RESUMO
Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.
Assuntos
Peixe-Zebra , Animais , Norepinefrina/metabolismo , Norepinefrina/farmacologia , Cor , Pigmentação/fisiologia , Microscopia Eletrônica de Varredura , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/químicaRESUMO
Controlling the morphology of crystalline materials is challenging, as crystals have a strong tendency toward thermodynamically stable structures. Yet, organisms form crystals with distinct morphologies, such as the plate-like guanine crystals produced by many terrestrial and aquatic species for light manipulation. Regulation of crystal morphogenesis was hypothesized to entail physical growth restriction by the surrounding membrane, combined with fine-tuned interactions between organic molecules and the growing crystal. Using cryo-electron tomography of developing zebrafish larvae, we found that guanine crystals form via templated nucleation of thin leaflets on preassembled scaffolds made of 20-nm-thick amyloid fibers. These leaflets then merge and coalesce into a single plate-like crystal. Our findings shed light on the biological regulation of crystal morphogenesis, which determines their optical properties.
Assuntos
Guanina , Peixe-Zebra , AnimaisRESUMO
Skin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish's color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.
Assuntos
Diferenciação Celular/fisiologia , Cromatóforos/fisiologia , Cromatóforos/ultraestrutura , Pigmentação da Pele/fisiologia , Pele/ultraestrutura , Peixe-Zebra/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/fisiologia , Fator de Transcrição Associado à Microftalmia , Mutagênese , Pele/metabolismo , Pigmentação da Pele/genética , Transcriptoma , Difração de Raios X , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
To maintain body homeostasis, endocrine systems must detect and integrate blood-borne peripheral signals. This is mediated by fenestrae, specialized permeable pores in the endothelial membrane. Plasmalemma vesicle-associated protein (Plvap) is located in the fenestral diaphragm and is thought to play a role in the passage of proteins through the fenestrae. However, this suggested function has yet to be demonstrated directly. We studied the development of fenestrated capillaries in the hypophysis, a major neuroendocrine interface between the blood and brain. Using a transgenic biosensor to visualize the vascular excretion of the genetically tagged plasma protein DBP-EGFP, we show that the developmental acquisition of vascular permeability coincides with differential expression of zebrafish plvap orthologs in the hypophysis versus brain. Ultrastructural analysis revealed that plvapb mutants display deficiencies in fenestral diaphragms and increased density of hypophyseal fenestrae. Measurements of DBP-EGFP extravasation in plvapb mutants provided direct proof that Plvap limits the rate of blood-borne protein passage through fenestrated endothelia. We present the regulatory role of Plvap in the development of blood-borne protein detection machinery at a neuroendocrine interface through which hormones are released to the general circulation.
Assuntos
Permeabilidade Capilar/fisiologia , Endotélio Vascular/metabolismo , Proteínas de Membrana/metabolismo , Hipófise/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Proteínas de Membrana/genética , Mutação , Transporte Proteico/fisiologia , Peixe-Zebra/genéticaRESUMO
Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of Danio fishes, including the zebrafish Danio rerio, we identified two populations of white pigment cells-leucophores-one of which arises by transdifferentiation of adult melanophores and another of which develops from a yellow-orange xanthophore or xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical, and ultrastructural analyses of zebrafish leucophores revealed cell-type-specific chemical compositions, organelle configurations, and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching, and we showed that leucophore complement influences behavior. Together, our studies reveal independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution.
Assuntos
Plasticidade Celular/genética , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Genética Populacional/métodos , Melanóforos/fisiologia , Mutação/genética , Crista Neural/fisiologia , Fenótipo , Pigmentação/genética , Transcriptoma/genéticaRESUMO
Many marine organisms have evolved a reflective iris to prevent unfocused light from reaching the retina. The fish iris has a dual function, both to camouflage the eye and serving as a light barrier. Yet, the physical mechanism that enables this dual functionality and the benefits of using a reflective iris have remained unclear. Using synchrotron microfocused diffraction, cryo-scanning electron microscopy imaging, and optical analyses on zebrafish at different stages of development, it is shown that the complex optical response of the iris is facilitated by the development of high-order organization of multilayered guanine-based crystal reflectors and pigments. It is further demonstrated how the efficient light reflector is established during development to allow the optical functionality of the eye, already at early developmental stages.
RESUMO
Organic crystals are of primary importance in pharmaceuticals, functional materials, and biological systems; however, organic crystallization mechanisms are not well-understood. It has been recognized that "nonclassical" organic crystallization from solution involving transient amorphous precursors is ubiquitous. Understanding how these precursors evolve into crystals is a key challenge. Here, we uncover the crystallization mechanisms of two simple aromatic compounds (perylene diimides), employing direct structural imaging by cryogenic electron microscopy. We reveal the continuous evolution of density, morphology, and order during the crystallization of very different amorphous precursors (well-defined aggregates and diffuse dense liquid phase). Crystallization starts from initial densification of the precursors. Subsequent evolution of crystalline order is gradual, involving further densification concurrent with optimization of molecular ordering and morphology. These findings may have implications for the rational design of organic crystals.
RESUMO
Vision mechanisms in animals, especially those living in water, are diverse. Many eyes have reflective elements that consist of multilayers of nanometer-sized crystalline plates, composed of organic molecules. The crystal multilayer assemblies owe their enhanced reflectivity to the high refractive indices of the crystals in preferred crystallographic directions. The high refractive indices are due to the molecular arrangements in their crystal structures. Herein, data regarding these difficult-to-characterize crystals are reviewed. This is followed by a discussion on the function of these crystalline assemblies, especially in visual systems whose anatomy has been well characterized under close to in vivo conditions. Three test cases are presented, and then the relations between the reflecting crystalline components and their functions, including the relations between molecular structure, crystal structure, and reflecting properties are discussed. Some of the underlying mechanisms are also discussed, and finally open questions in the field are identified.
RESUMO
This study investigates the structural basis for the red, silver and black coloration of the theridiid spider, Phoroncidia rubroargentea (Berland, 1913) from Madagascar. Specimens of this species can retain their colour after storage in ethanol for decades, whereas most other brightly pigmented spider specimens fade under identical preservation conditions. Using correlative optical, structural and chemical analysis, we identify the colour-generating structural elements and characterize their optical properties. The prominent silvery appearance of the spider's abdomen results from regularly arranged guanine microplatelets, similar to those found in other spiders and fish. The microplatelets are composed of a doublet structure twinned about the [[Formula: see text]] axis, as suggested by electron diffraction. The red coloration originates from chambered microspheres (approx. 1 µm in diameter), which contain structured fluorescent material. Co-localization of the red microparticles on top of the reflective guanine microplatelets appears to enhance the red coloration. The spider's thick cuticular layer, which encases its abdomen, varies in its optical properties, being transparent in regions where only guanine reflectors are present, and tanned, exhibiting light absorption where the red microspheres are found. Moreover, colour degradation in some preserved spider specimens that had suffered damage to the cuticular layer suggests that this region of the exoskeleton may play an important role in the stabilization of the red coloration.
Assuntos
Pigmentação/fisiologia , Aranhas/fisiologia , Animais , MadagáscarRESUMO
Scallops possess a visual system comprising up to 200 eyes, each containing a concave mirror rather than a lens to focus light. The hierarchical organization of the multilayered mirror is controlled for image formation, from the component guanine crystals at the nanoscale to the complex three-dimensional morphology at the millimeter level. The layered structure of the mirror is tuned to reflect the wavelengths of light penetrating the scallop's habitat and is tiled with a mosaic of square guanine crystals, which reduces optical aberrations. The mirror forms images on a double-layered retina used for separately imaging the peripheral and central fields of view. The tiled, off-axis mirror of the scallop eye bears a striking resemblance to the segmented mirrors of reflecting telescopes.
Assuntos
Pecten/fisiologia , Pecten/ultraestrutura , Retina/fisiologia , Retina/ultraestrutura , Visão Ocular/fisiologia , Animais , Microscopia CrioeletrônicaRESUMO
Guanine crystals are widely used in nature as components of multilayer reflectors. Guanine-based reflective systems found in the copepod cuticle and in the mirror of the scallop eye are unique in that the multilayered reflectors are tiled to form a contiguous packed array. In the copepod cuticle, hexagonal crystals are closely packed to produce brilliant colors. In the scallop eye, square crystals are tiled to obtain an image-forming reflecting mirror. The tiles are about 1â µm in size and 70â nm thick. According to analysis of their electron diffraction patterns, the hexagon and square tiles are not single crystals. Rather, each tile type is a composite of what appears to be three crystalline domains differently oriented and stacked onto one another, achieved through a twice-repeated twinning about their ⟨011⟩ and ⟨021⟩ crystal axes, respectively. By these means, the monoclinic guanine crystal mimics higher symmetry hexagonal and tetragonal structures to achieve unique morphologies.
RESUMO
Vibrational spectroscopy in the electron microscope would be transformative in the study of biological samples, provided that radiation damage could be prevented. However, electron beams typically create high-energy excitations that severely accelerate sample degradation. Here this major difficulty is overcome using an 'aloof' electron beam, positioned tens of nanometres away from the sample: high-energy excitations are suppressed, while vibrational modes of energies <1 eV can be 'safely' investigated. To demonstrate the potential of aloof spectroscopy, we record electron energy loss spectra from biogenic guanine crystals in their native state, resolving their characteristic C-H, N-H and C=O vibrational signatures with no observable radiation damage. The technique opens up the possibility of non-damaging compositional analyses of organic functional groups, including non-crystalline biological materials, at a spatial resolution of â¼10 nm, simultaneously combined with imaging in the electron microscope.
Assuntos
Guanina/química , Microscopia Eletrônica de Transmissão/instrumentação , Espectroscopia de Perda de Energia de Elétrons/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Vibração , Animais , Carpas , Microscopia Eletrônica/instrumentação , Análise Espectral/métodosRESUMO
Males of sapphirinid copepods use regularly alternating layers of hexagonal-shaped guanine crystals and cytoplasm to produce spectacular structural colors. In order to understand the mechanism by which the different colors are produced, we measured the reflectance of live individuals and then characterized the organization of the crystals and the cytoplasm layers in the same individuals using cryo-SEM. On the basis of these measurements, we calculated the expected reflectance spectra and found that they are strikingly similar to the measured ones. We show that variations in the cytoplasm layer thickness are mainly responsible for the different reflected colors and also that the copepod color strongly depends on the angular orientation relative to the incident light, which can account for its appearance and disappearance during spiral swimming in the natural habitat.
Assuntos
Cor , Copépodes/fisiologia , Animais , Microscopia Crioeletrônica , Cristalização , Citoplasma/metabolismo , Ecossistema , Guanina/química , Luz , Masculino , Microscopia Eletrônica de Varredura , FótonsRESUMO
The fresh water fish neon tetra has the ability to change the structural color of its lateral stripe in response to a change in the light conditions, from blue-green in the light-adapted state to indigo in the dark-adapted state. The colors are produced by constructive interference of light reflected from stacks of intracellular guanine crystals, forming tunable photonic crystal arrays. We have used micro X-ray diffraction to track in time distinct diffraction spots corresponding to individual crystal arrays within a single cell during the color change. We demonstrate that reversible variations in crystal tilt within individual arrays are responsible for the light-induced color variations. These results settle a long-standing debate between the two proposed models, the "Venetian blinds" model and the "accordion" model. The insight gained from this biogenic light-induced photonic tunable system may provide inspiration for the design of artificial optical tunable systems.
Assuntos
Cor , Luz , Óptica e Fotônica , Fótons , Animais , Peixes , Modelos Biológicos , Difração de Raios XRESUMO
In the title compound, disodium 2-amino-6-oxo-6,7-di-hydro-1H-purine-1,7-diide hepta-hydrate, 2Na(+)·C5H3N5O(2-)·7H2O, the structure is composed of alternating (100) layers of guanine mol-ecules and hydrated Na(+) ions. Within the guanine layer, the mol-ecules are arranged in centrosymmetric pairs, with a partial overlap between the guanine rings. In this compound, guanine exists as the amino-keto tautomer from which deprotonation from N1 and N7 has occurred (purine numbering). There are no direct inter-actions between the Na(+) cations and the guanine anions. Guanine mol-ecules are linked to neighboring water mol-ecules by O-Hâ¯N and O-Hâ¯O hydrogen bonds into a network structure.
RESUMO
Fish have evolved biogenic multilayer reflectors composed of stacks of intracellular anhydrous guanine crystals separated by cytoplasm, to produce the silvery luster of their skin and scales. Here we compare two different variants of the Japanese Koi fish; one of them with enhanced reflectivity. Our aim is to determine how biology modulates reflectivity, and from this to obtain a mechanistic understanding of the structure and properties governing the intensity of silver reflectance. We measured the reflectance of individual scales with a custom-made microscope, and then for each individual scale we characterized the structure of the guanine crystal/cytoplasm layers using high-resolution cryo-SEM. The measured reflectance and the structural-geometrical parameters were used to calculate the reflectance of each scale, and the results were compared to the experimental measurements. We show that enhanced reflectivity is obtained with the same basic guanine crystal/cytoplasm stacks, but the structural arrangement between the stack, inside the stacks, and relative to the scale surface is varied when reflectivity is enhanced. Finally, we propose a model that incorporates the basic building block parameters, the crystal orientation inside the tissue, and the resulting reflectance and explains the mechanistic basis for reflectance enhancement.
Assuntos
Citoplasma/química , Guanina/química , Prata/química , Pele/química , Animais , Peixes , Japão , Estrutura MolecularRESUMO
Starting from disorder: anhydrous guanine crystals compose the photonic arrays responsible for the skin and scale iridescence found in Japanese Koi fish. These guanine crystals were found to form in intracellular vesicles through an amorphous precursor phase. A combined cryo-SEM and synchrotron radiation X-ray diffraction study showed the evolution of the crystals in great detail.