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Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, the neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing their refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that the reduction in repulsive net charge of the purified, recombinant reflectin—either (for the first time) by generalized anionic screening with salt or by pH titration—drives a finely tuned, precisely calibrated increase in the size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. The precise proportionality between the assembly size and charge neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth by a continual, equilibrium tuning of the balance between the protein’s Coulombic repulsion and short-range interactive forces. The resulting stability of reflectin assemblies with time ensures a reciprocally precise control of the particle number concentration, encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The charge regulation of reflectin assembly precisely fine-tunes a colligative property-based nanostructured biological machine. A physical mechanism is proposed. 

Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple groups have reported that early endocytic proteins form flexible condensates, which provide a platform for efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic assemblies. In live cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitylase enzyme destabilized nascent endocytic sites within minutes. In both in vitro and live cell settings, dynamic exchange of Eps15 proteins, a measure of protein network stability, was decreased by Eps15-ubiquitin interactions and increased by loss of ubiquitin. These results collectively suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic internalization.