Abstract

Cephalopods have evolved the ability to alter their appearance rapidly, which functions in signaling and camouflage. Iridophores are cells that reflect light and are typically composed of stacks of purine crystals. Cephalopods, however, utilize a novel protein, reflectin. Upon iridophore activation, reflectin undergoes a conformational change causing an increase in the amount of light reflected and altering the wavelength of the light reflected as well (Mäthger & Hanlon, 2007). Having a deeper understanding of how reflectin functions has applications in the field of biomaterials (Ordinario et al., 2014). This review will elucidate the signal transduction mechanism governing how cephalopods differentiate light and then initiate the conformational change of reflectin to produce a change in reflected light. Upon detection of light, the central nervous system will send an electrical signal to iridophores, where acetylcholine is released and then binds to muscarinic receptors on the surface of iridophores. Acetylcholine induces a signal transduction cascade leading to the conformational change of reflectin. This allows it to alter the wavelength of incoming light and reflect light back that spans the entire visible spectrum and in turn alters cephalopod appearance (D. G. DeMartini, Krogstad, & Morse, 2013; Tao et al., 2010).

Introduction

Cephalopods are a class of species in phylum Mollusca. They are characterized by the presence of tentacles and containing bilateral symmetry. Common cephalopods are squid, cuttlefish, and octopi (Gebhardt & Knebelsberger, 2015). They are notorious for their dazzling ability to reversibly alter their appearance. The combination of altering light reflectance and manipulating pigment concentrations produce colors spanning the entire visible spectrum (DeMartini, Izumi, Weaver, Pandolfi, & Morse, 2015). Altering appearance has a multitude of purposes in cephalopods. Cryptic coloration allows cephalopods to match the color of the surrounding environment to prevent detection from potential predators. Many predators of cephalopods have color vision, thus the ability to effectively match the color of its surroundings helps cephalopods evade predation (Mäthger & Hanlon, 2007). In addition, modulating appearance may play an important role in signaling and communication between other cephalopods. Cephalopods reflect polarized light and may be able to detect polarized light as well. Thus, reflection of polarized light may be utilized to produce signals to other cephalopods, while still remaining camouflaged (Talbot & Marshall, 2011). Varieties of different cell types are involved in altering cephalopod appearance.

Chromatophores are cells containing pigment sacs (Mäthger & Hanlon, 2007). Cephalopods contain thousands of chromatophores, each containing one type of pigment, such as red, yellow, or orange (Mäthger & Hanlon, 2007). Radial muscles, which are neurally controlled, are attached to chromatophores and the voluntary contractions and relaxation of these muscles directly affects the surface area of the pigment containing sacs. This alters the concentration of the pigments in the sacs and leads to a change in body patterns and color of the organism (Mäthger & Hanlon, 2007). It is important to reiterate, pigment molecules are present in chromatophores, whereas iridophores lack pigments.

Iridophores are light reflecting structures adjacent to chromatophores that work in part through the process of thin-film interference. Thin-film interference is the formation of a new wave through the constructive or destructive interference of other reflecting light waves (Hanlon, Cooper, Budelmann, & Pappas, 1990). To reflect light, structures must meet two requirements. The first conditions are that there must be a difference in the refractive index of the plate reflecting light and the space between the plates. In addition, the thickness of the plates and distance between the plates must be similar in size to the wavelength of light (Mäthger & Hanlon, 2007). Iridophores in fish, amphibians, and reptiles contain purine crystal layers to generate iridescence. However, in cephalopods, iridophores contain a novel protein, reflectin, which alters the wavelength of light reflected by undergoing a conformational change (Izumi et al., 2010).

Reflectin proteins stack into platelets and function as Bragg reflectors, because they are composed of alternating high refractive index reflectin proteins and a lower refractive index space between the proteins, which is simply the extracellular space (Fig 1) (Mäthger & Hanlon, 2007;Ghosal, Demartini, Eck & Morse, 2013). As mentioned earlier, this Bragg reflector like protein functions similarly to the properties of thin-film interference. In contrast to organisms that utilize purine crystals, reflectin is a dynamic protein while purine crystals are typically static structures and thus cannot be modulated to directly alter the wavelength of light reflected (Izumi et al., 2010). Therefore, it appears reflectin proteins are under voluntary control (Wardill, Gonzalez-Bellido, Crook, & Hanlon, 2012).

Figure 1. Schematic of reflectin functioning like a Bragg reflector. When reflectin is in its active conformation, reflected light is in phase resulting in the amount of light reflected being amplified through constructive interference. When the protein is in its inactive conformation, reflected light is out of phase and thus reflectance is low, due to destructive interference (Tao et al., 2010).

 

 

Cephalopods must be able to detect and differentiate light in order to finely regulate their appearance. The perplexing fact is cephalopods are able to modulate their color, yet are color blind (Mäthger & Hanlon, 2007). The question remains, how are cephalopods able to sense differences in light, while being unable to detect color? If cephalopods contain photoreceptors that can differentiate light and contain cells with proteins that alter the wavelength of light reflected, then it should be able to modulate the color of its skin.

Results

Polarization vision in cephalopods:

All signal transduction processes begin with detection. Although cephalopods are color blind, we can assume they must be able to differentiate light in some capacity to be able to camouflage so effectively. Cephalopods have a camera-like eye, but still utilize common phototransduction components, such rhodoposin, TRP channels, and a heterotrimeric G-protein initiated pathway (DeMartini et al., 2015; Mäthger & Hanlon, 2007). Reverse transcriptase PCR was utilized to identify the presence of these proteins in the retina of cephalopods (Kingston, Kuzirian, Hanlon, & Cronin, 2015). Interestingly, these proteins were found in the skin of cephalopods as well, indicating the presence of dermal photoreceptors in cephalopods. These dermal photoreceptors appear to aid in the function of chromatophores, not iridophores (Kingston, Kuzirian, Hanlon, & Cronin, 2015). Dermal photoreceptors were not found in the iridophore layer of cephalopods, and these photoreceptors lacked afferent neurons indicating they are not integrated with the central nervous system (Kingston, Wardill, Hanlon, & Cronin, 2015). However, both retinal and dermal photoreceptors utilize a similar phototransduction pathway involving rhodopsin.

Rhodopsin is a G-protein coupled receptor protein embedded in the cell membrane of photoreceptor cells. It transverses the cell membrane seven times, each consisting of alpha helices (Ota, Furutani, Terakita, Shichida, & Kandori, 2006). It is a made of a single polypeptide, called opsin, and a chromophore, 11-cis-retinal, that absorbs light (Ota, Furutani, Terakita, Shichida, & Kandori, 2006). Rhodopsin is opsin when it is covalently 

bound to retinal. The chromophore is covalently bound to lysine 296 in opsin via a Schiff base linkage (Ota et al., 2006). The nitrogen present in the Schiff base linkage is protonated, but is paired with glutamic acid 181, a negatively charged amino acid, acting as a counter ion to lower the energy of the protonated Schiff-base linkage. In vertebrates, the glutamic acid counter ion is located at position 113 (Ota et al., 2006). In cephalopods, there is a water molecule bridging the protonated Schiff-base linkage and glutamic acid, via hydrogen bonding, to stabilize the ion pair (Ota et al., 2006). This bridging water molecule is not present in vertebrate rhodopsin (Ota et al., 2006). The protonated Schiff-base linkage has a pKa of about 10.5 in cephalopod rhodopsin, while it is 16 in vertebrae rhodopsin. Thus, the cephalopod protonated Schiff-base linkage is more acidic than in vertebrate rhodopsin, meaning it is of higher energy as well. The hydrogen bonding from the bridging water molecule is necessary to stabilize the pronated Schiff-base linkage in cephalopod rhodopsin to ensure the protein functions properly (Ota et al., 2006).

When rhodopsin is struck by a photon, a photoisomerization reaction occurs where 11-cis-retinal is converted to all-trans-retinal (Ota, Furutani, Terakita, Shichida, & Kandori, 2006). A G-protein pathway is stimulated, activating phospholipase C and thus the secondary messengers DAG and IP3 as well. Phospholipase C is an enzyme that cleaves phosphatidylinositol-4,5-bisphosphate, a phospholipid, into diacyl glycerol (DAG) and inositol-1,4,5-triphosphate (IP3). DAG remains in the membrane, while IP3 is released into the cytoplasm and binds to the endoplasmic reticulum to stimulate calcium release (D. G. DeMartini et al., 2013; Gomis, Soriano, Belmonte, & Viana, 2008). This pathway eventually leads to modulating the opening and closing of ion channels, such as TRP channels, and thus a cellular signal develops (Kingston, Kuzirian, et al., 2015; Mäthger & Hanlon, 2007). However, this pathway does not identify how cephalopods are able to identify different qualities of light.

The photoreceptors in the retinas of cephalopods are arranged to optimally differentiate light (Talbot & Marshall, 2011). Although cephalopods are unable to identify particular colors, it is believed they can differentiate between different polarizations of light (Talbot & Marshall, 2011). The photoreceptor arrangement of retinas from different cephalopods were analyzed and compared with the predicted arrangement, allowing for optimal sensitivity to different polarizations of light. It was predicted that an orthogonal arrangement of photoreceptors best allows for identifying different polarizations of light. The observed photoreceptor arrangements were similar to the predicted, ideal photoreceptor arrangements (Fig 2) (Talbot & Marshall, 2011). Thus, it appears that cephalopods lack color vision, but may contain polarization vision, which can be a substitute for color vision (Talbot & Marshall, 2011).

Polarization vision may even be more beneficial in bodies of water than color vision, because as the depth of water increases, less of the visible spectrum becomes available (Mäthger & Hanlon, 2007). Additionally, when light enters water, it results in the scattering of light such that most of the light becomes horizontally polarized. In this environment, organisms with polarization vision will better detect objects (Talbot & Marshall, 2011). Although it is not certain, it is highly possible that once different polarizations of light are detected, the information is sent to the central nervous system to process this information and then potentially elicit a response.

Figure 2. Orthogonal arrangement of photoreceptors in cephalopod retinas for ideal detection of polarized light. This arrangement was similar to the actual photoreceptor arrangement in cephalopods. Polarization vision could potentially serve as a substitute for color vision and thus allow cephalopods to still differentiate light (Talbot & Marshall, 2011).

Electrical stimulation of nerves activates iridophores:

The central nervous system of cephalopods regulates iridophore coloration (Wardill, Gonzalez-Bellido, Crook, & Hanlon, 2012). Nerves were tagged with fluorescent dyes and axons were observed to branch from these nerve bundles into iridophores, indicating iridophores are neurally regulated (Wardill et al., 2012). Previous research produced similar results, where nerves were stained with a silver dye and observed in the layer of iridophores as well (Cooper, Hanlon, & Budelmann, 1990). Color changes were observed in iridophores when nerves directly connected to an iridophore were electrically stimulated (Wardill et al., 2012). Before electrical stimulation, iridophores had a low reflectance, but after the electrical stimulus was induced reflectance increased on average by 80% and the wavelength of light reflected changed as well (Figs. 3A & 3B) (Wardill et al., 2012). This process occurs rapidly and occurs in less than thirty seconds. Interestingly, different iridophores reflected different wavelengths of light. The rate at which wavelength of light changed correlated directly with how much the wavelength reflected shifted. Thus, iridophores that shifted their color at the greatest rate, also shifted their wavelength the furthest in the visible spectrum (Wardill et al., 2012). Although an electrical stimulation via nerve fibers from the central nervous system initiates a change in iridophore color, acetylcholine initiates the signal transduction pathway within the iridophore (Cooper et al., 1990).

Figure 3AB. Electrical stimulation of skin nerves in D. pealeii activated fin iridophores and caused a change in reflectance. Iridophores reflected on average 80% of the normalized reflectance value upon activation (A). Once iridophores were activated, a color change was observed such that the larger the shift in wavelength of light reflected, the greater the rate of color change observed in the iridophore (B) (Wardill, Gonzalez-Bellido, Crook, & Hanlon, 2012).

Acetylcholine binds to muscarinic receptors to activate iridophores:

The electrical stimulation sent from the central nervous system causes acetylcholine to be released within the iridophore layer (Wardill et al., 2012). This was determined when all skin layers below and above iridophores were removed, yet iridophores were still activated when electrically stimulated (Wardill et al., 2012). Acetylcholine initiates both the change in reflectance and coloration in iridophores, in a dose-dependent manner (Fig 4) (Hanlon, Cooper, Budelmann, & Pappas, 1990).

There is a direct relationship between the amount of acetylcholine added to cephalopod iridophores and the measured increase of reflectance (Hanlon, Cooper, Budelmann, & Pappas, 1990). The acetylcholine dosage also affected the observed color. At lower dosages (10-7 M), the tissues appeared pink to red, while at higher dosages (10-6 M) tissues appeared gold, green, and sometimes blue (Hanlon, Cooper, Budelmann, & Pappas, 1990). The iridophores consistently changed in a similar manner, beginning non-iridescent and then changing pink to gold to green. Thus, this indicates cells underwent a similar mechanism and response upon acetylcholine application (Hanlon, Cooper, Budelmann, & Pappas, 1990). However, any concentration greater than 10-6 M acetylcholine appeared to have no further effect on iridophore color, indicating the minimum acetylcholine dosage needed to observe the complete activation of iridophores (Hanlon, Cooper, Budelmann, & Pappas, 1990). The color change of iridophores after acetylcholine application is caused by a conformational change in reflectin proteins present in iridophores (Cooper et al., 1990). Acetylcholine application altered the structure of reflectin platelets in iridophores. When acetylcholine was added to iridophores the platelet thickness decreased, indicating reflectin proteins were condensing. The space between the reflectin platelets increased as well (Cooper et al., 1990).Thus, reflectin condensation is also dependent on how much acetylcholine is added. The change in the state of the reflectin and modulation of iridophore color consistently occurring in a time sequence manner upon acetylcholine application indicates acetylcholine initiates the conformational change in reflectin, but the conformational change is responsible for alteration of color. However, acetylcholine merely initiates a signal transduction cascade that ends with reflectin changing its conformation.

Figure 4. Graph showing dose dependent relationship between acetylcholine and reflectance in active iridophores over time. LUX is an arbitrary unit of reflectance and ASW is artificial seawater used to wash away acetylcholine from the previous trial. Thus, it acts as a negative control. The length of the vertical bars indicates standard deviations of LUX measurements taken at different points during the trials (Hanlon et al., 1990).

Acetylcholine binds to the muscarinic receptor of iridopohres causing a signal transduction cascade (Mäthger, Collins, & Lima, 2004). Acetylcholine can bind to both the nicotinic and muscarinic receptors present on iridophore cell surfaces. Thus, which receptor acetylcholine was binding to for iridophore activation remained to be answered. The muscarinic receptor is part of a metabotropic pathway, while the nicotinic receptor pathway initiates an ionotropic pathway (Hanlon, Cooper, Budelmann, & Pappas, 1990) .

Literature demonstrated intracellular calcium is necessary for iridophore activation (Hanlon et al., 1990). The calcium ionophore, A23187, was added to iridophores and when calcium was added, it mimicked the effects of acetylcholine application on iridophores, causing both an increase in reflectance and changes in color (Hanlon et al., 1990). When calcium channels were blocked with verapamil, iridophore activation was inhibited (Hanlon et al., 1990). Extracellular calcium is also required for iridophore activation even though IP3 is activated upon acetylcholine binding to muscarinic receptors, leading to intracellular calcium release.

When acetylcholine was applied to cells when calcium was not present in the extracellular solution, iridophores were initially activated, but then reflectance sharply declined (Fig 5) (Hanlon et al., 1990). This indicates intracellular calcium initially was utilized to activate iridophores, but once intracellular calcium was depleted, the pathway was halted. Calcium acts a secondary messenger in this pathway, because when a calmodulin (CaM) antagonist was added with acetylcholine, iridophore activation was inhibited (Hanlon et al., 1990). Thus, the requirement of calcium increasing in the cytoplasm during iridophore activation allowed for the usage of Fura-2AM to determine which receptor acetylcholine is binding to, in order to induce color change in cephalopods (Mäthger et al., 2004).

Acetylcholine was utilized as a positive control and when it was utilized to stimulate iridophores, intracellular calcium increased as expected. Carbachol, a cholinergic agonist, was also utilized as a positive control, because it causes a release of intracellular calcium stores from the endoplasmic reticulum into the cytoplasm (Mäthger et al., 2004). When nicotine was utilized, which binds to the nicotinic receptor of cells, intracellular calcium did not increase and iridophores were not activated (Fig 6A). However, when muscarine was added, an agonist of acetylcholine that binds to muscarinic receptors, intracellular calcium increased as expected and iridophores were activated as well, producing a change in color (Fig 6B) (Mäthger et al., 2004). However, when atropine, an antagonist

Figure 5. Graph showing relationship between calcium and reflectance in active iridophores with acetylcholine present. LUX is an arbitrary unit of reflectance and ASW is artificial seawater used to wash away acetylcholine from the previous trial, thus it acts as a negative control. The length of the vertical bars indicates standard deviations of LUX measurements taken at different points during the trials (Hanlon, Cooper, Budelmann, & Pappas, 1990).

of muscarine was added after muscarine application, intracellular calcium levels did not increase and iridophores were not activated, because atropine was competitively inhibiting muscarine from binding to muscarinic receptors (Mäthger et al., 2004). Thus, acetylcholine must bind to muscarinic receptors, not nicotinic receptors to initiate color change in iridophores and cytoplasmic calcium is required as well.

The muscarinic receptor is a type-G protein coupled receptor, as expected, because an increase in reflectance and change in color was observed in cephalopod iridophores when a cholera toxin, which activates the G-protein, was added without acetylcholine present (Izumi et al., 2010). Reverse transcriptase PCR has confirmed the heterotrimeric G protein is a Gq protein (Kingston et al., 2015). As mentioned earlier, this G-protein activates phospholipase C, which cleaves PIP2 into DAG and IP3. As noted earlier, IP3 binds to the endoplasmic reticulum to release calcium, which then binds to calmodulin (D. G. DeMartini et al., 2013). One of the functions of calmodulin is to activate kinases. It is known reflectin undergoes a rapid conformational change, thus it makes sense to determine if phosphorylation, one of the most common inducers of conformational change, was involved.

 

Figure 6AB. Application of nicotine, which mimics ACh by binding to nicotinic receptors, but cannot bind to muscarinic receptors. ACh binding to muscarinic receptors produced an increase in intracellular calcium and activated iridophores (A) . Application of muscarine, which mimics ACh by binding to muscarinic receptors, causes an intracellular increase in calcium and iridophore activation, while atropine competitively inhibits this action when muscarine is added after atropine application (B) (Mäthger, Collins, & Lima, 2004)

Reflectin is phosphorylated to induce a conformational change:

Phosphorylation of reflectin proteins induces a conformational change (Tao et al., 2010). This was determined by measuring iridophore reflectance upon acetylcholine application with and without genistein, a potent inhibitor of tyrosine kinases (Izumi et al., 2010). When acetylcholine was applied concurrently with genistein, iridophore reflectance was greatly reduced in comparison to the positive control, where no genistein was present upon acetylcholine activation (Fig 7) (Izumi et al., 2010). Genistein inhibited reflectin reflectance in a dose-dependent manner (Izumi et al., 2010). Thus, when kinases remain active, iridophores can be activated, but cannot be stimulated when tyrosine kinase activity is inhibited. Phosphorylation of reflectin is necessary for iridophore activation and thus color change as well (Izumi et al., 2010). Interestingly, protein kinase C inhibitors did not inhibit iridophore activation as effectively as genistien, indicating tyrosine kinases play a pivotal role in phosphorylating reflectin (Izumi et al., 2010). Mass spectrometry has also provided evidence supporting tyrosine phosphorylation accompanying the conformational change of reflectin and iridophore activation (Tao et al., 2010).

Reflectin is a cationic protein and the addition of negatively charged phosphate groups can lessen electrostatic forces involved in maintaining the protein’s conformation. This increases the effect of hydrophobic forces on reflectin’s active conformation (Daniel G DeMartini, Izumi, Weaver, Pandolfi, & Morse, 2015; Tao et al., 2010). These short-range hydrophobic forces lead to the condensing of reflectin, termed hydrophobic collapse, resulting in an increase in protein concentration and the refractive index of reflectin (Daniel G DeMartini, Izumi, Weaver, Pandolfi, & Morse, 2015; Tao et al., 2010). Phosphatases are also activated by calmodulin to reverse the conformational change induced by phosphorylation (D. G. DeMartini et al., 2013). Although, phosphorylation of reflectin initiates its conformational change, the movement of water across the plasma membrane plays a pivotal role as well.

Figure 7. Effect of kinase inhibition on iridophore reflectance after ACh application. Upon genistein application with acetylcholine, iridophore reflectance was greatly reduced in comparison to the control where only ACh was added to iridophores. (Izumi et al., 2010)

Water is rapidly exchanged from reflectin through membrane invaginations:

Water plays an important role in reflectin condensation upon being phosphorylated (Izumi et al., 2010). Electron microscopy was utilized to study properties of iridophores and the study identified the presence of membrane invaginations, which increase the surface area of iridophores, and thus diffusion capabilities as well (D. G. DeMartini et al., 2013). The purpose of these membrane invaginations is for the rapid exchange of water during the conformational change of reflectin. This allows cephalopods to quickly change its appearance. Water is directly exchanged between the extracellular space and reflectin proteins, which prevents disrupting cytoplasmic concentrations of other chemicals so metabolic processes are not inhibited (D. G. DeMartini et al., 2013). Additionally, hydrated reflectin may also have hydrophilic water channels to improve the efficiency of removing water during its conformational change (Ordinario et al., 2014).

Mathematical models have determined the color changing capabilities of iridophores is directly related to the volume of water present in the reflectin platelets (D. G. DeMartini et al., 2013). To determine whether water was exchanged during reflectin’s conformational change, deuterated water was utilized, because deuterium is a hydrogen isotope which can be traced. Upon iridophore activation with acetylcholine, water is expelled from reflectins causing the volume of water in reflectin to decrease and, thus, the protein condenses and the protein concentration increases (Fig 8A, 8B) (D. G. DeMartini et al., 2013). As noted earlier, the increase in protein concentration amplifies the amount of light reflected due to increased constructive interference of reflected light waves (Tao et al., 2010). Additionally, the condensing of reflectin causes charged amino acids to be less exposed to the cytosol, resulting in ions to be effluxed across the cell to maintain osmotic equilibrium. After this mechanism is completed, refelectin proteins are rehydrated causing the protein platelets to expand, since its volume is increasing (D. G. DeMartini et al., 2013). Once water is expelled from reflectin proteins, the protein is in its active conformation. Iridophores are now activated and reflect varying wavelengths of light, based upon protein orientation (Tao et al., 2010).

Figure 8AB. Deuterated water was utilized to measure water uptake into reflectin. When deuterated water was initially added, it diffused into the extracellular space. Upon acetylcholine application, reflectin condensed, expelling water across the plasma membrane into the extracellular space. Deuterated water was taken up again once acetylcholine was removed and reflectin returned to its initial conformation. Once acetylcholine was reapplied, deuterated water uptake decreased, due to reflectin condensing and once again expelling water (DeMartini, Krogstad, & Morse, 2013).

Color change and reflectance amplification result from different mechanisms:

Reflectin appears to organize into nanoparticles that appear similar to beads on a string (Tao et al., 2010). Upon phosphorylation, both the reflectin platelet layers and the reflectin nanoparticles condense (Tao et al., 2010). However, each of these processes appears to regulate a separate mechanism in how light is reflected. Shifts in color and reflectance are temporally isolated processes (Ghoshal, DeMartini, Eck, & Morse, 2013; Izumi et al., 2010; Mäthger & Hanlon, 2007). Tao and associates (2010) obtained evidence of iriodphore color changing before peak reflectance. Half of the maximum color shift took eight seconds to achieve, while half the peak reflectance took fifteen seconds (Tao et al., 2010). Others argued reflectance amplification occurs first (Ghoshal et al., 2013; Wardill et al., 2012). Regardless, both agree the processes occur separately.

The change in reflectance can be attributed to a decrease in the thickness of reflectin platelets, thus causing an increase in the concentration of the proteins as they condense and causing an increase in the refractive index (Fig 9A & 9B) (Ghoshal et al., 2013). During reflectin platelet condensation, light reflecting off different reflectin layers become in phase, causing light to amplify from constructive interference. This leads to an increase in the amount of light reflected off reflectin (Ghoshal et al., 2013). However, condensation and organization of nanoparticles are primarily responsible for the observed change in color (Fig 9C) (Izumi et al., 2010). At a fixed reflectin concentration, there was a direct relationship between reflectin platelet thickness and wavelength of light reflected. Since protein concentration was a constant, the change in the wavelength of light reflected is attributed to nanoparticle condensation. Thus, reflectance amplification can be attributed to interparticle interactions, while change in the color reflected is due to intraparticle interactions (Tao et al., 2010). It is incredible that this intricate process is so finely tuned to match almost any color within the visible spectrum.

Figure 9AB. Refractive index and reflectance increases as protein concentration increases. There is a direct, linear relationship between refractive index and protein concentration. Thus, there is also a direct relationship between refractive index and reflectance as well (Tao et al., 2010).

Figure 9C. The wavelength of light reflected (x-axis) increases as platelet width increases. The protein concentration was held constant at 40 mg/mL, thus nanoparticle orientation is primarily responsible for the wavelength of light reflected (Tao et al., 2010).

Future Studies:

Although a great deal of work spanning multiple decades has been conducted regarding the pathway of iridophore activation, more work must be done to fully elucidate the mechanism. As discussed earlier in this review, tyrosine kinases phosphorylate tyrosines in reflectin to induce a conformational change in the protein (Izumi et al., 2010). However, even when tyrosine kinases were inhibited with genistein, some increase in reflectance was still observed, indicating other kinases or classes of enzymes may be involved in the conformational change of reflectin. Serines are phosphorylated during reflectin’s conformational change as well, thus other kinases must be involved in the mechanism (DeMartini et al., 2015). Serine and threonine are commonly phosphorylated residues, because like tyrosine, they each contain a hydroxyl group, which can make a nucleophilic attack on the electrophilic phosphorous in a phosphate group to make a phosphoester. Specific amino acids can then be identified to determine where the phosphorylation sites are in reflectin.

Color changing capabilities of cephalopods contain powerful applications in designing nanomaterials (Izumi et al., 2010). The military is particularly interested, because of the potential to produce materials that camouflage troops in a variety of different environments. This type of material would have to modulate its structure in order to change how light interacts with the material. Like many technologies utilized by man, evolution was the original inventor.

Conclusion

Cephalopods have the ability to rapidly alter their appearance to produce colors spanning the entire visible spectrum (Tao et al., 2010). The process is neurally controlled and finely tuned for signaling other organisms and to provide camouflage for evading predator detection (Wardill et al., 2012). Light is detected by photoreceptors in the eye of cephalopods, which are orthogonally arranged to detect differences in the polarization of light. This allows cephalopods, which are color blind, to differentiate between light (Talbot & Marshall, 2011). An electrical signal is then sent to the iridophore layer, causing acetylcholine to be released within the iridophore layer and bind to muscarinic receptors to induce a signal transduction cascade (Fig 10) (Mäthger et al., 2004; Wardill et al., 2012). Activation of muscarinic receptors initiates a Gq protein pathway, thus activating phospholipase C, which hydrolyzes PIP2 to produce DAG and IP3. IP3 binds to the endoplasmic reticulum, causing it to release intracellular calcium stores. Calcium binds to calmodulin, which then activates kinases and phosphatases (DeMartini et al., 2015). Kinases then phosphorylate reflectin to induce a conformational change in the protein, which then rapidly expels water out of the cell through membrane invaginations that facilitate water movement (D. G. DeMartini et al., 2013). Reflectin proteins condense and alter their orientation, such that reflectance is amplified and the wavelength of light reflected back is altered (Tao et al., 2010). Cephalopods remain masters of disguise which man is still unable to emulate.

Figure 10. Proposed pathway of how reflectin conformation is altered to induce color change. ACh binds to muscarinic receptors to initiate a Gq protein pathway leading to the activation of phospholipase C, which cleaves PIP2 into DAG and IP3. IP3 binds to the endoplasmic reticulum to release calcium, which then binds to calmodulin. Calmodulin activates kinases and phosphatases to alter whether specific amino acids are phosphorylated. This change in phosphorylation state induces a conformational change of reflectin where the protein condenses and expels water across the membrane invaginations surrounding reflectin (DeMartini, Krogstad, & Morse, 2013).

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