UC Santa Barbara

Reflectins are a unique class of proteins found in the specialized iridescent cells, iridocytes, in cephalopods. The colors reflected by dorsal iridocytes from the California market squid Doryteuthis opalescens are neuronally tunable, and these cells are enriched in reflectins A1, A2, B, and C. The phosphorylation of cationic reflectin proteins result in reflectin assembly: the degree of assembly controls the extent of dehydration of the light-scattering Bragg lamellae, adjusting their dimensions and therefore wavelengths of light that are reflected. I show that reflectin A1 assemblies are in dynamic exchange through a dilute monomeric phase and characterize the aging of dynamic exchange using DLS (dynamic light scattering) and FRET (Forster resonant energy transfer). Reflectin A1 phase behavior is determined by protein net charge density and ionic strength. At high net charge densities, increasing salt concentration drives liquid-liquid phase separation of reflectin A1 via nanoscale assembly intermediates. The hydrophobic effect and electrostatic screening of intra- and inter-protein Coulombic repulsion progressively drive reflectin A1 folding, assembly, and LLPS. Using fluorescence recovery after photobleaching (FRAP) and droplet fusion dynamics I demonstrate the liquidity of reflectin A1 condensates is tuned by protein NCD and ionic strength. Reflectin C co-phase separates with A1 and drastically increases the liquidity of reflectin A1. Condensates formed from physiologically relevant mixtures of reflectins A1, A2, B, and C show complex layered droplet formation in which the relative miscibility of each reflectin varies with pH, ionic strength, and proportions of reflectin species. The many inputs that define multi-reflectin phase behavior in vivo shows reflectins usefulness for creating soft biomaterials with tunable spatial organizations and liquid properties. These findings also demonstrate that LLPS of reflectins A1, A2, B and C could occur upon iridocyte activation, and that ratios of these reflectin species found in tunable iridocytes could enhance the tunability of lamellar dehydration induced by reflectin phosphorylation.