silica). The phosphate moieties on biomineralization proteins play an essential role in mineral formation, yet the kinases catalyzing the phosphorylation of these proteins have remained poorly characterized. Recent functional genomics studies on the diatom Thalassiosira pseudonana have revealed >100 proteins potentially involved in diatom silica formation. Here we have characterized the biochemical properties and biological function of one of these proteins, tpSTK1. Multiple tpSTK1-like proteins are encoded in diatom genomes, all of which exhibit low but significant sequence similarity to kinases from other organisms. We show that tpSTK1 has serine/threonine kinase activity capable of phosphorylating silaffins but not silacidins. Cell biological and biochemical analysis demonstrated that tpSTK1 is an abundant component of the lumen of the endoplasmic reticulum. The present study provides the first molecular structure of a kinase that appears to catalyze phosphorylation of biomineral forming proteins in vivo.     

Pentalysine Clusters Mediate Silica Targeting of Silaffins in Thalassiosira pseudonana

The biological formation of inorganic materials (biomineralization) often occurs in specialized intracellular vesicles. Prominent examples are diatoms, a group of single-celled eukaryotic microalgae that produce their SiO₂ (silica)-based cell walls within intracellular silica deposition vesicles (SDVs). SDVs contain protein-based organic matrices that control silica formation, resulting in species specifically nanopatterned biosilica, an organic-inorganic composite material. So far no information is available regarding the molecular mechanisms of SDV biogenesis. Here we have investigated by fluorescence microscopy and subcellular membrane fractionation the intracellular transport of silaffin Sil3. Silaffins are a group of phosphoproteins constituting the main components of the organic matrix of diatom biosilica. We demonstrate that the N-terminal signal peptide of Sil3 mediates import into a specific subregion of the endoplasmic reticulum. Additional segments from the mature part of Sil3 are required to reach post-endoplasmic reticulum compartments. Further transport of Sil3 and incorporation into the biosilica (silica targeting) require protein segments that contain a high density of modified lysine residues and phosphoserines. Silica targeting of Sil3 is not dependent on a particular peptide sequence, yet a lysine-rich 12–14-amino acid peptide motif (pentalysine cluster), which is conserved in all silaffins, strongly promotes silica targeting. The results of the present work provide the first insight into the molecular mechanisms for biogenesis of mineral-forming vesicles from an eukaryotic organism.     

Biomineralization in diatoms: characterization of novel polyamines associated with silica

Pattern formation during silica biomineralization in diatoms appears to depend on long-chain polyamines as well as proteins covalently modified with polyamines (silaffins). Recently, the complete genome of the diatom Thalassiosira pseudonana has been sequenced making this species an attractive model organism for future studies on biomineralization. Mass- and NMR-spectroscopic analysis of the long-chain polyamines from this diatom species reveals the existence of a complex population with as yet unknown structural features. These include complex methylation patterns, different attachment moieties as well as the existence of quaternary ammonium functionalities.     

Biomolecular Self-assembly and its Relevance in Silica Biomineralization

Biomineralization, which means the formation of inorganic materials by biological processes, currently finds increasing research interest. It involves the synthesis of calcium-based minerals such as bones and teeth in vertebrates, and of shells. Silica biomineralization occurs, for example, in diatoms and silica sponges. Usually, biominerals are made up of amorphous compounds or small microcrystalline domains embedded into an amorphous matrix. Nevertheless, they exhibit very regular shapes and, as in the case of diatoms, intricate nanopatterns of amazing beauty. It is, therefore, commonly assumed that biominerals are formed under the structure-directing influence of templates. However, single molecules are by far too small to direct the formation of the observed shapes and patterns. Instead, supramolecular aggregates are shown to be involved in the formation of templating superstructures relevant in biomineralization. Specific biomolecules were identified in both diatoms and silica sponges, which elegantly combine two indispensable functions: on the one hand, the molecules are capable of inducing silica precipitation from precursor compounds. On the other hand, these molecules are capable of self-assembling into larger, structure-directing template aggregates. Such molecules are the silaffins in the case of diatoms and the silicateins in sponges. Long-chain polyamines of similar composition have meanwhile been discovered in both organisms. The present review is especially devoted to the discussion of the self-assembly behavior of these molecules. Physico-chemical studies on a model compound, poly(allylamine), are discussed in detail in order to elucidate the nature of the interactions responsible for self-assembly of long-chain polyamines and the parameters controlling this process. Numerous biomimetic silica synthesis experiments are discussed and evaluated with respect to the observations made on the aforementioned "natural" biomolecules.     

BIOCHEMISTRY OF SILICA BIOMINERALIZATION IN DIATOMS

Diatoms are well known for the intricate patterns of their silica-based cell walls. The complex structures of diatom cell walls are species specific and become precisely reproduced during each cell division cycle, indicating a genetic control of silica biomineralization. Therefore, the formation of the diatom cell wall has been regarded as a paradigm for controlled production of nanostructured silica. However, the mechanisms allowing biosilicification to proceed at ambient temperature at high rates have remained enigmatic. Recently, we have shown that a set of highly cationic peptides (called silaffins) isolated from Cylindrotheca fusiformis shells are able to generate networks of silica nanospheres within seconds when added to a solution of silicic acid. Different silaffin species produce different morphologies of the precipitated silica. Silaffins contain covalently modified Lys-Lys elements. One of these lysine residues bears a novel type of protein modification, a polyamine consisting of 6–11 repeats of the N-methyl-propylamine unit. In addition to the silaffins, additional polyamine-containing substances have been isolated from a number of diatom species that may be involved in the control of biosilica morphology. Scanning electron microscopic analysis of diatom shells isolated in statu nascendi provide insights into the processes of pattern formation in biosilica. A model will be discussed that explains production of nanostructured biosilica in diatoms on the basis of these experimental results.     

Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis.     

Bioinspired synthesis of multifunctional inorganic and bio-organic hybrid materials

Owing to their physical and chemical properties, inorganic functional materials have tremendous impacts on key technologies such as energy generation and storage, information, medicine, and automotive engineering. Nature, on the other hand, provides evolution-optimized processes, which lead to multifunctional inorganic-bio-organic materials with complex structures. Their formation occurs under physiological conditions, and is goverened by a combination of highly regulated biological processes and intrinsic chemical properties. Nevertheless, insights into the molecular mechanisms of biomineralization open up promising perspectives for bioinspired and biomimetic design and the development of inorganic-bio-organic multifunctional hybrids. Therefore, biomimetic approaches may disclose new synthetic routes under ambient conditions by integrating the concept of gene-regulated biomineralization principles. The skeletal structures of marine sponges provide an interesting example of biosilicification via enzymatically controlled and gene-regulated silica metabolism. Spicule formation is initiated intracellularly by a fine-tuned genetic mechanism, which involves silica deposition in vesicles (silicassomes) under the control of the enzyme silicatein, which has both catalytic and templating functions. In this review, we place an emphasis on the fabrication of biologically inspired materials with silicatein as a biocatalyst.     

Diatom silicon biomineralization as an inspirational source of new approaches to silica production

The demand for new materials and products is still growing and the interest in naturally formed biopolymers and biominerals, such as chitin, calcium precipitates and silica is increasing. Photosynthesizing microalgae of the family Bacillariophyceae (diatoms) produce silica exoskeletons with a potential to be used in specific industrial or technological processes, they also are an excellent model in studies of silicon biomineralization. In contrast to geologically aged diatomaceous earth, the freshly prepared silica of cultured or harvested natural diatoms has been characterized insufficiently with respect to the properties (e.g. purity, specific surface area, porosity) required for technological and industrial application. In this contribution we summarize aspects of cellular processes that are involved in silicon biomineralization of diatoms and the current knowledge of the characterization of diatomaceous silica, following methods used for synthetically derived silica-based materials.     

The fascinating diatom frustule—can it play a role for attenuation of UV radiation?

Diatoms are ubiquitous organisms in aquatic environments and are estimated to be responsible for 20–25 % of the total global primary production. A unique feature of diatoms is the silica wall, called the frustule. The frustule is characterized by species-specific intricate nanopatterning in the same size range as wavelengths of visible and ultraviolet (UV) light. This has prompted research into the possible role of the frustule in mediating light for the diatoms’ photosynthesis as well as into possible photonic applications of the diatom frustule. One of the possible biological roles, as well as area of potential application, is UV protection. In this review, we explore the possible adaptive value of the silica frustule with focus on research on the effect of UV radiation on diatoms. We also explore the possible effect of the frustules on UV radiation, from a theoretical, biological, and applied perspective, including recent experimental data on UV transmission of diatom frustules.     

Biogeochemical silica mass balances in Lake Michigan and Lake Superior

Silica budgets for Lake Michigan and Lake Superior differ in several respects. Mass balance calculations for both lakes agree with previous studies in that permanent burial of biogenic silica in sediments may be only about 5% of the biogenic silica produced by diatoms. Because dissolution rates are large, good estimates of permanent burial of diatoms can not be obtained indirectly from the internal cycle of silica (silica uptake by diatoms and subsequent dissolution) but must be obtained from the sediment stratigraphy. The annual net production of biogenic silica in Lake Michigan requires 71% of the winter maximum silica reservoir which must be maintained primarily by internal cycling in this large lake whereas the comparable silica demand in Lake Superior is only 8.3%. The greater silica demand in Lake Michigan is the result of phosphorus enrichment which has increased diatom production. It is hypothesized that steady-state silica dynamics in Lake Michigan were disrupted by increased diatom production between 1955 and 1970 and that a new steady state based on silica-limited diatom production developed after 1970. Mass balance calculations for Lake Michigan show in contrast with previous work that the hypothesized water column silica depletion of 3.0 g · m^–3 could have occurred even though 90% or more of the biogenic silica production is recycled.     

Diversity of mineral cell coverings and their formation processes: a review focused on the siliceous cell coverings

Mineral cell coverings are found in various protists. Some macroalgae accumulate calcium carbonate in the intercellular space, and some unicellular organisms use calcium carbonate or silica for the construction of loricas, scales, and frustules. Diatoms are representatives of those utilizing silica for the material of the cell covering called a frustule. The development of the frustule is initiated in a silica-deposition vesicle (SDV), which occurs just beneath the plasma membrane and, subsequently, the silicified cell covering expands its area, following the expansion of the SDV from valve face to valve mantle. Sequential valve development with whole valves is reviewed in several diatoms placed in different phylogenetic positions. Every diatom commences its valve formation from its pattern center and then develops by means of individual procedures. The results indicate that the valve development reflects the phylogeny of diatoms. In addition, recent progress in silica biomineralization is briefly reviewed, and the phylogeny of ability concerning siliceous cell covering formation is inferred. Electronic Publication     

SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH

Diatoms are the world's largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting nutrient for diatom growth and hence is a controlling factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we are not fully knowledgeable about intracellular factors that may affect diatom productivity in the oceans. The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research. Numerous studies have characterized parameters of silicic acid uptake by diatoms, and molecular characterization of transport has begun with the isolation of genes encoding the transporter proteins. Multiple types of silicic acid transporter gene have been identified in a single diatom species, and multiple types appear to be present in all diatom species. The controlled expression and perhaps localization of the transporters in the cell may be factors in the overall regulation of silicic acid uptake. Transport can also be regulated by the rate of silica incorporation into the cell wall, suggesting that an intracellular sensing and control mechanism couples transport with incorporation. Sizable intracellular pools of soluble silicon have been identified in diatoms, at levels well above saturation for silica solubility, yet the mechanism for maintenance of supersaturated levels has not been determined. The mechanism of intracellular transport of silicon is also unknown, but this must be an important part of the silicification process because of the close coupling between silica incorporation and uptake. Although detailed ultrastructural analyses of silica deposition have been reported, we know little about the molecular details of this process. However, proteins occluded within silica that promote silicification in vitro have recently been characterized, and the application of molecular techniques holds the promise of great advances in this area. Cellular energy for silicification and transport comes from aerobic respiration without any direct involvement of photosynthetic energy. As such, diatom silicon metabolism differs from that of other major limiting nutrients such as nitrogen and phosphorous, which are closely linked to photosynthetic metabolism. Cell wall silicification and silicic acid transport are tightly coupled to the cell cycle, which results in a dependency in the extent of silicification on growth rate. Silica dissolution is an important part of diatom cellular silicon metabolism, because dissolution must be prevented in the living cell, and because much of the raw material for mineralization in natural assemblages is supplied by dissolution of dead cells. Perhaps part of the reason for the ecological success of diatoms is due to their use of a silicified cell wall, which has been calculated to impart a substantial energy savings to organisms that have them. However, the growth of diatoms and other siliceous organisms has depleted the oceans of silicon, such that silicon availability is now a major factor in the control of primary productivity. Much new progress in understanding silicon metabolism in diatoms is expected because of the application of molecular approaches and sophisticated analytical techniques. Such insight is likely to lead to a greater understanding of the role of silicon in controlling diatom growth, and hence primary productivity, and of the mechanisms involved in the formation of the intricate silicified structures of the diatom cell wall.     

Live diatom silica immobilization of multimeric and redox-active enzymes

Living organisms are adept in forming inorganic materials (biominerals) with unique structures and properties that exceed the capabilities of engineered materials. Biomimetic materials syntheses are being developed that aim at replicating the advantageous properties of biominerals in vitro and endow them with additional functionalities. Recently, proof-of-concept was provided for an alternative approach that allows for the production of biomineral-based functional materials in vivo. In this approach, the cellular machinery for the biosynthesis of nano-/micropatterned SiO₂ (silica) structures in diatoms was genetically engineered to incorporate a monomeric, cofactor-independent (“simple”) enzyme, HabB, into diatom silica. In the present work, it is demonstrated that this approach is also applicable for enzymes with “complex” activity requirements, including oligomerization, metal ions, organic redox cofactors, and posttranslational modifications. Functional expression of the enzymes β-glucuronidase, glucose oxidase, galactose oxidase, and horseradish peroxidase in the diatom Thalassiosira pseudonana was accomplished, and 66 to 78% of the expressed enzymes were stably incorporated into the biosilica. The in vivo incorporated enzymes represent approximately 0.1% (wt/wt) of the diatom biosilica and are stabilized against denaturation and proteolytic degradation. Furthermore, it is demonstrated that the gene construct for in vivo immobilization of glucose oxidase can be utilized as the first negative selection marker for diatom genetic engineering.     

A new calcium binding glycoprotein family constitutes a major diatom cell wall component.

Diatoms possess silica-based cell walls with species-specific structures and ornamentations. Silica deposition in diatoms offers a model to study the processes involved in biomineralization. A new wall is produced in a specialized vesicle (silica deposition vesicle, SDV) and secreted. Thus proteins involved in wall biogenesis may remain associated with the mature cell wall. Here it is demonstrated that EDTA treatment removes most of the proteins present in mature cell walls of the marine diatom Cylindrotheca fusiformis. A main fraction consists of four related glycoproteins with a molecular mass of approximately 75 kDa. These glycoproteins were purified to homogeneity. They consist of repeats of Ca2+ binding domains separated by polypeptide stretches containing hydroxyproline. The proteins in the EDTA extract aggregate and precipitate in the presence of Ca2+. Immunological studies detected related proteins in the cell wall of the freshwater diatom Navicula pelliculosa, indicating that these proteins represent a new family of proteins that are involved in the biogenesis of diatom cell walls.     

Possible role of ubiquitin in silica biomineralization in diatoms: identification of a homologue with high silica affinity

In diatom silicon biomineralization peptides are believed to play a role in silica precipitation and the consequent structure direction of the cell wall. Characterization of such peptides should reveal the nature of this organic-inorganic interaction, knowledge that may eventually well be used to expand the existing range of artificial silicas ("biomimicking"). Biochemical studies on Navicula pelliculosa revealed a set of proteins, which have a high affinity for a solid silica matrix; some were only eluted from the matrix when SDS-denaturation was applied. One of the proteins with an affinity for silica, about 8.5 kDa, is shown to be a homologue of ubiquitin on the basis of its N-terminal amino acid sequence; ubiquitin itself is a highly conserved 8.6 kDa protein that is involved in protein degradation. This finding is in line with a model of silica biomineralization in diatoms that implies the removal of templating polypeptides when pores in the growing cell wall develop. Western blotting with specific anti-ubiquitin antibodies confirmed cross-reactivity. Immunocytochemical localization of ubiquitin indicates that it is present along the diatom cell wall and inside pores during different stages of valve formation.     

Prospects in diatom research

Diatoms are unicellular photosynthetic eukaryotes that play a major role in the global cycling of carbon and silicon. They are believed to have arisen from a secondary endosymbiotic event between two eukaryotes, a red alga and a flagellated heterotroph. Recent analysis of a diatom genome indeed reveals a 'mosaic' nature, with genes derived from plant, animal and bacterial lineages. Advances in molecular genomics are facilitating the use of diatom-specific genes or pathways for biotechnology. Another interest is in understanding the artistry of the amorphous silica shell and the underlying biomineralization process. Materials scientists and chemists are now exploiting diatoms to develop new biomimetic approaches and to create silicon-based microdevices with specific features.     

3D imaging of diatoms with ion-abrasion scanning electron microscopy

Ion-abrasion scanning electron microscopy (IASEM) takes advantage of focused ion beams to abrade thin sections from the surface of bulk specimens, coupled with SEM to image the surface of each section, enabling 3D reconstructions of subcellular architecture at 30 nm resolution. Here, we report the first application of IASEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms have highly patterned silica-based cell wall structures that are unique models for the study and application of directed nanomaterials synthesis by biological systems. Our study provides new insights into the architecture and assembly principles of both the “hard” (siliceous) and “soft” (organic) components of the cell. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we show that both micro- and nanoscale siliceous structures can be visualized at specific stages in their formation. We show that not only are structures visualized in a whole-cell context, but demonstrate that fragile, early-stage structures are visible, and that this can be combined with elemental mapping in the exposed slice. We demonstrate that the 3D architectures of silica structures, and the cellular components that mediate their creation and positioning can be visualized simultaneously, providing new opportunities to study and manipulate mineral nanostructures in a genetically tractable system.     

Expression, purification, and reconstitution of a diatom silicon transporter

The synthesis and manipulation of silicon materials on the nanoscale are core themes in nanotechnology research. Inspiration is increasingly being taken from the natural world because the biological mineralization of silicon results in precisely controlled, complex silica structures with dimensions from the millimeter to the nanometer. One fascinating example of silicon biomineralization occurs in the diatoms, unicellular algae that sheath themselves in an ornate silica-based cell wall. To harvest silicon from the environment, diatoms have developed a unique family of integral membrane proteins that bind to a soluble form of silica, silicic acid, and transport it across the cell membrane to the cell interior. These are the first proteins shown to directly interact with silicon, but the current understanding of these specific silicon transport proteins is limited by the lack of in vitro studies of structure and function. We report here the recombinant expression, purification, and reconstitution of a silicon transporter from the model diatom Thalassiosira pseudonana. After using GFP fusions to optimize expression and purification protocols, a His(10)-tagged construct was expressed in Saccharomyces cerevisiae, solubilized in the detergent Fos-choline-12, and purified by affinity chromatography. Size-exclusion chromatography and particle sizing by dynamic light scattering showed that the protein was purified as a homotetramer, although nonspecific oligomerization occurred at high protein concentrations. Circular dichroism measurements confirmed sequence-based predictions that silicon transporters are α-helical membrane proteins. Silicic acid transport could be established in reconstituted proteoliposomes, and silicon uptake was found to be dependent upon an applied sodium gradient. Transport data across different substrate concentrations were best fit to the sigmoidal Hill equation, with a K(0.5) of 19.4 ± 1.3 μM and a cooperativity coefficient of 1.6. Sodium binding was noncooperative with a K(m)(app) of 1.7 ± 1.0 mM, suggesting a transport silicic acid:Na(+) stoichiometry of 2:1. These results provide the basis for a full understanding of both silicon transport in the diatom and protein-silicon interactions in general.     

New tools for labeling silica in living diatoms

Silicon biomineralization is a widespread mechanism found in several kingdoms that concerns both unicellular and multicellular organisms. As a result of genomic and molecular tools, diatoms have emerged as a good model for biomineralization studies and have provided most of the current knowledge on this process. However, the number of techniques available to study its dynamics at the cellular level is still rather limited. Here, new probes were developed specifically to label the pre-existing or the newly synthesized silica frustule of several diatoms species. It is shown that the LysoTracker Yellow HCK-123, which can be used to visualize silica frustules with common filter sets, presents an enhanced signal-to-noise ratio and allows details of the frustules to be imaged without of the use of ionophores. It is also demonstrated that methoxysilane derivatives can be coupled to fluorescein-5-isothiocyanate (FITC) to preferentially label the silica components of living cells. The coupling of labeling procedures might help to address the challenging question of the process of frustule exocytosis.