USING 3D TEM TOMOGRAPHY TO ELUCIDATE DIATOM MINERALIZATION

Diatoms are unicellular algae, abundant across marine ecological niches. The hallmark of diatoms is their ability to form intricately shaped cell walls made of silica. The formation of the silica elements is linked to the cell cycle and occurs intracellularly in specialized compartments called silica deposition vesicles (SDVs). When a mature cell divides, each daughter cell produces a new cell wall element in order to reconstruct a full coverage. Upon completion of the silicified element inside the SDV, the entire bio-silica structure is exocytosed to the outer surface of the cell membrane, forming the cell wall. Despite many years of research, the structural and chemical properties of the microenvironment inside the SDV, which controls the morphology and chemistry of the biosilica, are unclear. This is mostly due to the limitations of conventional TEM techniques in elucidating the structural motifs of the SDV. Here, we use state-of-the-art sample preparation procedures to preserve the cellular ultrastructure. Diatom cultures were synchronized to enrich cells actively producing SDVs. The cells were then cryo-immobilized by High-Pressure-Freezing, followed by freeze substitution and embedding in a resin. Since the width of the SDV is only tens of nanometers, we use electron tomography for high-resolution 3D reconstruction of the nanoscale mineralization process. The tomograms show, that in contradiction to the prevailing dogma, the SDV is completely filled with the inorganic phase at all developmental stages, gradually increasing in size over time. These in situ observations of the silicification process pave the road for establishing a mechanistic understanding of cellular control of diatoms over mineral formation.