Reflectins form multicompartment liquid-liquid phase separated condensates that mirror and may facilitate spatial organization in squid skin Bragg lamellae

Cationic reflectin proteins transduce neuronal signals tuning skin color for dynamic camouflage and communication in Loliginid squid. Neuronally released acetylcholine (ACh) activates phosphorylation of the reflectins, triggering their condensation, folding and hierarchical assembly. This causes osmotic and Gibbs-Donnan dehydration of membrane-enclosed Bragg lamellae containing these proteins in skin cells called iridocytes, changing their refractive index and spacing to finely tune the wavelength of reflected light. Reflectins B and C are enriched in ACh-responsive iridocytes, suggesting they are critical for the neuronally tuned protein phase transitions within the Bragg lamellae. Here, using pH titration as an in vitro surrogate for phosphorylation to reduce protein net charge density, we demonstrate with confocal microscopy that reflectins A1, A2, B and C individually undergo LLPS to form protein dense liquid condensates, while physiological mixtures of these proteins exhibit coordinated phase transitions to form multicompartment condensates whose internal spatial organization is tuned by the proportions and charge densities of the different reflectins. Our findings demonstrate that (i) the charge densities of the different reflectins control their spatial organization within liquid condensates, (ii) the relative proportions of the different reflectins found in the ACh-responsive iridocytes increase the sensitivity of their proteins liquid phase transitions to changes in reflectin charge density; and (iii) the spatial segregation of reflectins A1 and C we observe in their multiphasic liquid condensate mirror their spatial segregation in the Bragg lamellae in vivo, suggesting a mechanistic explanation for the spatial segregation of these reflectins across the iridocytes Bragg lamellae.