Daniel G. DeMartini

Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid

Many cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the light-organ iridescence is static and is due to reflectin protein-based platelets assembled into lamellar thin-film reflectors called iridosomes, contained within iridescent cells called iridocytes. Squid in the family Loliginidae appear to be unique in which the dermis possesses a dynamic iridescent component with reflective, coloured structures that are assembled and disassembled under the control of the muscarinic cholinergic system and the associated neurotransmitter acetylcholine (ACh). Here we present the sequences and characterization of three new members of the reflectin family associated with the dynamically changeable iridescence in Loligo and not found in static Euprymna iridophores. In addition, we show that application of genistein, a protein tyrosine kinase inhibitor, suppresses ACh- and calcium-induced iridescence in Loligo. We further demonstrate that two of these novel reflectins are extensively phosphorylated in concert with the activation of iridescence by exogenous ACh. This phosphorylation and the correlated iridescence can be blocked with genistein. Our results suggest that tyrosine phosphorylation of reflectin proteins is involved in the regulation of dynamic iridescence in Loligo.

Membrane invaginations facilitate reversible water flux driving tunable iridescence in a dynamic biophotonic system.

Squids have used their tunable iridescence for camouflage and communication for millions of years; materials scientists have more recently looked to them for inspiration to develop new “biologically inspired” adaptive optics. Iridocyte cells produce iridescence through constructive interference of light with intracellular Bragg reflectors. The cell’s dynamic control over the apparent lattice constant and dielectric contrast of these multilayer stacks yields the corresponding optical control of brightness and color across the visible spectrum. Here, we resolve remaining uncertainties in iridocyte cell structure and determine how this unusual morphology enables the cell’s tunable reflectance. We show that the plasma membrane periodically invaginates deep into the iridocyte to form a potential Bragg reflector consisting of an array of narrow, parallel channels that segregate the resulting high refractive index, cytoplasmic protein-containing lamellae from the low-index channels that are continuous with the extracellular space. In response to control by a neurotransmitter, the iridocytes reversibly imbibe or expel water commensurate with changes in reflection intensity and wavelength. These results allow us to propose a comprehensive mechanism of adaptive iridescence in these cells from stimulation to color production. Applications of these findings may contribute to the development of unique classes of tunable photonic materials.

The shell-eyes of the chiton Acanthopleura granulata (Mollusca, Polyplacophora) use pheomelanin as a screening pigment

Certain species of chiton (Mollusca, Polyplacophora) have hundreds of small (< 100 µm) eyes embedded in their dorsal shell plates. These eyes each contain a retina, a layer of screening pigment, and a lens. Previously, we demonstrated that the eyes of chitons provide spatial vision. As in other camera-type eyes, the screening pigments in the eyes of chitons absorb off-axis light in order to preserve the contrast of images formed on the retina. Our results indicate that the red-brown, alkali-soluble screening pigment associated with the eyes of the chiton Acanthopleura granulata (Gmelin, 1791) is pheomelanin. Using high-performance liquid chromatography (HPLC) and MALDI-TOF mass spectroscopy, we find that degrading A. granulata’s screening pigment with alkaline hydrogen peroxide produces 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole (BTCA), a diagnostic marker of pheomelanin. Chitons are the first molluscs demonstrated to use pheomelanin as a screening pigment in their eyes. Our results suggest that the image-forming eyes of chitons may have evolved separately from eyes that employ different types of screening pigment, such as those of most other invertebrates. Further, we hypothesize that change in the expression pattern of tyrosinase – an enzyme responsible for melanin synthesis in many other metazoans – may have contributed to the origin of screening pigments in chitons, a critical step in the evolution of their eyes.

The microscopic network structure of mussel (Mytilus) adhesive plaques

Marine mussels of the genus Mytilus live in the hostile intertidal zone, attached to rocks, bio-fouled surfaces and each other via collagen-rich threads ending in adhesive pads, the plaques. Plaques adhere in salty, alkaline seawater, withstanding waves and tidal currents. Each plaque requires a force of several newtons to detach. Although the molecular composition of the plaques has been well studied, a complete understanding of supra-molecular plaque architecture and its role in maintaining adhesive strength remains elusive. Here, electron microscopy and neutron scattering studies of plaques harvested from Mytilus californianus and Mytilus galloprovincialis reveal a complex network structure reminiscent of structural foams. Two characteristic length scales are observed characterizing a dense meshwork (approx. 100 nm) with large interpenetrating pores (approx. 1 µm). The network withstands chemical denaturation, indicating significant cross-linking. Plaques formed at lower temperatures have finer network struts, from which we hypothesize a kinetically controlled formation mechanism. When mussels are induced to create plaques, the resulting structure lacks a well-defined network architecture, showcasing the importance of processing over self-assembly. Together, these new data provide essential insight into plaque structure and formation and set the foundation to understand the role of plaque structure in stress distribution and toughening in natural and biomimetic materials.

Structures, Organization, and Function of Reflectin Proteins in Dynamically Tunable Reflective Cells

Background: ACh-induced phosphorylation drives assembly of reflectins, dynamically tuning iridescence from subcellular Bragg reflectors in squid iridocytes. Results: Reflectin sequences and phosphorylation sites are characterized from iridocytes with different photonic behaviors. Conclusion: Differences in reflectin structures and phosphorylation determine the emergent photonic behavior of reflective squid tissues. Significance: Biomolecular mechanisms of adaptive iridescence provide new insights into protein-dependent energy transduction and approaches to tunable optical materials. The reversible assembly of reflectin proteins drives dynamic iridescence in cephalopods. Squid dynamically tune the intensity and colors of iridescence generated by constructive interference from intracellular Bragg reflectors in specialized skin cells called iridocytes. Analysis of the tissue specificity of reflectin subtypes reveals that tunability is correlated with the presence of one specific reflectin sequence. Differential phosphorylation and dephosphorylation of the reflectins in response to activation by acetylcholine, as well as differences in their tissue-specific and subcellular spatial distributions, further support the suggestion of different roles for the different reflectin subtypes.

A cohort of new adhesive proteins identified from transcriptomic analysis of mussel foot glands

The adaptive attachment of marine mussels to a wide range of substrates in a high-energy, saline environment has been explored for decades and is a significant driver of bioinspired wet adhesion research. Mussel attachment relies on a fibrous holdfast known as the byssus, which is made by a specialized appendage called the foot. Multiple adhesive and structural proteins are rapidly synthesized, secreted and moulded by the foot into holdfast threads. About 10 well-characterized proteins, namely the mussel foot proteins (Mfps), the preCols and the thread matrix proteins, are reported as representing the bulk of these structures. To explore how robust this proposition is, we sequenced the transcriptome of the glandular tissues that produce and secrete the various holdfast components using next-generation sequencing methods. Surprisingly, we found around 15 highly expressed genes that have not previously been characterized, but bear key similarities to the previously defined mussel foot proteins, suggesting additional contribution to byssal function. We verified the validity of these transcripts by polymerase chain reaction, cloning and Sanger sequencing as well as confirming their presence as proteins in the byssus. These newly identified proteins greatly expand the palette of mussel holdfast biochemistry and provide new targets for investigation into bioinspired wet adhesion.

Molecular mechanism of reflectin’s tunable biophotonic control: Opportunities and limitations for new optoelectronics

Discovery that reflectin proteins fill the dynamically tunable Bragg lamellae in the reflective skin cells of certain squids has prompted efforts to design new reflectin-inspired systems for dynamic photonics. But new insights into the actual role and mechanism of action of the reflectins constrain and better define the opportunities and limitations for rationally designing optical systems with reflectin-based components. We and our colleagues have discovered that the reflectins function as a signal-controlled molecular machine, regulating an osmotic motor that tunes the thickness, spacing, and refractive index of the tunable, membrane-bound Bragg lamellae in the iridocytes of the loliginid squids. The tunable reflectin proteins, characterized by a variable number of highly conserved peptide domains interspersed with positively charged linker segments, are restricted in intra- and inter-chain contacts by Coulombic repulsion. Physiologically, this inhibition is progressively overcome by charge-neutralizati...

Sparkling Reflective Stacks of Purine Crystals in the Nudibranch Flabellina iodinea

Although pigments contribute to much of the brilliant purple and orange coloration of the aeolid nudibranch Flabellina iodinea, the optical appearance of the animal was found to be augmented by dynamically sparkling, brightly reflective material in cells located throughout its epidermis. Electron microscopy revealed that specialized cells most abundant near the epithelial basal lamina contain numerous multilayer stacks of crystals, each within a fragile membrane capsule. High-resolution light microscopy of tissue sections showed that these crystalline stacks intermittently reflect light, with a temporally dynamic, sparkling appearance, suggesting that they are free to move—a phenomenon also observed in the live, intact whole animal and in the purified crystal stacks as well. Thin-layer chromatography and ultraviolet spectrometry show that the crystals isolated from all epithelial tissues are identical in composition, with guanine being the major component and its derivative, hypoxanthine, a minor component, regardless of the tissue’s pigmentary color. Electron diffraction of the crystals purified separately from the orange and purple tissues exhibits nearly identical lattice parameters that closely match those measured for guanine crystals, which are widely distributed in other biophotonic systems ranging from marine invertebrates to terrestrial vertebrates. Heterogeneity of the thickness and spacing of the crystals within their stacks accounts for their broadband silvery reflectance. The optical appearance of the epidermis of this nudibranch thus results from the interaction of incident light with mobile stacks of purine crystals, augmenting the effects of its pigmentary colors.

Intertidal exposure favors the soft-studded armor of adaptive mussel coatings

The mussel cuticle, a thin layer that shields byssal threads from environmental exposure, is a model among high-performance coatings for being both hard and hyper-extensible. However, despite avid interest in translating its features into an engineered material, the mechanisms underlying this performance are manifold and incompletely understood. To deepen our understanding of this biomaterial, we explore here the ultrastructural, scratch-resistant, and mechanical features at the submicrometer scale and relate our observations to individual cuticular components. These investigations show that cuticle nanomechanics are governed by granular microinclusions/nanoinclusions, which, contrary to previous interpretations, are three-fold softer than the surrounding matrix. This adaptation, which is found across several related mussel species, is linked to the level of hydration and presumed to maintain bulk performance during tidal exposures. Given the interest in implementing transfer of biological principles to modern materials, these findings may have noteworthy implications for the design of durable synthetic coatings.There is interest in the development of mussel inspired materials; however, this requires an understanding of the materials. Here, the authors report on an investigation into the properties of mussel cuticle from different species that challenges conventional wisdom about particle filled composites.