ISM2019 (Microscopy)


Santosh Kumar 1 Katya Rechav 2 Ifat Kaplan-Ashiri 2 Assaf Gal 1
1Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel
2Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

Diatoms are unicellular algae with cell walls made of silica. During cell division, the daughter cell inherits half of the cell wall from the mother cell, and synthesizes the other half in specialized, intracellular compartments called silica deposition vesicles (SDVs). Once silica deposition in an SDV is complete, it is exocytosed and deposited to the cell surface. Silicic acid is the monomeric building block of silica that is taken up by diatoms from their aqueous environment. Environmental concentrations of silicic acid are far below its solubility limit of ~2 mM. Thus, the diatom cell needs to concentrate silicon to facilitate silica deposition inside the SDV. Several hypotheses, including the existence of an intracellular silicon-pool have been proposed to explain silicification in diatoms. However, no direct observation of these putative silicon pools has been presented.

Thalassiosira pseudonana is a model diatom species with sequenced genome and extensive studies into its cell cycle and cell wall formation. We investigated intracellular silicon-pools in T. pseudonana using cryo-focused ion beam scanning electron microscopy (cryo-FIB SEM). Batches of T. pseudonana cultures were high-pressure-frozen in their pristine native state. Using slice-and-view approach, the frozen cells were milled using the ion beam and their interior imaged with the SEM. The 3D image stacks revealed that the space inside the cells is mostly filled with membrane bound organelles, leaving little space for the cytosol. We used energy dispersive X-Ray spectroscopy (EDX), to detect silicon signal within the cells. In cells growing in abundant silicon supply, some cells showed high intracellular silicon signal, while in others the silicon signal was indistinguishable from the growth medium. To investigate the silicon-pool in relation to the cell cycle, we tested cells recovering from Si-starvation. We found that the frequency of cells with detectable silicon-pool was higher 1.5 hours after Si-repletion compared to 4 hours after Si-repletion. This first observation of an internal silicon pool within the diatom cell suggests a flexible physiological response to environmental conditions and cell cycle stage. In addition, our results demonstrate the applicability of cryoFIB-SEM combined with cryoEDS to elucidate the presence and dynamics of mineral pools within live cells.