Biology subjects

Levenson, R.

Publications and source records attributed to Levenson, R..

5 recordsLinked to original sources

bioRxiv2026

Heterotypic interactions and sequence features modulate cellular reflectin condensate dynamics

Reflectin proteins drive dynamic structural coloration in cephalopods by organizing into dense intracellular lamellar structures that dictate local refractive index. While reconstituted reflectins readily undergo liquid-liquid phase separation in vitro, these assemblies frequently undergo dynamic arrest, vitrifying into non-dynamic condensates. Here, we investigate how primary sequence features, post-translational modifications, and heterotypic interactions regulate the material properties of reflectin condensates within the environment of mammalian HeLa cells. Using confocal microscopy and fluorescence recovery after photobleaching (FRAP), we demonstrate that canonical block copolymeric A-type reflectins readily form dynamically arrested condensates, with the linker blocks primarily responsible for the observed arrest. In contrast, non-canonical B/C reflectin variants exhibit significantly greater fluidity and rapid recovery kinetics. We show that phosphomimetic substitutions progressively fluidize some reflectin condensates. Lastly, we find that heterotypic condensates composed of canonical and non-canonical reflectins in combinations associated with reversible iridescence in squid substantially enhance canonical mobility. Our findings establish a biophysical framework in which phosphorylation and heterotypic mixing cooperatively suppress dynamic arrest, enabling the reversible material transitions required for active cephalopod camouflage and communication.

biophysics↗

bioRxiv2026

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.

biophysics↗

bioRxiv2024

Charge screening and hydrophobicity drive progressive assembly and liquid-liquid phase separation of reflectin protein

The intrinsically disordered reflectin proteins drive tunable reflectivity for dynamic camouflage and communication in the recently evolved Loliginidae family of squid. Previous work revealed that reflectin A1 forms discrete assemblies whose size is precisely predicted by protein net charge density (NCD) and charge screening by the local anion concentration. Using dynamic light scattering (DLS), Forster resonant energy transfer (FRET) and confocal microscopy, we show that these assemblies, of which 95-99% of bulk protein in solution is partitioned into, are dynamic intermediates to liquid protein-dense condensates formed by liquid-liquid phase separation (LLPS). Increasing salt concentration drives this progression by anionic screening of the cationic proteins Coulombic repulsion, and by increasing the hydrophobic effect which tips the balance between short-range attraction and long-range repulsion (SALR) to drive protein assembly and ultimately LLPS. Measuring fluorescence recovery after photobleaching (FRAP) and droplet fusion dynamics, we demonstrate that reflectin diffusivity in condensates is tuned by salt concentration and protein NCD. These results illuminate the physical processes governing reflectin A1 assembly and LLPS, and demonstrate the potential for reflectin A1 condensate-based tunable biomaterials. They also compliment previous observations of liquid phase separation in the Bragg lamellae of activated iridocytes and suggest that LLPS behavior may serve a critical role in governing the tunable and reversible dehydration of the membrane-bounded Bragg lamellae and vesicles containing reflectin in biophotonically active cells.

biophysics↗

bioRxiv2021

Protein Charge Neutralization is the Proximate Driver Dynamically Tuning a Nanoscale Bragg Reflector

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, 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 its refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that 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 size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. X-ray scattering analyses confirm the sphericity, size and low polydispersity of the assemblies. Precise proportionality between assembly size and charge-neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth. The resulting stability of reflectin assemblies with time ensures reciprocally precise control of the particle number concentration, thereby encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The results presented here strongly suggest that it is charge neutralization, rather than any change in aromatic content, that is the proximate driver of assembly, fine-tuning a colligative property-based nanostructured biological machine. A physical mechanism is proposed.

molecular biology↗

bioRxiv2018

Neutralization of a Distributed Coulombic Switch Precisely Tunes Reflectin Assembly

Reflectin proteins are widely distributed in reflective structures in cephalopods, but only in Loliginid squids are they and the sub-wavelength photonic structures they control dynamically tunable, driving changes in skin color for camouflage and communication. The reflectins are block copolymers with repeated canonical domains interspersed with cationic linkers. Neurotransmitter-activated signal transduction culminates in catalytic phosphorylation of the tunable reflectins cationic linkers, with the resulting charge-neutralization overcoming Coulombic repulsion to progressively allow condensation and concommitant assembly to form multimeric spheres of tunable size. Structural transitions of reflectins A1 and A2 were analyzed by dynamic light scattering, transmission electron microscopy, solution small angle x-ray scattering, circular dichroism, atomic force microscopy, and fluorimetry. We analyzed the assembly behavior of phospho-mimetic, deletion, and other mutants in conjunction with pH-titration as an in vitro surrogate of phosphorylation to discover a predictive relationship between the extent of neutralization of the proteins net charge density and the size of resulting multimeric protein assemblies of narrow polydispersity. Comparison of mutants shows this sensitivity to neutralization resides in the linkers and is spatially distributed along the protein. These results are consistent with the behavior of a charge-stabilized colloidal system, while imaging of large particles, and analysis of sequence composition, suggest that assembly may proceed through a transient liquid-liquid phase separated intermediate. These results offer insights into the basis of reflectin-based tunable biophotonics and open new paths for the design of new reflectin mutants with tunable properties.

molecular biology↗