1. Identification of the Muscarinic Acetylcholine Receptor in D. opalescens
Specialized skin cells in photonic tissue of the squid, Doryteuthis opalescens, create
dynamically tunable camouflage. The first cell type responsible for these abilities are iridocytes,
which use tunable intracellular Bragg reflectors to produce iridescent colors through constructive
interference of reflected light. The reflector stack is formed by the unique structure of the
extracellular membrane, which repeatedly invaginates to form pleats of high refractive index
intracellular lamellae filled with reflectin proteins alternating with low refractive index
extracellular space filled with seawater (1, 2). Switchable leucophores, the other cell type, are
found exclusively in female squid and contain intracellular vesicles called leucosomes that
contain high refractive index reflectins; these vesicles scatter light of all visible wavelengths to
produce a white color (3). Both types of cells are hypothesized to contain muscarinic
acetylcholine receptors (mAChRs) that are activated by the neurotransmitter acetylcholine (ACh)
(2). These G protein coupled receptors convert external ACh stimuli into intracellular signals.
When activated, these receptors trigger a signal transduction cascade involving a heterotrimeric
G-protein subunit (Gαq) and Phospholipase C (PLC-4), ultimately causing a release of calcium
from the endoplasmic reticulum. Calcium then binds calmodulin (CALM), activating protein
kinases and phosphatases, subsequently changing phosphorylation of the cationic reflectin
proteins, causing them to overcome their Coulombic repulsion and condense (1, 2) This
condensation increases the refractive index of the membrane bound lamellae containing the
reflectin proteins, thereby activating reflectance, while simultaneously triggering expulsion of
water from the lamellae, reducing their thickness and spacing and thus changing the wavelength
of their reflected light (2).

2. The goal of my research is to determine if iridocytes and leucophores from D. opalescens
express a mAChR and other components of the signaling pathway to validate the model
presented above. So far, I have found that DNA encoding a mAChR with homology to that in D.
pealei can be PCR-amplified from cDNA generated from D. opalescens iridocyte and
leucophore mRNA. A ClustalW alignment of the mAChR transmembrane domains exhibited
49% identity and 67% similarity between human and squid. Of residues in the ACh-binding
pocket (4), 12 of the 13 amino acid residues that interact with ACh are conserved. I now am
analyzing transcripts in these tissues for three downstream signaling proteins, Gαq, PLC-β4, and
CALM because these are known to function in the mammalian ACh transduction system. PCR
successfully amplified all three cDNAs from iridocytes but not from leucophores.
My research has resulted in discovery of the mAChR protein sequence from the target
cells, allowing future researchers to produce antibodies that will help pinpoint the location of the
receptors in relation to the iridocyte lamellae and leucosomes vesicles. I further hope to develop
a model of the signal transduction pathway in leucophores, identifying the signal transducers and
localization of the mAChR to gain new insights into switchable broadband photonic scattering
system.
1. Tao AR, et al. (2010) The Role of Protein Assembly in Dynamically Tunable Bio-optical
Tissues. Biomaterials 31 (5): 793-801
2. DeMartini, DG, Krogstad, DV, and Morse, DE (2013) Membrane Invaginations Facilitate
Reversible Water Flux Driving Tunable Iridescence in a Dynamic Biophotonic System. Proc.
Natl. Acad. Sci. USA 110, 2552-2556.
3. DeMartini DG, et al. (2013) Dynamic Biophotonics: Female Squid Exhibit Sexually
Dimorphic Tunable Leucophores and Iridocytes. The Journal of Experimental Biology 216:
3733-741.
4. Chin SP, et al. (2014) Toward Activated Homology Models of the Human M1 Muscarinic
Acetylcholine Receptor. Journal of Molecular Graphics and Modeling 49: 91-98.