Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 "template" complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.

Structure, self-assembly, and properties of a truncated reflectin variant.

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue-enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, we elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant's hierarchical assembly, and establish a direct correlation between the protein's structural characteristics and intrinsic optical properties. Altogether, our findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells' color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

Squid leucophore-inspired engineering of optically dynamic human cells.

Cephalopods (e.g., squids, octopuses, and cuttlefishes) possess remarkable dynamic camouflage abilities and therefore have emerged as powerful sources of inspiration for the engineering of dynamic optical technologies. Within this context, we have focused on the development of engineered living systems that can emulate the tunable optical characteristics of some squid skin cells. Herein, we expand our ability to controllably incorporate reflectin-based structures within mammalian cells via genetic engineering methods, and demonstrate that such structures can facilitate holotomographic and standard microscopy imaging of the cells. Moreover, we show that the reflectin-based structures within our cells can be reconfigured with a straightforward chemical stimulus, and we quantify the stimulus-induced changes observed for the structures at the single cell level. The reported findings may enable a better understanding of the color- and appearance-changing capabilities of some cephalopod skin cells and could afford opportunities for reflectins as molecular probes in the fields of cell biology and biomedical optics.

Squid Skin Cell-Inspired Refractive Index Mapping of Cells, Vesicles, and Nanostructures.

The fascination with the optical properties of naturally occurring systems has been driven in part by nature's ability to produce a diverse palette of vibrant colors from a relatively small number of common structural motifs. Within this context, some cephalopod species have evolved skin cells called iridophores and leucophores whose constituent ultrastructures reflect light in different ways but are composed of the same high refractive index material─a protein called reflectin. Although such natural optical systems have attracted much research interest, measuring the refractive indices of biomaterial-based structures across multiple different environments and establishing theoretical frameworks for accurately describing the obtained refractive index values has proven challenging. Herein, we employ a synergistic combination of experimental and computational methodologies to systematically map the three-dimensional refractive index distributions of model self-assembled reflectin-based structures both in vivo and in vitro. When considered together, our findings may improve understanding of squid skin cell functionality, augment existing methods for characterizing protein-based optical materials, and expand the utility of emerging holotomographic microscopy techniques.

Novel Roles for Diacylglycerol in Synaptic Vesicle Priming and Release Revealed by Complete Reconstitution of Core Protein Machinery.

Here we introduce the full functional reconstitution of genetically-validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca 2+ . Using this novel setup, we discover new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca 2+- triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca 2+ -dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of ready-release vesicles. Dynamic single-molecule imaging of Complexin binding to ready-release vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by Munc13 and Munc18 chaperones. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 'template' complex is a functional intermediate in the production of primed, ready-release vesicles, which requires the coordinated action of Munc13 and Munc18. SIGNIFICANCE STATEMENT: Munc13 and Munc18 are SNARE-associated chaperones that act as "priming" factors, facilitating the formation of a pool of docked, release-ready vesicles and regulating Ca 2+ -evoked neurotransmitter release. Although important insights into Munc18/Munc13 function have been gained, how they assemble and operate together remains enigmatic. To address this, we developed a novel biochemically-defined fusion assay which enabled us to investigate the cooperative action of Munc13 and Munc18 in molecular terms. We find that Munc18 nucleates the SNARE complex, while Munc13 promotes and accelerates the SNARE assembly in a DAG-dependent manner. The concerted action of Munc13 and Munc18 stages the SNARE assembly process to ensure efficient 'clamping' and formation of stably docked vesicles, which can be triggered to fuse rapidly (∼10 msec) upon Ca 2+ influx.

Two successive oligomeric Munc13 assemblies scaffold vesicle docking and SNARE assembly to support neurotransmitter release.

The critical presynaptic protein Munc13 serves numerous roles in the process of docking and priming synaptic vesicles. Here we investigate the functional significance of two distinct oligomers of the Munc13 core domain (Munc13C) comprising C1-C2B-MUN-C2C. Oligomer interface point mutations that specifically destabilized either the trimer or lateral hexamer assemblies of Munc13C disrupted vesicle docking, trans-SNARE formation, and Ca 2+ -triggered vesicle fusion in vitro and impaired neurotransmitter secretion and motor nervous system function in vivo. We suggest that a progression of oligomeric Munc13 complexes couples vesicle docking and assembly of a precise number of SNARE molecules to support rapid and high-fidelity vesicle priming.

At the Intersection of Natural Structural Coloration and Bioengineering.

Most of us get inspired by and interact with the world around us based on visual cues such as the colors and patterns that we see. In nature, coloration takes three primary forms: pigmentary coloration, structural coloration, and bioluminescence. Typically, pigmentary and structural coloration are used by animals and plants for their survival; however, few organisms are able to capture the nearly instantaneous and visually astounding display that cephalopods (e.g., octopi, squid, and cuttlefish) exhibit. Notably, the structural coloration of these cephalopods critically relies on a unique family of proteins known as reflectins. As a result, there is growing interest in characterizing the structure and function of such optically-active proteins (e.g., reflectins) and to leverage these materials across a broad range of disciplines, including bioengineering. In this review, I begin by briefly introducing pigmentary and structural coloration in animals and plants as well as highlighting the extraordinary appearance-changing capabilities of cephalopods. Next, I outline recent advances in the characterization and utilization of reflectins for photonic technologies and and discuss general strategies and limitations for the structural and optical characterization of proteins. Finally, I explore future directions of study for optically-active proteins and their potential applications. Altogether, this review aims to bring together an interdisciplinary group of researchers who can resolve the fundamental questions regarding the structure, function, and self-assembly of optically-active protein-based materials.

Reconfigurable Micro- and Nano-Structured Camouflage Surfaces Inspired by Cephalopods.

Wrinkled surfaces and materials are found throughout the natural world in various plants and animals and are known to improve the performance of emerging optical and electrical technologies. Despite much progress, the reversible post-fabrication tuning of wrinkle sizes and geometries across multiple length scales has remained relatively challenging for some materials, and the development of comprehensive structure-function relationships for optically active wrinkled surfaces has often proven difficult. Herein, by drawing inspiration from natural cephalopod skin and leveraging methodologies established for artificial adaptive infrared platforms, we engineer systems with hierarchically reconfigurable wrinkled surface morphologies and dynamically tunable visible-to-infrared spectroscopic properties. Specifically, we demonstrate architectures for which mechanical actuation changes the surface morphological characteristics; modulates the reflectance, transmittance, and absorptance across a broad spectral window; controls the specular-to-diffuse reflectance ratios; and alters the visible and thermal appearances. Moreover, we demonstrate the incorporation of these architectures into analogous electrically actuated appearance-changing devices that feature competitive figures of merit, such as reasonable maximum areal strains, rapid response times, and good stabilities upon repeated actuation. Overall, our findings constitute another step forward in the continued development of cephalopod-inspired light- and heat-manipulating systems and may facilitate advanced applications in the areas of sensing, electronics, optics, soft robotics, and thermal management.

Cephalopod-inspired optical engineering of human cells.

Although many animals have evolved intrinsic transparency for the purpose of concealment, the development of dynamic, that is, controllable and reversible, transparency for living human cells and tissues has remained elusive to date. Here, by drawing inspiration from the structures and functionalities of adaptive cephalopod skin cells, we design and engineer human cells that contain reconfigurable protein-based photonic architectures and, as a result, possess tunable transparency-changing and light-scattering capabilities. Our findings may lead to the development of unique biophotonic tools for applications in materials science and bioengineering and may also facilitate an improved understanding of a wide range of biological systems.

Growth and Spatial Control of Murine Neural Stem Cells on Reflectin Films.

Stem cells have attracted significant attention due to their regenerative capabilities and their potential for the treatment of disease. Consequently, significant research effort has focused on the development of protein- and polypeptide-based materials as stem cell substrates and scaffolds. Here, we explore the ability of reflectin, a cephalopod structural protein, to support the growth of murine neural stem/progenitor cells (mNSPCs). We observe that the binding, growth, and differentiation of mNSPCs on reflectin films is comparable to that on more established protein-based materials. Moreover, we find that heparin selectively inhibits the adhesion of mNSPCs on reflectin, affording spatial control of cell growth and leading to a >30-fold change in cell density on patterned substrates. The described findings highlight the potential utility of reflectin as a stem cell culture material.

An introduction to color-changing systems from the cephalopod protein reflectin.

Cephalopods possess unrivaled camouflage and signaling abilities that are enabled by their sophisticated skin, wherein multiple layers contain chromatophore pigment cells (as part of larger chromatophore organs) and different types of reflective cells called iridocytes and leucophores. The optical functionality of these cells (and thus cephalopod skin) critically relies upon subcellular structures partially composed of unusual structural proteins known as reflectins. Herein, we highlight studies that have investigated reflectins as materials within the context of color-changing coatings. We in turn discuss these proteins' multi-faceted properties, associated challenges, and future potential. Through our presentation of selected case studies, we hope to stimulate additional dialogue and spur further research on photonic technologies based on and inspired by reflectins.