Introduction
Cephalopods such as squid and octopuses possess an optically dynamic epithelium, enabling complex camouflage and communication (
1Cephalopod dynamic camouflage.
,
2- Hanlon R.T.
- Chiao C.-C.
- Mäthger L.M.
- Barbosa A.
- Buresch K.C.
- Chubb C.
Cephalopod dynamic camouflage: bridging the continuum between background matching and disruptive coloration.
). In addition to pigmentary chromatophores, these animals possess reflective cells, leucophores and iridocytes, that act as structural reflectors through the interaction of light with their subwavelength nanostructures (
3- Kreit E.
- Mäthger L.M.
- Hanlon R.T.
- Dennis P.B.
- Naik R.R.
- Forsythe E.
- Heikenfeld J.
Biological versus electronic adaptive coloration: how can one inform the other?.
,
4- Mäthger L.M.
- Senft S.L.
- Gao M.
- Karaveli S.
- Bell G.R.R.
- Zia R.
- Kuzirian A.M.
- Dennis P.B.
- Crookes-Goodson W.J.
- Naik R.R.
- Kattawar G.W.
- Hanlon R.T.
Bright white scattering from protein spheres in color changing, flexible cuttlefish skin.
). Although leucophores are broadband scatterers of white light, iridocytes reflect specifically colored, iridescent light by angle- and wavelength-dependent constructive interference from intracellular Bragg reflectors. The lamellae of these reflectors are densely filled with cationic block copolymer-like proteins called reflectins and are separated from the low-refractive-index extracellular fluid by regular invaginations of the cell membrane (
5- Crookes W.J.
- Ding L.-L.
- Huang Q.L.
- Kimbell J.R.
- Horwitz J.
- McFall-Ngai M.J.
Reflectins: the unusual proteins of squid reflective tissues.
6- Izumi M.
- Sweeney A.M.
- Demartini D.
- Weaver J.C.
- Powers M.L.
- Tao A.
- Silvas T.V.
- Kramer R.M.
- Crookes-Goodson W.J.
- Mäthger L.M.
- Naik R.R.
- Hanlon R.T.
- Morse D.E.
Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.
,
7- Tao A.R.
- DeMartini D.G.
- Izumi M.
- Sweeney A.M.
- Holt A.L.
- Morse D.E.
The role of protein assembly in dynamically tunable bio-optical tissues.
,
8- DeMartini D.G.
- Krogstad D.V.
- Morse D.E.
Membrane invaginations facilitate reversible water flux driving tunable iridescence in a dynamic biophotonic system.
9- DeMartini D.G.
- Izumi M.
- Weaver A.T.
- Pandolfi E.
- Morse D.E.
Structures, organization, and function of reflectin proteins in dynamically tunable reflective cells.
).
Although the structural reflectors of the iridocytes and leucophores of most cephalopods are static, those in the Loliginidae squid family uniquely possess reversibly tunable versions of these reflectors (
10- Levenson R.
- DeMartini D.G.
- Morse D.E.
Molecular mechanism of reflectin's tunable biophotonic control: opportunities and limitations for new optoelectronics.
). Ultrastructural characterization of unactivated tunable iridocytes showed their intracellular lamellae to contain a heterogeneous mixture of discontinuous ∼10–20-nm nanoparticles and nanofibrils, suggesting that the cationic reflectins within exist in a predominantly unassembled state dominated by interparticle charge repulsion (
10- Levenson R.
- DeMartini D.G.
- Morse D.E.
Molecular mechanism of reflectin's tunable biophotonic control: opportunities and limitations for new optoelectronics.
,
11- Cooper K.M.
- Hanlon R.T.
- Budelmann B.U.
Physiological color change in squid iridophores. II. Ultrastructural mechanisms in Lolliguncula brevis.
). However, upon iridocyte activation initiated by binding of the neurotransmitter acetylcholine, released from nearby nerve cells, to cell-surface muscarinic receptors, a signal transduction cascade culminates in enzymatic phosphorylation of the reflectins, consequently neutralizing their cationic charge and driving assembly of the reflectins to form homogenous densely staining Bragg lamellae (
6- Izumi M.
- Sweeney A.M.
- Demartini D.
- Weaver J.C.
- Powers M.L.
- Tao A.
- Silvas T.V.
- Kramer R.M.
- Crookes-Goodson W.J.
- Mäthger L.M.
- Naik R.R.
- Hanlon R.T.
- Morse D.E.
Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.
,
8- DeMartini D.G.
- Krogstad D.V.
- Morse D.E.
Membrane invaginations facilitate reversible water flux driving tunable iridescence in a dynamic biophotonic system.
,
11- Cooper K.M.
- Hanlon R.T.
- Budelmann B.U.
Physiological color change in squid iridophores. II. Ultrastructural mechanisms in Lolliguncula brevis.
,
12Correlation of iridescence with changes in iridophore platelet ultrastructure in the squid Lolliguncula brevis.
). Measurements demonstrating the reversible efflux of D
2O and its reuptake revealed that condensation of the reflectins drives the expulsion of H
2O from the membrane-bounded lamellae, simultaneously increasing the refractive index contrast between the intracellular and extracellular layers of the Bragg reflector while shrinking their thickness and spacing, thus activating reflectance and progressively tuning the color of the reflected light across the visible spectrum (
7- Tao A.R.
- DeMartini D.G.
- Izumi M.
- Sweeney A.M.
- Holt A.L.
- Morse D.E.
The role of protein assembly in dynamically tunable bio-optical tissues.
,
8- DeMartini D.G.
- Krogstad D.V.
- Morse D.E.
Membrane invaginations facilitate reversible water flux driving tunable iridescence in a dynamic biophotonic system.
,
13- Ghoshal A.
- Demartini D.G.
- Eck E.
- Morse D.E.
Optical parameters of the tunable Bragg reflectors in squid.
,
14- Ghoshal A.
- DeMartini D.G.
- Eck E.
- Morse D.E.
Experimental determination of refractive index of condensed reflectin in squid iridocytes.
).
The role of the reflectins as tunable drivers of this biophotonic behavior has generated interest in understanding the principles underlying their responsiveness to signal-activated phosphorylation and their resulting changes in conformation and assembly. The reflectins are essentially block copolymers, composed of highly conserved reflectin domains (“reflectin motifs” (RMs))
2The abbreviations used are: RM
reflectin motif
DLS
dynamic light scattering
TEM
transmission EM
AFM
atomic force microscopy
L
linker
LLPS
liquid–liquid phase segregation.
interspersed with cationic linkers, as seen for reflectins A1 and A2 of the loliginid squid
Doryteuthis opalescens (see
Fig. 1A). The reflectins exhibit a unique amino acid composition with some heterogeneity across their sequences; they are highly enriched in methionine, arginine, and tyrosine residues but possess almost no (<1%) aliphatic residues (
5- Crookes W.J.
- Ding L.-L.
- Huang Q.L.
- Kimbell J.R.
- Horwitz J.
- McFall-Ngai M.J.
Reflectins: the unusual proteins of squid reflective tissues.
,
9- DeMartini D.G.
- Izumi M.
- Weaver A.T.
- Pandolfi E.
- Morse D.E.
Structures, organization, and function of reflectin proteins in dynamically tunable reflective cells.
,
10- Levenson R.
- DeMartini D.G.
- Morse D.E.
Molecular mechanism of reflectin's tunable biophotonic control: opportunities and limitations for new optoelectronics.
). The sequence composition of the reflectins suggests that attractive interactions are likely to be primarily driven by tyrosine–aromatic (π–π), arginine–tyrosine (cation–π), and methionine–tyrosine (sulfur–π) interactions, forming a complex interaction network of both intra- and interstrand noncovalent bonding (
10- Levenson R.
- DeMartini D.G.
- Morse D.E.
Molecular mechanism of reflectin's tunable biophotonic control: opportunities and limitations for new optoelectronics.
,
15- Vernon R.M.
- Chong P.A.
- Tsang B.
- Kim T.H.
- Bah A.
- Farber P.
- Lin H.
- Forman-Kay J.D.
π-π contacts are an overlooked protein feature relevant to phase separation.
).
Recently, we demonstrated an
in vitro assay that examined the assembly of purified recombinant
D. opalescens reflectins as a function of progressive neutralization, as a surrogate of
in vivo phosphorylation, through dilution of the H
2O-solubilized proteins into low-ionic-strength buffers of varying pH (
16- Levenson R.
- Bracken C.
- Bush N.
- Morse D.E.
Cyclable condensation and hierarchical assembly of metastable reflectin proteins, the drivers of tunable biophotonics.
). Analyzing the tunable reflectins and a variety of mutant derivatives, we now show
in vitro a predictive relationship between the extent of charge neutralization of the positively charged linker peptides and the size of the resulting assembled reflectin multimers. This discovery further elucidates the mechanistic origin of the synergistic effects of reflectin neutralization on the color and brightness of light reflected
in vivo. Mutational analyses reveal that the “switch” controlling the neutralization-dependent structural transitions underlying tunability is not localized but instead is spatially distributed in the multiple linkers along the reflectin's length.
Discussion
Reflectins assemble within cephalopod cells to form complex condensed nanostructures of high refractive index that produce diverse biophotonic effects (
5- Crookes W.J.
- Ding L.-L.
- Huang Q.L.
- Kimbell J.R.
- Horwitz J.
- McFall-Ngai M.J.
Reflectins: the unusual proteins of squid reflective tissues.
6- Izumi M.
- Sweeney A.M.
- Demartini D.
- Weaver J.C.
- Powers M.L.
- Tao A.
- Silvas T.V.
- Kramer R.M.
- Crookes-Goodson W.J.
- Mäthger L.M.
- Naik R.R.
- Hanlon R.T.
- Morse D.E.
Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.
,
7- Tao A.R.
- DeMartini D.G.
- Izumi M.
- Sweeney A.M.
- Holt A.L.
- Morse D.E.
The role of protein assembly in dynamically tunable bio-optical tissues.
,
8- DeMartini D.G.
- Krogstad D.V.
- Morse D.E.
Membrane invaginations facilitate reversible water flux driving tunable iridescence in a dynamic biophotonic system.
9- DeMartini D.G.
- Izumi M.
- Weaver A.T.
- Pandolfi E.
- Morse D.E.
Structures, organization, and function of reflectin proteins in dynamically tunable reflective cells.
,
14- Ghoshal A.
- DeMartini D.G.
- Eck E.
- Morse D.E.
Experimental determination of refractive index of condensed reflectin in squid iridocytes.
,
16- Levenson R.
- Bracken C.
- Bush N.
- Morse D.E.
Cyclable condensation and hierarchical assembly of metastable reflectin proteins, the drivers of tunable biophotonics.
). In most cephalopods, these structures are static, arising early during cellular development and remaining fixed for the lifetime of the cell (
23- Andouche A.
- Bassaglia Y.
- Baratte S.
- Bonnaud L.
Reflectin genes and development of iridophore patterns in Sepia officinalis embryos (Mollusca, Cephalopoda).
). However, within the loliginid squids, some iridocyte (iridescent/narrowband) and leucophore (broadband) cells demonstrate tunable, cyclable, acetylcholine-dependent reflectivity (
6- Izumi M.
- Sweeney A.M.
- Demartini D.
- Weaver J.C.
- Powers M.L.
- Tao A.
- Silvas T.V.
- Kramer R.M.
- Crookes-Goodson W.J.
- Mäthger L.M.
- Naik R.R.
- Hanlon R.T.
- Morse D.E.
Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.
,
24- DeMartini D.G.
- Ghoshal A.
- Pandolfi E.
- Weaver A.T.
- Baum M.
- Morse D.E.
Dynamic biophotonics: female squid exhibit sexually dimorphic tunable leucophores and iridocytes.
). Ultrastructural and immunohistochemical characterization of iridocyte Bragg lamellae shows that unactivated tunable iridocytes are filled with a heterogenous network of small reflectin nanoparticles (∼10–20 nm in diameter) and nanofibers (
7- Tao A.R.
- DeMartini D.G.
- Izumi M.
- Sweeney A.M.
- Holt A.L.
- Morse D.E.
The role of protein assembly in dynamically tunable bio-optical tissues.
,
9- DeMartini D.G.
- Izumi M.
- Weaver A.T.
- Pandolfi E.
- Morse D.E.
Structures, organization, and function of reflectin proteins in dynamically tunable reflective cells.
,
11- Cooper K.M.
- Hanlon R.T.
- Budelmann B.U.
Physiological color change in squid iridophores. II. Ultrastructural mechanisms in Lolliguncula brevis.
). Upon exposure to acetylcholine, phosphorylation of reflectin A1 and A2 drives neutralization and concomitant condensation and assembly, resulting in expulsion of water from the membrane-bounded Bragg lamellae, thus increasing the intralamellar protein density while shrinking the thickness and spacing of the lamellae. These physical changes increase the refractive index contrast between the reflectin-containing lamellae and the interspersed extracellular medium to activate reflectance while simultaneously tuning the reflected color (
6- Izumi M.
- Sweeney A.M.
- Demartini D.
- Weaver J.C.
- Powers M.L.
- Tao A.
- Silvas T.V.
- Kramer R.M.
- Crookes-Goodson W.J.
- Mäthger L.M.
- Naik R.R.
- Hanlon R.T.
- Morse D.E.
Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.
,
8- DeMartini D.G.
- Krogstad D.V.
- Morse D.E.
Membrane invaginations facilitate reversible water flux driving tunable iridescence in a dynamic biophotonic system.
,
9- DeMartini D.G.
- Izumi M.
- Weaver A.T.
- Pandolfi E.
- Morse D.E.
Structures, organization, and function of reflectin proteins in dynamically tunable reflective cells.
,
24- DeMartini D.G.
- Ghoshal A.
- Pandolfi E.
- Weaver A.T.
- Baum M.
- Morse D.E.
Dynamic biophotonics: female squid exhibit sexually dimorphic tunable leucophores and iridocytes.
). Inspired by this unique biological function, studies have investigated the assembly and resulting optical behavior of purified recombinant reflectin and reflectin-based peptides. Purified reflectins and their peptides have been processed into a variety of optically active materials, including thin films, gratings, and fibers (
18- Qin G.
- Dennis P.B.
- Zhang Y.
- Hu X.
- Bressner J.E.
- Sun Z.
- Crookes-Goodson W.J.
- Naik R.R.
- Omenetto F.G.
- Kaplan D.L.
Recombinant reflectin-based optical materials.
,
25- Kramer R.M.
- Crookes-Goodson W.J.
- Naik R.R.
The self-organizing properties of squid reflectin protein.
). These thin films have optical properties that are reversibly sensitive to both hydration and pH (
18- Qin G.
- Dennis P.B.
- Zhang Y.
- Hu X.
- Bressner J.E.
- Sun Z.
- Crookes-Goodson W.J.
- Naik R.R.
- Omenetto F.G.
- Kaplan D.L.
Recombinant reflectin-based optical materials.
,
19- Phan L.
- Walkup 4th, W.G.
- Ordinario D.D.
- Karshalev E.
- Jocson J.-M.
- Burke A.M.
- Gorodetsky A.A.
Reconfigurable infrared camouflage coatings from a cephalopod protein.
20- Naughton K.L.
- Phan L.
- Leung E.M.
- Kautz R.
- Lin Q.
- Van Dyke Y.
- Marmiroli B.
- Sartori B.
- Arvai A.
- Li S.
- Pique M.E.
- Naeim M.
- Kerr J.P.
- Aquino M.J.
- Roberts V.A.
- et al.
Self-assembly of the cephalopod protein reflectin.
,
25- Kramer R.M.
- Crookes-Goodson W.J.
- Naik R.R.
The self-organizing properties of squid reflectin protein.
26- Phan L.
- Ordinario D.D.
- Karshalev E.
- Iv W.G.W.
- Shenk M.A.
- Gorodetsky A.A.
Infrared invisibility stickers inspired by cephalopods.
,
27- Phan L.
- Kautz R.
- Leung E.M.
- Naughton K.L.
- Van Dyke Y.
- Gorodetsky A.A.
Dynamic materials inspired by cephalopods.
28- Dennis P.B.
- Singh K.M.
- Vasudev M.C.
- Naik R.R.
- Crookes-Goodson W.J.
Research update: a minimal region of squid reflectin for vapor-induced light scattering.
). In a nonoptical context, reflectin-based thin films have also been investigated as proton conductors and transistors as well as substrates for neural cell growth (
29- Ordinario D.D.
- Phan L.
- Walkup 4th, W.G.
- Jocson J.-M.
- Karshalev E.
- Hüsken N.
- Gorodetsky A.A.
Bulk protonic conductivity in a cephalopod structural protein.
30- Ordinario D.D.
- Phan L.
- Iv W.G.W.
- Dyke Y.V.
- Leung E.M.
- Nguyen M.
- Smith A.G.
- Kerr J.
- Naeim M.
- Kymissis I.
- Gorodetsky A.A.
Production and electrical characterization of the reflectin A2 isoform from Doryteuthis (Loligo) pealeii.
,
31- Ordinario D.D.
- Phan L.
- Van Dyke Y.
- Nguyen T.
- Smith A.G.
- Nguyen M.
- Mofid N.M.
- Dao M.K.
- Gorodetsky A.A.
Photochemical doping of protonic transistors from a cephalopod protein.
32- Phan L.
- Kautz R.
- Arulmoli J.
- Kim I.H.
- Le D.T.
- Shenk M.A.
- Pathak M.M.
- Flanagan L.A.
- Tombola F.
- Gorodetsky A.A.
Reflectin as a material for neural stem cell growth.
).
Starting from the highly disordered, H
2O-solubilized reflectin monomers, we found that progressive neutralization drives the assembly of a collection of
D. opalescens A1 WT and mutationally altered variants, with the resulting multimeric assembly sizes being predictively tuned by the normalized net charge density of the spatially distributed, cationic linkers that act as an electrostatic sensor and switch. Assembly size is indifferent to the mode of charge neutralization (whether by genetic alteration, pH titration, or both, acting as surrogates of phosphorylation
in vivo) and indifferent to the location of the cationic linkers remaining after deletion. TEM analyses reveal the multimeric assemblies to be spherical with small size variation. Assembly sizes measured by DLS and TEM agree closely with each other as well as with small angle X-ray scattering measurements of assemblies.
3R. Levenson, P. Kohl, Y. Li, and D. Morse, unpublished data.
The consistency of spherical morphology and internal structure (indicated by tryptophan fluorescence) between WT and mutant reflectins indicates that the assembly of these mutant reflectins is not the result of aberrant or off-pathway aggregation but lies on the common continuum, as also suggested by the colinearity of data for the mutants and WT (
Figure 3,
Figure 4). Coulombic repulsion presumably contributes to maintenance of the positively charged reflectins in an extended, disordered, and monomeric state, with progressive neutralization progressively overcoming that repulsion to permit condensation and assembly. The strong predictive relationship observed between the net charge density of the cationic linkers and reflectin's final assembly size indicates that electrostatic interactions in some way limit that size.
To undergo assembly, the disordered reflectin monomers must pass through a net charge neutralization threshold, at which point they form multimers exhibiting an apparently exponential relationship between assembly size and calculated net charge density. Size control of reflectin assembly, tuned by changes in histidine protonation within the pH range tested here, is governed by a spatially distributed switch spread across the reflectin linkers. This behavior is consistent with the location of the previously identified sites of
in vivo phosphorylation, almost exclusively found within the cationic linkers (
6- Izumi M.
- Sweeney A.M.
- Demartini D.
- Weaver J.C.
- Powers M.L.
- Tao A.
- Silvas T.V.
- Kramer R.M.
- Crookes-Goodson W.J.
- Mäthger L.M.
- Naik R.R.
- Hanlon R.T.
- Morse D.E.
Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.
,
9- DeMartini D.G.
- Izumi M.
- Weaver A.T.
- Pandolfi E.
- Morse D.E.
Structures, organization, and function of reflectin proteins in dynamically tunable reflective cells.
). The tunable and reversible assembly of the reflectin proteins resembles that of the intrinsically disordered amelogenin proteins, which have been observed to form monomers, oligomers, and larger nanoparticle multimers in a pH-controlled manner and for which oligomers are thought to serve as the building blocks for larger nanoparticles (
33- Margolis H.C.
- Beniash E.
- Fowler C.E.
Role of macromolecular assembly of enamel matrix proteins in enamel formation.
,
34- Bromley K.M.
- Kiss A.S.
- Lokappa S.B.
- Lakshminarayanan R.
- Fan D.
- Ndao M.
- Evans J.S.
- Moradian-Oldak J.
Dissecting amelogenin protein nanospheres.
). However, unlike the amelogenins, the reflectins form assemblies of tunable size with low polydispersity as a function of neutralization, suggesting that some aspects of the unique reflectin amino acid composition and sequence enable a finer degree of control and tunability.
Comparison of
D. opalescens reflectin A1 with similarly sized representative reflectins from other cephalopods that have nontunable iridescence shows that the N-terminal half of the reflectins generally possesses greater conservation (∼50%) than the C-terminal half (∼30%) (
Fig. 7A). Interestingly, the N-terminal region, L
N, possesses a well-conserved, strongly cationic character across all reflectins, being composed of ∼19–26% arginine compared with 11–12% arginine in the reflectins' full sequences overall. The conserved cationic character of L
N, combined with our finding that the ΔL
NRM
N mutant forms large assemblies, suggests that this linker may promote solubility during reflectin translation by inhibiting potential off-pathway aggregation through charge repulsion. Overall, our results show that linker electrostatics control reflectin condensation and assembly, consistent with the
in vivo regulation by phosphorylation. In these respects, the behavior of the reflectins is consistent with that of a charge-stabilized colloidal system, although additional complexity is apparent (
35- Klein R.
- von Grünberg H.H.
Charge-stabilized colloidal suspensions. Phase behavior and effects of confinement.
,
36- Hierrezuelo J.
- Sadeghpour A.
- Szilagyi I.
- Vaccaro A.
- Borkovec M.
Electrostatic stabilization of charged colloidal particles with adsorbed polyelectrolytes of opposite charge.
).
Reflectins are highly enriched in conserved arginine (11%), tyrosine (20%), and methionine (15%) residues and relatively deficient in lysines and residues with simple aliphatic side chains (<1%). Enrichment in these residues is associated with the liquid–liquid phase segregation (LLPS) of multivalent, intrinsically disordered proteins that form biomolecular condensates, also known as coacervates, mediated through the formation of extensive webbed networks of relatively weak, short-range cation–π, sulfur–π, and π–π associative interactions (
37- Brangwynne C.P.
- Tompa P.
- Pappu R.V.
Polymer physics of intracellular phase transitions.
,
38Liquid phase condensation in cell physiology and disease.
39- Banani S.F.
- Lee H.O.
- Hyman A.A.
- Rosen M.K.
Biomolecular condensates: organizers of cellular biochemistry.
). Arginine and tyrosine residues, abundant in the reflectins, have been recognized as particularly strong drivers of protein LLPS, often paired with glycine or serine (
39- Banani S.F.
- Lee H.O.
- Hyman A.A.
- Rosen M.K.
Biomolecular condensates: organizers of cellular biochemistry.
40- Qamar S.
- Wang G.
- Randle S.J.
- Ruggeri F.S.
- Varela J.A.
- Lin J.Q.
- Phillips E.C.
- Miyashita A.
- Williams D.
- Ströhl F.
- Meadows W.
- Ferry R.
- Dardov V.J.
- Tartaglia G.G.
- Farrer L.A.
- et al.
FUS phase separation Is modulated by a molecular chaperone and methylation of arginine cation-π interactions.
,
41- Thandapani P.
- O'Connor T.R.
- Bailey T.L.
- Richard S.
Defining the RGG/RG motif.
,
42- Chong P.A.
- Vernon R.M.
- Forman-Kay J.D.
RGG/RG motif regions in RNA binding and phase separation.
43- Lin Y.
- Currie S.L.
- Rosen M.K.
Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs.
). An analysis of reflectin proteins from diverse cephalopods for di- and tripeptide sequence motifs associated with LLPS reveals a high occurrence of sequentially biased Arg-Gly, Tyr-Gly, and Tyr-Ser dipeptide sequences (
Fig. 7,
A and
B), comprising ∼20% of the entire reflectin sequence (
40- Qamar S.
- Wang G.
- Randle S.J.
- Ruggeri F.S.
- Varela J.A.
- Lin J.Q.
- Phillips E.C.
- Miyashita A.
- Williams D.
- Ströhl F.
- Meadows W.
- Ferry R.
- Dardov V.J.
- Tartaglia G.G.
- Farrer L.A.
- et al.
FUS phase separation Is modulated by a molecular chaperone and methylation of arginine cation-π interactions.
,
42- Chong P.A.
- Vernon R.M.
- Forman-Kay J.D.
RGG/RG motif regions in RNA binding and phase separation.
,
43- Lin Y.
- Currie S.L.
- Rosen M.K.
Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs.
44Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity.
). Notably, these dipeptide motifs are substantially clustered together to from a significant number of Tyr-Gly-Arg and Gly-Arg-Tyr tripeptides (
Fig. 7,
A–C). Given their high frequency and conservation in the reflectins, we suggest that these di- and tripeptides may play a critical role in driving reflectin LLPS. Based on the distribution of these associative peptide motifs throughout the reflectins, it is likely that both the conserved domains and the linkers robustly and directly participate in noncovalent bond formation during neutralization-triggered condensation.
Although the sphericity of the large reflectin A1 assemblies formed
in vitro also is consistent with the suggestion that a transient phase-segregated liquid state may be formed upon sufficient charge neutralization and subsequent formation of reflectin condensates, the stability of reflectin assemblies and absence of coalescence and macroscopic phase separation (as observed by DLS and microscopy) indicate that these particles do not persist in this state of low interfacial energy but apparently undergo rapid dynamic arrest of further assembly, preventing further coalescence or phase separation (
16- Levenson R.
- Bracken C.
- Bush N.
- Morse D.E.
Cyclable condensation and hierarchical assembly of metastable reflectin proteins, the drivers of tunable biophotonics.
,
38Liquid phase condensation in cell physiology and disease.
,
45- Woodruff J.B.
- Hyman A.A.
- Boke E.
Organization and function of non-dynamic biomolecular condensates.
). Such arrest (variously also described as hardening, gelation, solidification, or vitrification) of LLPS condensates has been widely observed in other systems and in several cases is associated with pathology; in some instances, it is accompanied by deformations of the initially spherical assemblies (
38Liquid phase condensation in cell physiology and disease.
,
45- Woodruff J.B.
- Hyman A.A.
- Boke E.
Organization and function of non-dynamic biomolecular condensates.
). We have observed such gel-like deformation with compaction of reflectin assemblies (
Fig. 5F). Formally, dynamic arrest of assembly is determined by the balance of short-range (weak) attractive forces and long-range (strong) repulsive forces, as well-understood for many colloidal systems. In the case of reflectin assembly, the weak attractive forces potentially include a combination of hydrogen and hydrophobic bonding, β-stacking, coil–coil interactions, cation–π, sulfur–π, π–π, van der Waals and other forms of noncovalent bonding, whereas coulombic repulsion, which we have experimentally tuned here by pH titration of histidine and by mutation, likely comprises the dominant counteracting repulsive interaction.
It is interesting that it is the dynamic arrest of reflectin assembly that is responsible for the precise and finely tunable relationship between the extent of charge neutralization and the resulting size and number of reflectin assemblies. This dynamic arrest of neutralization-driven assembly is thus in turn responsible for the precise control of the colligatively triggered osmotic motor that proportionally drives the cyclable dehydration of the membrane-bounded subcellular Bragg lamellae containing the reflectins, proportionally and simultaneously increasing refractive index and shrinking the dimensions of the lamellae to tune the reflected light (
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Membrane invaginations facilitate reversible water flux driving tunable iridescence in a dynamic biophotonic system.
). The precise relationship between the extent of charge neutralization and size of reflectin assembly (with its reciprocal control of particle number concentration), followed by rapid dynamic arrest as described here, thus ensures a precise calibration between the triggering neuronal signal (and its consequent phosphorylation of reflectin) and the resulting change in color in squid skin. The reflectin's unique sequence composition, rich in aromatic residues and sulfur-containing methionine, apparently enables a further synergistic enhancement of this photonic tuning, providing the reflectins with one of the highest known incremental refractive indices (d
n/d
c) and thus allowing the reflectins to generate higher-refractive-index contrast with the extracellular fluid in the physiological Bragg reflectors while also driving their tunable assembly (
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Molecular mechanism of reflectin's tunable biophotonic control: opportunities and limitations for new optoelectronics.
,
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On the distribution of protein refractive index increments.
).
Genomic sequencing of cephalopods is now revealing an ever-increasing family of unique reflectin sequences, suggesting that a rich network of inter-reflectin interactions may occur within cephalopod Bragg reflectors (
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Molecular mechanism of reflectin's tunable biophotonic control: opportunities and limitations for new optoelectronics.
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The octopus genome and the evolution of cephalopod neural and morphological novelties.
). The results reported here highlight the unique properties of the tunable reflectins that enable them to function as a finely tuned molecular machine that precisely regulates an osmotic motor to mechanically tune the color and intensity of light reflected from intracellular nanophotonic structures. Extending the reflectin-based thin-film applications described above (
18- Qin G.
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Research update: a minimal region of squid reflectin for vapor-induced light scattering.
), the unique structure–function relationships of the highly evolved tunable reflectins may provide inspiration and guidance for future classes of tunably reconfigurable optical and other materials, open further possibilities for exploration of the design principles underlying reflectin assembly, and suggest new pathways toward the rational design of tunable assembly (
48- Dzuricky M.
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).
Article info
Publication history
Published online: September 26, 2019
Received in revised form:
September 14,
2019
Received:
July 31,
2019
Edited by Joseph M. Jez
Footnotes
This work was supported by United States Department of Energy, Basic Energy Sciences Grant DE-SC0015472; the Institute for Collaborative Biotechnologies through Grant W911NF-09-0001 from the United States Army Research Office; and Army Research Office Grant W911NF-17-1-0160. The authors declare that they have no conflicts of interest with the contents of this article.
This article contains Figs. S1–S9.
Copyright
© 2019 Levenson et al.