Intra- to extracellular crystallization of calcite in the freshwater green algae Phacotus lenticularis

Phacotus lenticularis is a freshwater unicellular green alga that forms lens-shaped calcitic shells around the cell. We documented P. lenticularis biomineralization pathways in live daughter cells while still within the reproductive complex, using scanning confocal microscopy and after vitrification using cryo-scanning electron microscopy (cryo-SEM). We show that some or all of the calcium ions required for mineral formation enter the cell through endocytosis, as inferred from the uptake of calcein fluorescent dye. Ions first concentrate inside intracellular vesicles to form small crystals that were detected by birefringence, reflectance, and cryo-SEM of cells in near-native, hydrated state. The crystals later exit the cell and build up the lens-shaped shell. The small crystals first cover the outer lorica surface and later fuse to form a thin continuous shell. This is most likely followed by a second shell maturation phase in which the shell undergoes thickening and crystal reorganization. Crystal assembly within the confined protected volume of the reproduction complex allows controlled shell formation outside the daughter cell. Only two other unicellular marine calcifiers, coccolithophores and miliolid foraminifera, are known to perform intracellular crystal formation. STATEMENT OF SIGNIFICANCE: Calcium carbonate (CaCO₃) deposition in aquatic environments is a major component of the global carbon cycle, which determines the CO₂ content of the atmosphere. In freshwater ecosystems, the green alga Phacotus lenticularis is considered the main contributor of autochthonous calcite precipitation and the only algal species known to form its shell through a controlled process. The chemical and ecological effects of P. lenticularis are intensively investigated, but our understanding of its shell formation is limited. We used advanced confocal laser scanning microscopy and cryo-scanning electron microscopy (cryo-SEM) to provide new insights into mineral formation and trafficking in the calcifying P. lenticularis cells.