Microscopic Algae as New Emerging Models for Studies of Organic Biocrystallization and Vision

Résumé fourni par la source

Purines are the building blocks of nucleic acids (DNA, RNA), carriers of chemical energy (ATP, GTP), and signaling molecules (cAMP, cGMP) [1,2]. Crystals of purine also play key roles in various biological systems, being the end-product of nitrogen metabolism in animals [3] and acting as high-capacity nitrogen stores in unicellular organisms [4]. They also have excellent optical properties, which is why they can be found in the eyeballs of invertebrates and vertebrates, where they are used to enhance night vision [5], and the skin of cephalopods and chameleons, where they are used for camouflage [6]. In this work, we employ a wide range of microscopic techniques to study the structure of eyespots in Effrenium voratum (Symbiodiniaceae, Dinoflagellata, Alveolata, SAR). Previous studies have suggested that the eyespot consists of crystals of uric acid [7–9]. However, our Raman microscopy measurements indicated that these crystals were β-polymorphic anhydrous guanine (Fig. 1 A–E) [4,10]. Guanine crystals have a high refractive index (1.83) and can interlace with low-refractive-index cytoplasm (1.34) to form a structure that exhibits constructive light interference depending on the thickness of the layer [6,11], which our transmission electron microscopy (TEM) and EM tomography measurements suggest is approximately 70 nm thick. (Fig. 1 F,G). This structural information enables us to simulate the reflectance spectra using finite-difference time-domain optical simulations [11]. We also employed 3D electron diffraction and 4D scanning transmission electron microscopy to map grains and defects in these crystals in unprecedented detail for organic biocrystals grown by biological entities. Optical super-resolution imaging of highly reflective guanine crystals using interference reflection microscopy and ring total internal reflection fluorescence microscopy (IRM ring-TIRF) provided us with real-time kinetics of crystals moving within the eyespot (Fig. 2). Based on the data from soft-X-ray tomography of flagellated cells (Fig. 2 F–J), dividing cells, and vegetative coccoid cells, we propose the mechanisms of the formation of crystalline layers in the eyespot in the posterior part of the cell adjacent to the flagella and its functional connection to cellular vision and cellular phototaxy. Raman and transmission electron microscopy of a single flagellated cell of E. voratum. A – polarization microscopy, B,C,D – Raman chemical maps: B – distribution of guanine, C – distribution of lipids, D – merged, arrow highlighting the eyespot, E – Raman spectra of guanine, F – transmission electron microscopy of a cell cross-section, scalebar 2 μm, G – focus on the eyespot, scalebar 0.5 μm, H – the schematic representation of four-layered narrow-band reflector made of darker layer of material with high refractive index (i.e., guanine nA=1.83) and lighter layer of material with low refractive index (i.e., cytoplasm nB=1.34) where θA and θB are angles of the incident light, m is an integer, and λ is the wavelength of the reflected light. Laser scanning confocal microscopy and cryo-soft-X-ray tomography (cryoSXT) of E. voratum. A – reflectance of the single optical section depicting the eyespot, B – 3D projection of the reflection of guanine crystals in the cell, C – maximum projection of the Z-stack of the plastid, D – transmission illumination, E – merged, scalebar 2 μm, arrow pointing at the eyespot; F–J – cryoSXT of flagellated cells – guanine crystals are the most absorbing particles on the tomograms: F – single optical section of a flagellate, G – 3D reconstruction of the cell shape with two flagella in blue, nucleus in white and guanine crystals in red, H – flagellate cell with guanine crystals in cyan, magenta and the eyespot in green, plastids in red, nucleus in pink; I, J – dividing flagellated cells covered by the mother cell wall.

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