The role of muscarinic receptors and intracellular Ca2+ in the spectral reflectivity changes of squid iridophores

Abstract

SUMMARYIn this paper we describe changes in spectral reflectivity of the light reflectors (iridophores) of the squid Alloteuthis subulata. The spectral changes that can be seen in living squid, can also be brought about by superfusing whole skin preparations with acetylcholine (ACh) (20 μmol l-1) and muscarine (30 μmol l-1) but not nicotine (up to 50 mmol l-1), suggesting that cholinergic muscarinic receptors are involved. Changing the osmolarity of the external solution had no effect on spectral reflectivity. To study the iridophores at the cellular level,iridophores were isolated enzymatically. Lucifer Yellow filled the iridophores uniformly, showing cellular individuality. Isolated iridophore cells were loaded with Fura-2 AM and cytoplasmic Ca2+ was recorded ratiometrically. Intracellular Ca2+ (resting concentration at 66.16 nmol l-1) increased transiently after addition of ACh (50 μmol l-1), muscarine (25 μmol l-1), but not nicotine (up to 5 mmol l-1). Ca2+ also increased when superfused with potassium chloride (10 mmol l-1) and caffeine (2.5 mmol l-1). Hypo- and hyperosmotic solutions had no effects on the cytoplasmic Ca2+. By presenting direct evidence that iridophores are polarised cellular structures containing Ca2+ stores and that they are activated via cholinergic muscarinic receptors, we demonstrate that Ca2+ is involved in the reflectivity changes of the iridophores of A. subulata. Specimens were prepared for transmission electron microscopy. It was found that the orientations of the plates with respect to the skin surface are in good agreement with the expected orientations based on the prediction that the iridophores act as multilayer reflectors.

Membrane invaginations facilitate reversible water flux driving tunable iridescence in a dynamic biophotonic system

“…In this case, we observed significant water expulsion from the dorsal iridocytes, whereas the ventral iridocytes showed relatively poor response compared with the control. Correspondingly, the dorsal iridocytes displayed the most significant photonic response, changing color from the initial nonreflecting state to red and then progressively to blue, whereas the ventral cells showed only small changes (7,21,26). Several important assumptions and approximations are made in analyzing the water exchange experiments of this study.…”

“…We began by isolating single adaptive iridocytes from the squid, Doryteuthis opalescens, by careful dissection and enzymatic digestion to liberate the tunably reflective cells from the surrounding connective tissue (Fig. 1B); methods are similar to those reported for the related squid, Alloteuthis subulata (21). The isolated iridocyte cells were confirmed to be intact via the exclusion of trypan blue and propidium iodide.…”

“…The color changing ability of these squid cells is much more pronounced in the dorsal iridocytes, which can change sequentially from nonreflecting to red, then green, and finally blue. The ventral cells have a much weaker response, showing only minor changes, if any at all (7,21). Regularity of the membrane folding is more clearly seen in iridocytes from the ventral dermis than from the dorsal dermis because the platelets of the ventral iridocytes are oriented perpendicular to the flat plane of the cells (21), have greater thicknesses and spacing, and are less convoluted ( Fig.…”

“…4): ACh is known to bind a muscarinic type G protein-coupled receptor (7). The G protein is then released and activates the signaling cascade (as confirmed by stimulation of brightness and color with cholera toxin) (13), resulting in calcium release from the endoplasmic reticulum (21). Ca 2+ acts as a second messenger, binding calmodulin (24) and activating protein kinases and phosphatases (29).…”

“…Similar membrane invagination/lamination morphology has been observed in photophore (i.e., bioluminescent organ) iridocytes of Pterygioteuthis microlampas (17) and the dermal iridocytes of Octopus dolfini (16) (but here, the Bragg stacks are arranged outside the cell body). It has been unclear, however, whether these unusual invaginations are artifactual (18), and recent articles have suggested that the low refractive index space of the tunable iridocytes may instead be wholly intracellular (19)(20)(21). Although TEM analyses have provided great insights, efforts to extrapolate a 3D model from such 2D images have been unsuccessful (18), and a full understanding of the tunable reflective structure through optical microscopy and TEM alone has remained elusive (15,18,22).…”