Background to the project

How to disappear completely (or stand out in a crowd) - Reflectins in cephalopods

The beautiful optical properties which reflectin makes possible are used for different purposes in nature. Manipulation of reflectance may allow squid to communicate through polarised light. Altering refractive index to match that of the water column allows cephalopods to hide from their predators - a living invisibility cloak.

Reflectin was first identified as such in the Hawaiian bobtail squid Euprymna scolopes as the protein responsible for a reflective layer in the "light organ". This allows light emitted by symbiotic bacteria to be reflected downwards away from the squid, like a car headlamp.

The Longfin inshore squid Loligo pealei shows dynamic iridescence controlled through signals from the nervous system which turn on a protein kinase, an enzyme which adds negatively charged phosphate groups to the positively charged protein. This alters the attraction/repulsion between the platelet arrangement of reflectins, changing the spacing of the iridophore layers and therefore the colour of light reflected. (Read more about structural colour)

Squid skin contains multiple specialised cell types designed to allow highly controlled changes of colour and reflectance to optimise camouflage. The beautiful optical properties in light reflecting tissues occur due to a hierarchy of structural arrangements - the structure of reflectin protein itself, the complex platelets which the protein forms, and the shape and layering of reflective cells. A thin tissue layer made up of iridocytes close to the surface of squid skin is made up of ~40% reflectin (out of the total protein content). ² Within iridocytes, reflectins self assemble to form membrane associated platelets. The changes in refractive index as light moves through the layers of reflectin and cytoplasm forms a natural 'Bragg reflector'^[3].

Iridophores which appear similar in structure to those found in cephalopods are also seen in other members of mollusc phylum - giant clams^[4]. They are responsible for the stunning iridescent colours of the mantle, and may play a role in protecting against harmful UV and maximising capture of sunlight for the photosynthetic symbionts which live alongside the clam.

Reflectins as novel polymers

Reflectins have unique properties in part due to their unique amino acid composition - residues which are normally common in proteins (alanine, isoleucine, leucine and lysine) are nowhere to be seen in any reflectins identified so far, whilst typically rare residues (arginine, methionine, tryptophan and tyrosine) make up ~57% of the protein. The family of reflectin proteins share a repeated domain which may also possess unique optical properties.

The 3D protein structure of reflectin has not yet been characterised. It may be natively unstructured - Weiss et al hypothesised that it may form a beta barrel-like structure when interacting with membranes, as recombinant reflectin-like proteins associated strongly with artificial membrane structures after cell-free expression.

Kramer et al and Tao and DeMartini et al have demonstrated the remarkable capability of reflectin proteins to self assemble in vitro to create complex structures. Recombinant reflectin, refolded in vitro, can be carefully spread along a silicon slide to make thin films with intense structural colours from thin film interference. Measuring the refractive index of this in vitro arrangement of the protein reveals that it possesses the highest refractive index of any known protein. Kramer et al also demonstrated the ability of reflectin to form a diffraction grating when the ionic solvents used to dissolve it were diffused away in a water bath. This ultrastructural arrangement showed iridescence.

Light and interference - The physics behind structural colour

Structural colour is a common occurrence in nature - butterfly wings, fish scales and even layer which makes cats's eyes shine at night contain light reflecting components rather than pigments. It occurs due to the phenomenon of thin film interference.

The difference in refractive index between a thin film and the substance above and below it leads to some light being reflected (bouncing off) and some passing through the top surface of the film. When the light which passes through hits the bottom boundary of the film, again some will be reflected. When the two light waves meet, they will no longer be 'in sync' and interference will occur. Some wavelengths of light will have destructive interference and be removed from the white light, so not all colours in white light will be reflected from the surface, giving effects like the rainbow colours reflected by oil droplets on the surface of water.

As the viewing angle or the angle of incidence of light is varied the colour seen will change, giving an iridescent effect. Structural colour can also be altered by changing the spacing of the layers with different refractive indices as this will change the peak wavelengths where constructive and destructive interference occur. This is believed to be the principle by which colour is altered in the skin of squid - the thickness of and spacing between reflectin layers has been observed to change in vivo due to post translational modification.

References

[1] Kramer et al. The self-organizing properties of squid reflectin protein Nature Materials 533-538 VOL6 JULY 2007

[2] Crookes et al. Reflectins: The Unusual Proteins of Squid Reflective Tissues SCIENCE 235-238 VOL303 9 JANUARY 2004

[3] Morse et al. The role of protein assembly in dynamically tunable bio-optical tissues Biomaterials 793-801 VOL31 FEBRUARY 2010

[4]Iridophores in the mantle of giant clams