Background: Biomineralization, e.g., in sea urchins or mollusks, includes the assembly of mesoscopic superstructures from inorganic crystalline components and biopolymers. The resulting mesocrystals inspire biophysicists and material scientists alike, because of their extraordinary physical properties. Current efforts to replicate mesocrystal synthesis in vitro require understanding the principles of their self-assembly in vivo. One question, not addressed so far, is whether intracellular crystals of proteins can assemble with biopolymers into functional mesocrystal-like structures. During our electron microscopy studies into Artemia franciscana (Crustacea: Branchiopoda), we found initial evidence of such proteinaceous mesostructures.
Results: EM preparations with high-pressure freezing and accelerated freeze substitution revealed an extraordinary intracellular source of mesostructured inclusions in both the cyto-and nucleoplasm of the epidermal lining of ovisacs of A. franciscana. Confocal reflection microscopy not only confirmed our finding; it also revealed reflective, light dispersing activity of these flake-like structures, their positioning and orientation with respect to the ovisac inside. Both the striation of alternating electron dense and electron-lucent components and the sharp edges of the flakes indicate self-assembly of material of yet unknown origin under supposed participation of crystallization. However, selected area electron diffraction could not verify the status of crystallization. Energy dispersive X-ray analysis measured a marked increase in nitrogen within the flake-like inclusion, and the almost complete absence of elements that are typically involved in inorganic crystallization. This rise in nitrogen could possibility be related to higher package density of proteins, achieved by mesostructure assembly.
Conclusions: The ovisac lining of A. franciscana is endowed with numerous mesostructured inclusions that have not been previously reported. We hypothesize that their self-assembly was from proteinaceous polycrystalline units and carbohydrates. These mesostructured flakes displayed active optical properties, as an umbrella-like, reflective cover of the ovisac, which suggests a functional role in the reproduction of A. franciscana. In turn, studies into ovisac mesostructured inclusions could help to optimizing rearing Artemia as feed for fish farming. We propose Artemia ovisacs as an in vivo model system for studying mesostructure formation.
Keywords: Artemia franciscana; Brine shrimps; Cellular inclusions; Confocal reflection microscopy; Mesocrystals; Ovisac; Rapid freeze substitution; Reproduction; Transmission electron microscopy.