Hollergschwandtner E et al. (2017), Novel mesostructured inclusions in the epiderma...

ECB-ART-45838

PeerJ 2017 Jan 01;5:e3923. doi: 10.7717/peerj.3923.

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Novel mesostructured inclusions in the epidermal lining of Artemia franciscana ovisacs show optical activity.

Hollergschwandtner E , Schwaha T , Neumüller J , Kaindl U , Gruber D , Eckhard M , Stöger-Pollach M , Reipert S .

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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.

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Genes referenced: LOC100887844 LOC115919910 LOC115925415

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	Figure 1. Life cycle of Artemia Franciscana.Two distinctly different paths of reproduction are possible: Under favorable environmental conditions embryos develop directly inside the ovisac, and nauplii are released from there (ovoviviparity). Under harsh environmental conditions the ovisac produces diapausing eggs that dry out after their release (oviparity). Once the cysts are rehydrated, larvae (nauplii) will hatch. Notably, light has been identified as a factor for inducing hatching (Sorgeloos, 1973). The development of nauplii into sub-adults occurs within 1–3 weeks. Adult females release oocytes from their two oviducts into the ovisac where they become fertilized by males.
	Figure 2. Ovisac of A. franciscana visualized by using light microscopy.Depending upon the orientation of the incident illumination, the ovisac lining displays a glitter that is either inconspicuous (A) or very evident (B) under the stereomicroscope. For (B), the LED illumination source was oriented in favor of reflection caused by glittering flakes. (C) Autofluorescence microscopy of maximum intensity projections of CLSM-stack images. The ovisac lining shows variation of the autofluorescence over the surface area, especially in the region of the two characteristic spines, but no modulation of contrasts that could indicate the presence of glittering, flake-like entities observed in the stereomicroscope. (D) Confocal reflection microscopy. Overlay of three wavelengths images generated from a z-stack that covers a large field of view of the ovisac. The z-stack contains irregularly shaped inclusions in focus regions, which indicate almost complete covering of the ovisac by multicolored, reflective flake-like structures. Bars in (A–C), 0.5 mm. Bar in (D), 25 µm.
	Figure 3. Light dispersion by mesostructured flakes in the ovisac lining of A. franciscana.(A) Multicolored confocal reflection microscopy generated from a z-stack at high resolution reveals striations within flakes that vary in number and width. (B–D) display the contributions of three individual wavelengths (633 nm, 561 nm and 488 nm, respectively) to the overlay picture (A). The boxed areas mark one and the same region for comparison at different wave lengths. Notably, the image content is wave length-dependent. Bars, 10 µm.
	Figure 4. Transmission electron microscopy of mesostructured cytoplasmic inclusion in the epidermal cells of the ovisac lining of A. franciscana.(A) Sample chemically fixed and embedded in epoxy resin at room temperature. Rhomboidal inclusions are organized in groups, with individual rhomboids aligned in parallel and proximal to each other. The cytoplasm is severely washed out during sample processing, in particular in areas marked with an asterisk. (B) and (C) Samples high-pressure frozen, followed by freeze substitution and embedding in epoxy resin. (B) shows an epidermal cell detail at low magnification that is well-preserved by cryopreparation, inclusively of a fold in the ovisac lining. Cell organelles, ribosomes and glycogen are apparent. The cuticle (Cu) displays a continuous, brush-like surface layer (arrows), which is not preserved by conventional chemical processing. Given the folding of the epidermis, inclusions are cut at various angles. The impression that groups of aligned electron-dense rhomboids and electron-lucent material in-between are integral parts of common flake-like entities is confirmed at higher magnification in (C). Inclusions cut longitudinally resemble light microscopic observation of native tissues in Fig. 2D. The flakes are slightly curved and they display an edged interface with the cytoplasm (see arrowheads), and a smooth interface between alternating electron-dense and electron-lucent striations. The surrounding cytoplasm is densely filled with glycogen rosettes (asterisks). Essentially, no glycogen or other cytoplasmic content is incorporated in the electron-lucent material of the putative flakes. Bars: (A), 2 µm; (B) and (C), 1 µm.
	Figure 5. Electron tomography of a mesostructured flake-like inclusion in the cytoplasm of A. franciscana.(A) Individual virtual section of a slice thickness of ca. 0.5 nm generated from a 3D-reconstructed tilt series. The sectioned flake contains alternating electron-opaque and electron-lucent striations. Note a faint contrast between the cytoplasm (Cy) and the more electron-lucent material of the striations. (B) 3D reconstruction of the flake from virtual sections indicates the incorporation of the electron-lucent material between rhomboid electron-dense materials. Bar in (A) 200 nm.
	Figure 6. EDX-analysis of the element composition within cellular inclusions.The spectra of encircled crystal-like inclusions (free draw 2–4) were compared with the spectrum of the overall area (underlaid red). The Kα-peaks of nitrogen are all significantly higher in the free-draw regions. They are not affected by Ca- Lα-radiation, since calcium, a major candidate for inorganic crystallization, is not enriched in the free-draw regions; there are no Kα- and Kβ peaks present at the marked positions of the spectra. The enrichment in nitrogen, possibly caused by a protein concentration higher than in the surrounding cytoplasm, coincides with the enrichment in sulfur indicated by S-Kα∕β peaks. The chlorine peaks, in contrast, display variations in their intensity. Note also that a Si Kα-peak is generated within the overall area, because of the excitation of Si wafer material underneath the section (see ‘Material and Methods’). Moreover, the crystal-like inclusions (free-draw 2–4) show enrichment in osmium that is indicated by more prominent M-peaks in comparison with the overall area. Contrasting with osmium is the precondition for visibility of the cellular inclusions in the electron backscattering mode of the scanning electron microscope, notably, without resolving their characteristic striated patterns (boxed insert).
	Figure 7. Preliminary ultrastructural evidence for the formation of mesostructured inclusions inside both the cytoplasm and nucleoplasm of A. franciscana obtained by cryopreparation.(A) Epidermal cell displaying small inclusion at low abundance both in its cytoplasm (arrowheads) and its nucleus (arrows). (B) Detail displaying inclusions within a nucleus showing small electron-dense rhomboids with an electron-lucent coat (arrowheads) and small, flake-like inclusions in their neighborhood (arrows). (C) Cytoplasmic cluster (Cl) of small, putative precursors surrounded by small rhomboids similar to those observed in the cell nucleus in (B). The cytoplasm of the cell in (C) contains areas with numerous electron-lucent spots. Given the rarity of the observation it could not be decided whether these spots are related to the assembly process, or not. Artifact by ice crystals might have caused or at least influenced them. N- nucleus, Cy- cytoplasm, h- heterochromatin, m- mitochondria. Bar in (A) 2 µm. Bars in (B) and (C) 1 µm.

References [+] :

Asem, Morphological differentiation of seven parthenogenetic Artemia (Crustacea: Branchiopoda) populations from China, with special emphasis on ploidy degrees. 2016, Pubmed