Key objectives in the 'CRYSTALEYES' grants were; (i) to determine the chemical composition of ‘guanine’ crystals, (ii) to understand whether amorphous phases are utilized in crystallization (i.e. non-classical crystallization) and, (iii) to elucidate the mechanism of crystal morphology control in organisms.
The first major milestone of the project was finding that biogenic 'guanine' crystals are not made only of guanine. Pinsk et. al. (Pinsk et. al., J. Am. Chem. Soc., 2022) determined that many biogenic guanine crystals contain high quantities (<20%) of other purine metabolites as dopants within the crystal framework. This work answered a long-standing question on the composition of organic crystals in biology. The ability of guanine crystals to host other molecules enables animals to build physiologically “cheaper” crystals from mixtures of metabolites present in the crystal forming cells, without impeding their optical functionality. The amount of foreign molecules inside the crystals was surprising and raises several fundamental structural chemistry questions which we are currently investigating.
The second major milestone was the elucidation of a crystallization mechanism for biogenic guanine. By following guanine crystallization in a spider undergoing development, we showed that crystallization occurs in a non-classical manner, involving the crystallization of an amorphous phase via gradual orientational ordering and relaxation of structural defects (Wagner et. al., Adv. Mater., 2022). This was the first time a detailed crystallization mechanism of guanine had been revealed, which was a major challenge in the field.
Perhaps the most important question in the field was to understand how organisms control the morphology of guanine crystals. By controlling crystal shape, organisms can generate many different optical effects. Specifically, plate-like crystals expressing highly reflective, but thermodynamically unstable crystal faces are produced by organisms, which cannot be achieved synthetically. By studying guanine formation in developing scallops using cryogenic electron microscopy, we found that crystallization is controlled by macromolecular templates in the crystal-forming iridosome organelle (Wagner et. al. Nat. Commun., 2023). These templates guide crystal growth to generate highly reflective platelets. Similar observations were also made on developing lizard models, indicating that these may be universal features of guanine bio-crystallization (Zhang et. al., PNAS, 2023).
Another critical aspect of the CRYSTALEYES grant was to understand the biological regulation behind the crystallization process. Our long-term objective was to find the genes and proteins controlling crystal nucleation and growth. Initial studies focused on model species of crustaceans M. rosenbergii (freshwater prawn) and C. quadricarinatus (crayfish). These organisms utilize highly reflective crystals of isoxanthopterin in their eyes for enhancing photon capture. Using a similar approach to the aforementioned studies on guanine, we first elucidated key time points for crystal formation during embryo development. These studies led to several surprising findings and the serendipitous discovery of a novel optical structure in the eyes of larval crustaceans. Larval crustaceans are transparent pelagic organisms that utilize transparency to conceal themselves in the open ocean – a defense against predation. The organisms have evolved ways to remove pigment from almost all of their tissues. However, to see, the animals require conspicuous eye pigments required for vision. These opaque pigments represent an ‘achilleas heal’ to being spotted by predators. We discovered a reflective device overlying the opaque pigments in the eyes. The reflector is composed of isoxanthopterin crystals, similar to those found in adult eyes, and the color of reflector is tuned to the water color in native habitat of the animals, rendering them invisible to predators (Shavit et. al., Science 2023). This finding has stimulated further studies in the lab on the optical properties of isoxanthopterin crystals, including the discovery of an ultra-efficient white reflector in shrimp chromatophore cells (Lemcoff et. al., Nature Photonics, 2023).
