How to build a glass house: Revealing fundamental components of diatom cell wall biomineralization

Biomineralization, the formation of complex inorganic structures by organisms, is a widely distributed process in nature. One of the most spectacular examples of biomineralization is the diatom cell wall (frustule), which is a three-dimensional silica structure with intricate, species-specific patterns ranging from nano- to micrometer scale. While a number of proteins have been identified that take part in the deposition and patterning of silica within a specialized compartment (the silica deposition vesicle, SDV), the process as a whole is poorly understood. There is extensive interaction between the inside of the SDV and cytosolic factors such as the cytoskeleton, but the mechanisms of these interactions are unknown. We have identified a gene family restricted to diatoms that encodes predicted transmembrane proteins with a yet uncharacterised domain. Several lines of evidence suggest that members of this protein family, termed Silicanins, are localized to the SDV membrane, and that they are involved in frustule biosynthesis. In this project, we aim to investigate the roles of the Silicanin-D subfamily in cell wall biomineralization, using state-of-the-art molecular, biochemical and imaging techniques. We have generated a collection of mutants of Silicanin-D subfamily members in two diatom species using CRISPR/Cas9-based genome editing. Silica production and frustule patterning, structure and chemical composition will be analysed in these mutants as well as overexpression lines. We are currently developing a protocol for three-dimensional electron microscopy of diatom frustules, which will facilitate a holistic characterization of mutant cells. The intracellular localization and dynamics of different silicanin subfamilies has been analysed. We are in process of investigating their possible interaction with the cytoskeleton and other proteins. Recombinant Silicanin-D proteins have been produced and will be analysed for direct or indirect effect on silica formation activity. Furthermore, we are analysing two kinases with putative roles related to silicon transport. Mutants for these kinases will be characterised with regard to changes in the proteome, and may provide information on which proteins and processes that are affected or regulated by the kinases. The results from this project will increase knowledge on a fundamental process in the ecologically important diatoms, and could form the basis for a system to customize frustule structures for commercial applications.

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Biomineralization, the biological formation of complex inorganic structures, is a common process in nature. One of the most spectacular examples of biomineralization is the diatom cell wall (frustule), which is a three-dimensional silica structure with intricate, species-specific patterns ranging from nano- to micrometer scale. While a number of proteins have been identified that take part in the deposition and patterning of silica within a specialized compartment (the silica deposition vesicle, SDV), the process as a whole is poorly understood. There is extensive interaction between the SDV lumen and cytosolic factors such as the cytoskeleton, but the mechanisms of these interactions are unknown. We have identified a gene family restricted to diatoms encoding predicted transmembrane proteins with a yet uncharacterised domain. Several lines of evidence suggest that members of this protein family, termed Silica Matrix 7-Like (SMLs), are localized to the SDV membrane, and that they are involved in frustule biosynthesis. In this project, we aim to investigate the roles of the SML-D subfamily in cell wall biomineralization, using state-of-the-art molecular, biochemical and imaging techniques. Using CRISPR/Cas9-based genome editing, we will generate deletion series of the SML-D subfamily members. Silica production and frustule patterning, structure and chemical composition will be analysed in these mutants as well as overexpression lines. The intracellular localization and dynamics of SML-D members and their possible interaction with the cytoskeleton will be studied. Immunoprecipitation will be performed to identify possible interacting proteins. Finally, recombinant SML-D proteins will be analysed for direct or indirect effect on silica formation activity. The results from this project will increase knowledge on a fundamental process in the ecologically important diatoms, and could form the basis for a system to customize frustule structures for commercial applications.

Publications from Cristin

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Functional studies of fundamental processes in diatoms

Diatom responses to nitrogen and phosphorus limitation

How to build a glass house: Revealing fundamental components of diatom cell wall biomineralization

Analysis of short-term transcriptome responses to silicon addition in Thalassiosira pseudonana

Functional studies of a diatom-specific cell wall synthesis protein

Gene editing as a tool for functional studies of the diatom cell wall

How do you build a glass house? Using transcriptomics and gene editing to elucidate silica nanopatterning in diatom cell walls

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Creation of Gene Knockouts for Silicic Acid Transporter-Associated Kinases and Study of Alternative Selection Markers in the Diatom Thalassiosira pseudonana

Functional studies of Group II silicanins in the diatom Thalassiosira pseudonana cell wall biomineralization

Development of molecular tools in the diatom Coscinodiscus wailesii

Functional studies of two silicon transporter-associated kinases in the diatom Thalassiosira pseudonana

Functional studies of two genes encoding closely related group II silicanins in the diatom Thalassiosira pseudonana

Morphological changes in the frustule of Thalassiosira pseudonana by genetic manipulation and cultivation conditions

Functional studies of a family of ankyrin repeat-containing proteins in the diatom Thalassiosira pseudonana hypothesized to be involved in frustule morphogenesis

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