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, the aim was to investigate the roles of a Silicanin subfamily in cell wall biomineralization, using state-of-the-art molecular, biochemical and imaging techniques. In order to perform functional studies on selected genes, establisment of an efficient method for genetic transformation is necessary. Since we were not satisfied with the current protocols for genetic transformation of the diatom T. pseudonana, we established a protocol for electroporation, where genetic material is introduced into the cell by the use of an electric pulse. We also improved the ekspressjon of the selection marker by optmising the promoter. We generated a number of mutants of Silicanin members in T. pseudonana using CRISPR/Cas9-based genome editing. Analyses of silica production and frustule patterning, structure and chemical composition in these mutants was initiated. We developed 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 was analysed. We also prepared investigation of the direct and indirect interaction partners of a silicanin using so-called proximity proteomics. Furthermore, we analysed two kinases with putative roles related to silicon transport. Characterisation of mutants for these kinases with regard to changes in the proteome was initiated, 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.

I alt har åtte masterstudenter fullført sin masteroppgaver i tilknytning til prosjektet. PhD-studenten som har vært ansatt på prosjektet (Annika Messemer) planlegger å disputere i løpet av 2024. I tillegg har en PhD-student ansatt gjennom NTNU-midler (Marthe Hafskjold) arbeidet nært opp mot prosjektet, og har dratt fordeler av samarbeid. Hun forventes også å levere avhandlingen i løpet av 2024. I sammenheng med disse to PhD-avhandlingene forventer vi fire til seks publikasjoner. Manuskript vil trolig sendes inn for publikasjon i 2024 og 2025. Et bokkapittel vil bli publisert i mars 2024. Gjennom en av masteroppgavene tilknyttet prosjektet gjorde vi noen pilotforsøk med en annen kiselalge, Coscinodiscus wailesii. Dette arbeidet, sammen andre resultater direkte eller indirekte knyttet til prosjektet, førte til et samarbeid om prosjektsøknader med Martin Lopez-Garcia ved International Iberian Nanotechnology Laboratory i Portugal. I 2023 fikk vi innvilget midler fra Konvergerende Teknologier-programmet ved NFR for et fireårig prosjekt som skal studere muligheten for å manipulere kiselalger genetisk for å celleveggens egenskaper som en fotonisk krystall.

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.