Periodic Reporting for period 3 - CRYSTALEYES (Biogenic Organic Crystals: From Crystal Formation to Genetically Engineered Optical Materials)
Periodo di rendicontazione: 2023-03-01 al 2024-08-31
The aim of the 'CRYSTALEYES' project was to reveal the mechanisms underlying the formation of optically functional organic crystals. Many organisms use highly reflective organic crystalline materials to manipulate light in a variety of coloration and visual systems. Examples include the silvery reflectance of fish scales, the colors of chameleons and crustaceans and the unusual mirrored eyes of scallops and other marine organisms. This field of 'organic biomineralization' is now emerging as an exciting new field at the interface of materials chemistry, cell biology and optics.
The most common organic crystal used by organisms in this context is guanine, better known as one of the nucleotide bases. Many organisms form crystals of guanine in specialized reflective cells called iridophores. The crystals are highly reflective because of the extremely high refractive index of guanine. By exquisitely controlling the shape and ultrastructural assembly of these crystals, animals produce a range of different light scattering phenomena resulting in extremely vivid colors. Many of these optical phenomena are produced by the physical interference of light from the interfaces between the guanine crystals and the cytoplasm in the cell. A striking observation in these systems is how organisms can produce an enormous variety of different crystal shapes from a single molecule. This ultimately determines the optics of the biological device.
The overall objective of the grant was to understand how organisms control crystallization of organic molecules in biology - to uncover 'biology's crystallization tricks'. Until the 'CRYSTALEYS' grant the field had focused on the optical function of these systems, rationalizing how crystal assembly can give rise to different optical phenomena. The 'CRYSTALEYES' grant represented the first concerted attempt to understand crystal formation. To do this, our approach was to study model, crystal-forming organisms during development and to unveil the crystallization mechanisms using a combination of cryogenic electron microscopy, and in situ diffraction and spectroscopy techniques. A variety of organisms were chosen which produce guanine crystals. Additionally, we are also studying crustacean models which generate newly discovered pterdine crystals.
Understanding the 'tricks' which organisms use to control crystallization will reveal new strategies for synthesizing artificial crystalline materials with designer properties. Biological crystallization strategies are far more powerful and sophisticated than those currently employed by chemists. Thus, understanding the fundamental rules underlying biological crystallization paves the way for a new type of materials science, inspired by biology.
The most common organic crystal used by organisms in this context is guanine, better known as one of the nucleotide bases. Many organisms form crystals of guanine in specialized reflective cells called iridophores. The crystals are highly reflective because of the extremely high refractive index of guanine. By exquisitely controlling the shape and ultrastructural assembly of these crystals, animals produce a range of different light scattering phenomena resulting in extremely vivid colors. Many of these optical phenomena are produced by the physical interference of light from the interfaces between the guanine crystals and the cytoplasm in the cell. A striking observation in these systems is how organisms can produce an enormous variety of different crystal shapes from a single molecule. This ultimately determines the optics of the biological device.
The overall objective of the grant was to understand how organisms control crystallization of organic molecules in biology - to uncover 'biology's crystallization tricks'. Until the 'CRYSTALEYS' grant the field had focused on the optical function of these systems, rationalizing how crystal assembly can give rise to different optical phenomena. The 'CRYSTALEYES' grant represented the first concerted attempt to understand crystal formation. To do this, our approach was to study model, crystal-forming organisms during development and to unveil the crystallization mechanisms using a combination of cryogenic electron microscopy, and in situ diffraction and spectroscopy techniques. A variety of organisms were chosen which produce guanine crystals. Additionally, we are also studying crustacean models which generate newly discovered pterdine crystals.
Understanding the 'tricks' which organisms use to control crystallization will reveal new strategies for synthesizing artificial crystalline materials with designer properties. Biological crystallization strategies are far more powerful and sophisticated than those currently employed by chemists. Thus, understanding the fundamental rules underlying biological crystallization paves the way for a new type of materials science, inspired by biology.
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).
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).
Our approach of studying crystallization in developing biological organisms has proven to be extremely powerful. By studying a broad variety of non-typical organisms we are consistently revealing surprising new findings on biogenic organic crystals. Overall, these findings reveal the remarkable complexity and 'sophistication' of the cellular machinery underling biological crystallization. The 6 aforementioned scientific articles have each revealed information beyond the state of the art on (i) the formation mechanisms of biogenic crystals and (ii) previously unexplored optical phenomena of organic crystals.
The next stage of the project will focus on: (i) performing synthetic crystallization experiments utilizing small molecule and macromolecular additives inspired by biology, (ii) expanding the range of model organisms used to study biological crystallization, (iii) finding the genes and macromolecules responsible for isoxanthopterin crystal formation. In relation to aim (iii), we composed transcriptomic libraries on M. rosenbergii eyes during embryogenesis. These analyses revealed hundreds of candidate genes transcribed during crystallization. To refine the search for functional genes implicated in the crystallization process, proteins expressed in the crystal-forming organelles were found and characterized by Mass Spectrometry. Together, the transcriptomic and proteomic studies have generated approximately 20 gene candidates. We are currently performing CRISPR-CAS knockout of these genes to test their functionality and their effect on crystal phenotype. Several manuscripts on this topic are now being prepared. These represents the first step towards our long-term vision for ‘genetic engineering of crystals’ outlined in the CRYSTALEYES grant.
The next stage of the project will focus on: (i) performing synthetic crystallization experiments utilizing small molecule and macromolecular additives inspired by biology, (ii) expanding the range of model organisms used to study biological crystallization, (iii) finding the genes and macromolecules responsible for isoxanthopterin crystal formation. In relation to aim (iii), we composed transcriptomic libraries on M. rosenbergii eyes during embryogenesis. These analyses revealed hundreds of candidate genes transcribed during crystallization. To refine the search for functional genes implicated in the crystallization process, proteins expressed in the crystal-forming organelles were found and characterized by Mass Spectrometry. Together, the transcriptomic and proteomic studies have generated approximately 20 gene candidates. We are currently performing CRISPR-CAS knockout of these genes to test their functionality and their effect on crystal phenotype. Several manuscripts on this topic are now being prepared. These represents the first step towards our long-term vision for ‘genetic engineering of crystals’ outlined in the CRYSTALEYES grant.