Dynamic pigmentary and structural coloration within cephalopod chromatophore organs

Abstract

Chromatophore organs in cephalopod skin are known to produce ultra-fast changes in appearance for camouflage and communication. Light-scattering pigment granules within chromatocytes have been presumed to be the sole source of coloration in these complex organs. We report the discovery of structural coloration emanating in precise register with expanded pigmented chromatocytes. Concurrently, using an annotated squid chromatophore proteome together with microscopy, we identify a likely biochemical component of this reflective coloration as reflectin proteins distributed in sheath cells that envelop each chromatocyte. Additionally, within the chromatocytes, where the pigment resides in nanostructured granules, we find the lens protein Ω- crystallin interfacing tightly with pigment molecules. These findings offer fresh perspectives on the intricate biophotonic interplay between pigmentary and structural coloration elements tightly co-located within the same dynamic flexible organ - a feature that may help inspire the development of new classes of engineered materials that change color and pattern.

Fig. 1

Iridescence produced in chromatophores of live squid. a Low power of mantle skin; arrows indicate narrow zones of iridescence that coincide with yellow chromatocytes (white arrows) and typical iridophores (green arrows). Scale bar is 3 mm. b, c Arrows show yellow chromatophores under different lighting angles expressing different iridescence. Scale bars are 600 µm. d Yellow chromatophores showing iridescence that coincides with the exact expansion of the chromatocyte (arrow shows concentric variation in hue seen frequently). Scale bar is 300 µm. e Contrasting iridescence from a typical subjacent iridophore (green arrow) compared to a yellow chromatophore (white arrow). Scale bar is 1 mm. f A single yellow chromatophore showing granular and patchy iridescence. Scale bar is 100 µm

Fig. 2

Specular reflection from chromatophores. Still images collected from dynamic Movies of intact adult squid skin through a dissecting microscope using a movable light-pipe illuminator showing that specular reflection from chromatophores requires both specific illumination conditions and expansion of the chromatophores. a A subset of chromatophores (white dotted circles) in expanded configurations with off-axis illumination, where perceived color is attributed to pigment absorption and scattering; and b moments later, the same expanded chromatophores showing strong specular reflections (interpreted as structural coloration) that are seen restricted precisely to regions within each chromatophore. Only the angle of illumination was changed. When the same chromatophores are captured before c and after d actuation (black dotted circles), coloration varies depending on the angle of illumination, where there is intense specular reflectance for the expanded chromatophores but not for the compacted ones. (Movie frame time points all in seconds are as follows: A = 35 and B = 38, C = 4, and D = 10). Scale bars are 1 mm

Fig. 3

Dynamic visible color emanating from a single chromatophore over time Consecutive Movie frames (a–i) taken from Supplementary Movie 2 (7.60–7.87 s, where each frame represents 0.03 s) showing the relaxation of a yellow chromatophore in the lower right-hand side. The white outline in a (20 × 20 pixel square) shows the location of the selected area used to measure RGB profiles in each frame. These frames were selected because they encompass the visible spectrum, giving unique red, green, and blue (RGB) values during the actuation cycle. j Using ImageJ software, the RGB values over the selected area were measured, averaged, and plotted from each frame, Here, the line graph shows the changes in RGB value intensities over time with the red triangles anchored by solid red lines representing red values, the green circles anchored by dashed green lines representing green values, and the blue squares anchored by dotted blue lines representing blue values. The changing RGB values shown here comprise a much wider range of colors than previously attributable to absorbance by any yellow chromatophores and constitute strong argument that dynamic structural color mechanisms must be present

Fig. 4

Spectrometric analysis of chromatophore proteins. a Enzymatically isolated squid dorsal mantle chromatophores, retaining the plasma membrane of the variously colored chromatocytes and their surrounding sheath cells (sc). Scale bar is 6.5 µm. b Proteins identified and categorized by function in the yellow, red, and brown pigment cells in squid D. pealeii dorsal and ventral mantle. c Typical morphological arrangement of chromatophores in squid skin; note the radial muscles visible around the central retracted brown chromatophore. Scale bar is 500 µm. d Categorized proteins identified within the squid chromatophore granules, pigment-extracted granules (e.g., the granule shell), and the extracted pigment along with representative SEMs (scale bar = 500 nm) and optical image of extracted pigment

Fig. 5

Anatomical localization of reflectin to sheath cells. a Reflectin (confocal, secondary antibody in green, arrows) was present surrounding the edge of the chromatocyte and the apical portions of the radial muscles (M). Scale bar is 50 µm; b Reflectin (confocal, dual secondary antibodies in lavender; 405 nm and 568 nm fluorophores were applied simultaneously to reduce the potential ambiguity associated with autofluorescence of the tissue) often extended into the spaces between adjacent apical radial muscles. Scale bar is 25 µm; c Reflectin distribution was frequently punctate, and in addition to coating the muscles and the edge of the chromatocyte, reflectin was also present over its surface. Note that the chromatocyte section was very thin (in comparison with the confocal depth of field of the 0.45 NA x20 objective used here), but the label consistently showed on its outside, rather than within its sacculus. Scale bar is 25 µm; d Confocal section of a chromatophore imaged by autofluorescence with the sheath cells highlighted in yellow for better visualization (scale bar is 50 µm). Sheath cells completely enveloped the pigment sac in all dimensions, as illustrated in the electron micrograph in e; scale bar is 12 µm

Fig. 6

Binding poses elucidated from molecular docking. a, b Most energetically favorable binding poses from flexible molecular docking of Xa in two of the top-ranked binding pockets in crystallin. In each panel, the figures on the left show the orientation and size of the pigment molecule (space-filling beads) within the binding pockets of the respective proteins (cartoons colored by secondary structure). The inset provides closeup views of the molecule within the pockets. The figures on the right are schematics of the pigment molecule interacting with the amino acid sidechains in the respective pockets. Dotted lines indicate polar interactions between acceptor–donor pairs. Eyelashes (in red) denote sidechains and atoms that are in contact with each other. Solid circles represent carbon (black), oxygen (red), nitrogen (blue), and sulfur (yellow). c Top and side views of the five top-ranked poses of Xa binding from rigid docking (space-filling beads) within the central cavity of the crystallin tetramer (cartoon colored by chain number), where the five molecules are in disparate regions of the cavity. Inset provides a closeup view of these poses. Top and side views of the tetramer’s molecular electrostatic potential map are shown at the bottom

Fig. 7

pH-dependent color change of compound Xa. a Variations in absorptive behaviors of Xa associated with an increasing pH. b A bright-field image of the two visible colors displayed by compound Xa at high (>5.00) and low (<5.00) pH values. c Proposed molecular structures of Xa and their corresponding absorption spectra calculated under neutral (designated with a neutral charge, black line) and acidic (designated with a + 3 charge, blue line) conditions. d The absorbance intensity (collected at 430 nm) associated with varying the pH in the synthetic Xa compared to granules and the pigment-extracted from the granules (squid Xa)

Fig. 8

Predicted optical effects within and around the chromatophore organs. The associated optical phenomena proposed based on our current and previous (see ref. ) findings are represented as a back scatter, b refraction, c forward scatter, d absorption (of non-yellow wavelengths), e multilayer interference, and f diffuse scattering, where g represents the radial muscle fibers; h represents the sheath cells; i represents the cytoplasm of the sheath cells; j represents individual granules; k represents the a collection of granules within the yellow chromatocyte; and l represents an iridophore, deeper in the dermis. For simplicity, only yellow colored chromatophores are illustrated here