Experimental determination of refractive index of condensed reflectin in squid iridocytes

Abstract

Loliginid squid dynamically tune the structural iridescence of cells in their skin for active camouflage and communication. Bragg reflectors in these cells consist of membrane-bound lamellae periodically alternating with low refractive index extracellular spaces; neuronal signalling induces condensation of the reflectin proteins that fill the lamellae, consequently triggering the expulsion of water. This causes an increase in refractive index within the lamellae, activating reflectance, with the change in lamellar thickness and spacing progressively shifting the wavelength of reflected light. We used microspectrophotometry to measure the functionally relevant refractive index of the high-index lamellae of the Bragg reflectors containing the condensed reflectins in chemically fixed dermal iridocytes of the squid, Doryteuthis opalescens. Our high-magnification imaging spectrometer allowed us to obtain normalized spectra of optically distinct sections of the individual, subcellular, multi-layer Bragg stacks. Replacement of the extracellular fluid with liquids of increasing refractive index allowed us to measure the reflectivity of the Bragg stacks as it decreased progressively to 0 when the refractive index of the extracellular medium exactly matched that of the reflectin-filled lamellae, thus allowing us to directly measure the refractive index of the reflectin-filled lamellae as n condensed lamellae % 1.44. The measured value of the physiologically relevant n condensed lamellae from these bright iridocytes falls within the range of values that we recently determined by an independent optical method and is significantly lower than values previously reported for dehydrated and air-dried reflectin films. We propose that this directly measured value for the refractive index of the squid's Bragg lamellae containing the condensed reflectins is most appropriate for calculations of reflectivity in similar reflectin-based high-index layers in other molluscs.

Cyclable Condensation and Hierarchical Assembly of Metastable Reflectin Proteins, the Drivers of Tunable Biophotonics

“…We hypothesized that the reflectins are therefore in a state of Coulombic repulsion in the non-reflective state, with phosphorylation overcoming this repulsion, allowing the reflectins to condense and assemble. This explanation is consistent with the electron micrographic images first obtained by Hanlon and coworkers (12), showing that ACh activation of reflectance proceeds with condensation of the intralamellar proteins and subsequent shrinkage of the Bragg lamellae and confirmed by our subsequent microspectrophotometric analyses showing that the refractive index inside the reflectin-containing Bragg lamellae increases progressively, therefore activating and progressively increasing the intensity of reflectance at each of the membrane-bound interfaces with the extralamellar spaces (13,14). …”

Neutralization of a Distributed Coulombic Switch Precisely Tunes Reflectin Assembly

“…Reflectins assemble within cephalopod cells to form complex condensed nanostructures of high refractive index that produce diverse biophotonic effects [4][5][6][7][8]12,14 . In most cephalopods these structures are static, arising early during cellular development and remaining fixed for the lifetime of the cell 27 .…”

“…However, upon iridocyte activation initiated by binding of the neurotransmitter acetylcholine (ACh), 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 homogenously densely staining Bragg lamellae 5,7,10 . Measurements demonstrating the reversible efflux of D 2 O and its re-uptake revealed that condensation of the reflectins drives the expulsion of H 2 O 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 6,7,11,12 .…”

Photosymbiotic giant clams are transformers of solar flux

“…Therefore, for our DDA calculation, we simulated a single iridocyte by building a grid of one million dipoles into 50 alternating high-index (n ¼ 1.55, densely packed protein) and low-index (n ¼ 1.33, water) layers, each 100 nm thick, with less than 1% variance, within a 5-mm figure S4). The most accurately measured values of the high-refractive index platelets in protein-based iridocytes vary from 1.44 to 1.55 [17][18][19]. We chose the high end of this possible index range due to the extremely densely packed protein platelets we observed in TEM.…”