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.     

Extensive and intimate association of the cytoskeleton with forming silica in diatoms: control over patterning on the meso- and micro-scale

BACKGROUND: The diatom cell wall, called the frustule, is predominantly made out of silica, in many cases with highly ordered nano- and micro-scale features. Frustules are built intracellularly inside a special compartment, the silica deposition vesicle, or SDV. Molecules such as proteins (silaffins and silacidins) and long chain polyamines have been isolated from the silica and shown to be involved in the control of the silica polymerization. However, we are still unable to explain or reproduce in vitro the complexity of structures formed by diatoms. METHODS/PRINCIPAL FINDING: In this study, using fluorescence microscopy, scanning electron microscopy, and atomic force microscopy, we were able to compare and correlate microtubules and microfilaments with silica structure formed in diversely structured diatom species. The high degree of correlation between silica structure and actin indicates that actin is a major element in the control of the silica morphogenesis at the meso and microscale. Microtubules appear to be involved in the spatial positioning on the mesoscale and strengthening of the SDV. CONCLUSIONS/SIGNIFICANCE: These results reveal the importance of top down control over positioning of and within the SDV during diatom wall formation and open a new perspective for the study of the mechanism of frustule patterning as well as for the understanding of the control of membrane dynamics by the cytoskeleton.     

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.     

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.     

Prescribing diatom morphology: toward genetic engineering of biological nanomaterials

The formation of inorganic materials with complex form is a widespread biological phenomenon (biomineralization). Among the most spectacular examples of biomineralization is the production by diatoms (a group of eukaryotic microalgae) of intricately nanopatterned to micropatterned cell walls made of silica (SiO2). Understanding the molecular mechanisms of diatom silica biomineralization is not only a fundamental biological problem, but also of great interest in materials engineering, as the biological self-assembly of three-dimensional (3D) inorganic nanomaterials has no man-made analog. Recently, insight into the molecular mechanism of diatom silica formation has been obtained by structural and functional analysis of biomolecules that are involved in this process. Furthermore, the rapid development of diatom molecular genetics has provided new tools for investigating the silica forming machinery of diatoms and for manipulating silica biogenesis. This has opened the door for the production, through genetic engineering, of unique 3D nanomaterials with designed structures and functionalities.     

Silica morphogenesis by alternative processing of silaffins in the diatom Thalassiosira pseudonana

For almost 200 years scientists have been fascinated by the ornate cell walls of the diatoms. These structures are made of amorphous silica, exhibiting species-specific, mostly porous patterns in the nano- to micrometer range. Recently, from the diatom Cylindrotheca fusiformis unusual phosphoproteins (termed silaffins) and long chain polyamines have been identified and implicated in biosilica formation. However, analysis of the role of silaffins in morphogenesis of species-specific silica structures has so far been hampered by the difficulty of obtaining structural data from these extremely complex proteins. In the present study, the five major silaffins from the diatom Thalassiosira pseudonana (tpSil1H, -1L, -2H, -2L, and -3) have been isolated, functionally analyzed, and structurally characterized, mak- ing use of the recently available genome data from this organism. Surprisingly, the silaffins of T. pseudonana and C. fusiformis share no sequence homology but are similar regarding amino acid composition and post-translational modifications. Silaffins tpSil1H and -2H are higher molecular mass isoforms of tpSil1L and -2L, respectively, generated in vivo by alternative processing of the same precursor polypeptides. Interestingly, only tpSil1H and -2H but not tpSil1L and -2L induce the formation of porous silica patterns in vitro, suggesting that the alternative processing event is an important step in morphogenesis of T. pseudonana biosilica.     

Proper Actin Ring Formation and Septum Constriction Requires Coordinated Regulation of SIN and MOR Pathways through the Germinal Centre Kinase MST-1

Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Δmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.     

Identification of proteins from a cell wall fraction of the diatom Thalassiosira pseudonana: insights into silica structure formation

Diatoms are unicellular eucaryotic algae with cell walls containing silica, intricately and ornately structured on the nanometer scale. Overall silica structure is formed by expansion and molding of the membrane-bound silica deposition vesicle. Although molecular details of silica polymerization are being clarified, we have limited insight into molecular components of the silica deposition vesicle, particularly of membrane-associated proteins that may be involved in structure formation. To identify such proteins, we refined existing procedures to isolate an enriched cell wall fraction from the diatom Thalassiosira pseudonana, the first diatom with a sequenced genome. We applied tandem mass spectrometric analysis to this fraction, identifying 31 proteins for further evaluation. mRNA levels for genes encoding these proteins were monitored during synchronized progression through the cell cycle and compared with two previously identified silaffin genes (involved in silica polymerization) having distinct mRNA patterns that served as markers for cell wall formation. Of the 31 proteins identified, 10 had mRNA patterns that correlated with the silaffins, 13 had patterns that did not, and seven had patterns that correlated but also showed additional features. The possible involvements of these proteins in cell wall synthesis are discussed. In particular, glutamate acetyltransferase was identified, prompting an analysis of mRNA patterns for other genes in the polyamine biosynthesis pathway and identification of those induced during cell wall synthesis. Application of a specific enzymatic inhibitor for ornithine decarboxylase resulted in dramatic alteration of silica structure, confirming the involvement of polyamines and demonstrating that manipulation of proteins involved in cell wall synthesis can alter structure. To our knowledge, this is the first proteomic analysis of a diatom, and furthermore we identified new candidate genes involved in structure formation and directly demonstrated the involvement of one enzyme (and its gene) in the structure formation process.     

Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca²⁺-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg²⁺-dependent, Ca²⁺-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.     

A water-soluble precursor for efficient silica polymerization by silicateins

Silicateins, the spicule-forming proteins from marine demosponges capable to polymerize silica, are popular objects of biomineralization studies due to their ability to form particles varied in shape and composition under physiological conditions. Despite the occurrence of the many approaches to nanomaterial synthesis using silicateins, biochemical properties of this protein family are poorly characterized. The main reason for this is that tetraethyl orthosilicate (TEOS), the commonly used silica acid precursor, is almost insoluble in water and thus is poorly available for the protein. To solve this problem, we synthesized new water-soluble silica precursor, tetra(glycerol)orthosilicate (TGS), and characterized biochemical properties of the silicatein A1 from marine sponge Latrunculia oparinae. Compared to TEOS, TGS ensured much greater activity of silicatein and was less toxic for the mammalian cell culture. We evaluated optimum conditions for the enzyme - pH range, temperature and TGS concentration. We concluded that TGS is a useful silica acid precursor that can be used for silica particles synthesis and in vivo applications.     

A human kinase yeast array for the identification of kinases modulating phosphorylation‐dependent protein–protein interactions

Protein kinases play an important role in cellular signaling pathways and their dysregulation leads to multiple diseases, making kinases prime drug targets. While more than 500 human protein kinases are known to collectively mediate phosphorylation of over 290,000 S/T/Y sites, the activities have been characterized only for a minor, intensively studied subset. To systematically address this discrepancy, we developed a human kinase array in Saccharomyces cerevisiae as a simple readout tool to systematically assess kinase activities. For this array, we expressed 266 human kinases in four different S. cerevisiae strains and profiled ectopic growth as a proxy for kinase activity across 33 conditions. More than half of the kinases showed an activity‐dependent phenotype across many conditions and in more than one strain. We then employed the kinase array to identify the kinase(s) that can modulate protein–protein interactions (PPIs). Two characterized, phosphorylation‐dependent PPIs with unknown kinase–substrate relationships were analyzed in a phospho‐yeast two‐hybrid assay. CK2α1 and SGK2 kinases can abrogate the interaction between the spliceosomal proteins AAR2 and PRPF8, and NEK6 kinase was found to mediate the estrogen receptor (ERα) interaction with 14‐3‐3 proteins. The human kinase yeast array can thus be used for a variety of kinase activity‐dependent readouts.     

Molecular Evolution Allows Bypass of the Requirement for Activation Loop Phosphorylation of the Cdc28 Cyclin-Dependent Kinase

Many protein kinases are regulated by phosphorylation in the activation loop, which is required for enzymatic activity. Glutamic acid can substitute for phosphothreonine in some proteins activated by phosphorylation, but this substitution (T169E) at the site of activation loop phosphorylation in the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28p blocks biological function and protein kinase activity. Using cycles of error-prone DNA amplification followed by selection for successively higher levels of function, we identified mutant versions of Cdc28p-T169E with high biological activity. The enzymatic and biological activity of the mutant Cdc28p was essentially normally regulated by cyclin, and the mutants supported normal cell cycle progression and regulation. Therefore, it is not a requirement for control of the yeast cell cycle that Cdc28p be cyclically phosphorylated and dephosphorylated. These CDC28 mutants allow viability in the absence of Cak1p, the essential kinase that phosphorylates Cdc28p-T169, demonstrating that T169 phosphorylation is the only essential function of Cak1p. Some growth defects remain in suppressed cak1 cdc28 strains carrying the mutant CDC28 genes, consistent with additional nonessential roles for CAK1.     

Regulation of neuronal plasticity in the central nervous system by phosphorylation and dephosphorylation

Neuronal plasticity can be defined as adaptive changes in structure and function of the nervous system, an obvious example of which is the capacity to remember and learn. Long-term potentiation and long-term depression are the experimental models of memory in the central nervous system (CNS), and have been frequently utilized for the analysis of the molecular mechanisms of memory formation. Extensive studies have demonstrated that various kinases and phosphatases regulate neuronal plasticity by phosphorylating and dephosphorylating proteins essential to the basic processes of adaptive changes in the CNS. These proteins include receptors, ion channels, synaptic vesicle proteins, and nuclear proteins. Multifunctional kinases (cAMP-dependent protein kinase, Ca²⁺/phospholipid-dependent protein kinase, and Ca²⁺/calmodulin-dependent protein kinases) and phosphatases (calcineurin, protein phosphatases 1, and 2A) that specifically modulate the phosphorylation status of neuronal-signaling proteins have been shown to be required for neuronal plasticity. In general, kinases are involved in upregulation of the activity of target substrates, and phosphatases downregulate them. Although this rule is applicable in most of the cases studied, there are also a number of exceptions. A variety of regulation mechanisms via phosphorylation and dephosphorylation mediated by multiple kinases and phosphatases are discussed.     

Protein kinases and phosphatases involved in the acclimation of the photosynthetic apparatus to a changing light environment

Photosynthetic organisms are subjected to frequent changes in light quality and quantity and need to respond accordingly. These acclimatory processes are mediated to a large extent through thylakoid protein phosphorylation. Recently, two major thylakoid protein kinases have been identified and characterized. The Stt7/STN7 kinase is mainly involved in the phosphorylation of the LHCII antenna proteins and is required for state transitions. It is firmly associated with the cytochrome b₆f complex, and its activity is regulated by the redox state of the plastoquinone pool. The other kinase, Stl1/STN8, is responsible for the phosphorylation of the PSII core proteins. Using a reverse genetics approach, we have recently identified the chloroplast PPH1/TAP38 and PBPC protein phosphatases, which counteract the activity of STN7 and STN8 kinases, respectively. They belong to the PP2C-type phosphatase family and are conserved in land plants and algae. The picture that emerges from these studies is that of a complex regulatory network of chloroplast protein kinases and phosphatases that is involved in light acclimation, in maintenance of the plastoquinone redox poise under fluctuating light and in the adjustment to metabolic needs.     

Genome-Wide Analysis and Experimentation of Plant Serine/ Threonine/Tyrosine-Specific Protein Kinases

Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.     

Fyn kinase regulates translation in mammalian mitochondria

BackgroundMitochondrial translation machinery solely exists for the synthesis of 13 mitochondrially-encoded subunits of the oxidative phosphorylation (OXPHOS) complexes in mammals. Therefore, it plays a critical role in mitochondrial energy production. However, regulation of the mitochondrial translation machinery is still poorly understood. In comprehensive proteomics studies with normal and diseased tissues and cell lines, we and others have found the majority of mitochondrial ribosomal proteins (MRPs) to be phosphorylated. Neither the kinases for these phosphorylation events nor their specific roles in mitochondrial translation are known.MethodsMitochondrial kinases are responsible for phosphorylation of MRPs enriched from bovine mitoplasts by strong cation-exchange chromatography and identified by mass spectrometry-based proteomics analyses of kinase rich fractions. Phosphorylation of recombinant MRPs and 55S ribosomes was assessed by in vitro phosphorylation assays using the kinase-rich fractions. The effect of identified kinase on OXPHOS and mitochondrial translation was assessed by various cell biological and immunoblotting approaches.ResultsHere, we provide the first evidence for the association of Fyn kinase, a Src family kinase, with mitochondrial translation components and its involvement in phosphorylation of 55S ribosomal proteins in vitro. Modulation of Fyn expression in human cell lines has provided a link between mitochondrial translation and energy metabolism, which was evident by the changes in 13 mitochondrially encoded subunits of OXPHOS complexes.Conclusions and general significanceOur findings suggest that Fyn kinase is part of a complex mechanism that regulates protein synthesis and OXPHOS possibly by tyrosine phosphorylation of translation components in mammalian mitochondria.