Luminescent activity and ultrastructural characterization of photocytes dissociated from the coelenterate Renilla köllikeri

Dissociation and Percoll sedimentation techniques were used to separate and pool the autofluorescent luminescent cells (photocytes) of the pennatulid anthozoan Renilla k?llikeri. Photometric recordings of luminescent activity of photocyte suspensions show that activation of flashing and glowing by KCl depolarization is suppressed in calcium-free sea water and by cobalt but enhanced by trifluoperazine, thus suggesting that luminescence excitation is dependent on extracellular calcium and calmodulin-mediated mechanisms. Of several neuroactive substances tested, adrenaline, dopamine, N-methyl-N-phenylethanolamine, serotonin and the native neuropeptide Antho-RFamide all induced photocyte responses at high concentrations (0.1-1 mM) only, whereas lower concentrations of adrenaline and Antho-RFamide are known to activate or enhance luminescence or muscular contractions in intact Renilla tissues (Anctil et al., 1982; Anctil, 1987). Hence, none of these substances is a likely neurotransmitter candidate for direct photocyte activation. Ultrastructural observations of dissociated photocytes reveal that they are musculo-epithelial cells containing numerous 0.2-mum vesicles resembling previously extracted and light-emitting lumisomes (Anderson and Cormier, 1973). Similar cells were traced ultrastructurally in situ in the endodermal luminescent zones, but not in non-luminescent endoderm.     

Cytological changes during bioluminescence production in dissociated photocytes from the ophiuroid Amphipholis squamata (Echinodermata)

Bioluminescence in the ophiuroid Amphipholis squamata is produced by photocytes located within the spinal ganglia of arm spines. Ganglionic cells were dissociated (pronase digestion) and photocytes separated from other cell types by using a continuous density Percoll gradient. Aliquots from a stock suspension of photocytes in artificial sea water were stimulated to produce light by using KCl or acetylcholine and fixed for ultrastructural observation at different times of the luminous process. Preluminescent, luminescent, and postluminescent photocytes contained various intracytoplasmic structures, such as Golgi, flat and distended rough endoplasmic reticulum cisternae, bundles of fibrils, and up to six types of membrane-bounded vesicles. These structures either co-occurred or succeeded one another during the process of light production, indicating that they were most probably participating in the luminescence reaction. Two types of vesicles, sharing some ultrastructural features, probably represented the microsources of the photocytes. One type occurred almost exclusively in luminescent photocytes, and the other almost exclusively in postluminescent photocytes, suggesting that one may be transformed into the other. The latter type of vesicle contained densely packed fibro-tubular units, giving a characteristic paracrystalline appearance to postluminescent photocytes.     

The presence of vasoactive intestinal polypeptide (VIP)-like-immunoreactive nerve fibres and VIP-receptors in the pineal gland of the Mongolian gerbil (Meriones unguiculatus)

Summary The photocytes and other endodermal cells composing the wall of the meridional canals of the comb-jelly, Mnemiopsis leidyi, were investigated by transmission electron microscopy. Although many of these cells possess distinctive features such as a ciliary apparatus, lysosome-like bodies or vacuoles, they share with photocytes the presence of a network of rough endoplasmic reticulum (RER) whose cisternae enwrap large mitochondria and are aligned along the subsurface of the plasma membrane. A stereological analysis of organelle content in photocytes confirms the prominence of the RER in these cells and a shift of RER from mitochondria to plasma membrane subsurface in photocytes induced to luminesce by the mitochondrial inhibitor dinitrophenol. Photocytes and other endodermal cells of the meridional canals are interconnected by numerous gap junctions which, among photocytes, often form symmetrical triads with cortical cisternae and mitochondria. The gap junctions and RER/mitochondria assemblages are interpreted as possible substrates for, respectively, conduction of luminescence excitation along the canals and for excitation-luminescence coupling. Neuntes occasionally make synapses with photocytes and other endodermal cells lying adjacent to the mesoglea.     

Reflector of the body photophore in lanternfish is mechanistically tuned to project the biochemical emission in photocytes for counterillumination

Lanternfish, a family Myctophidae, use ventro-lateral body photophores for camouflage of the ventral silhouette, a strategy called counterillumination. While other deep-sea fishes possess pigmented filters and silver reflectors to match sunlight filtering down through the depths, myctophids developed a blue-green reflector for this purpose. In this study, we showed in a lanternfish Diaphus watasei that the reflector comprised monolayered iridophores containing multilayered guanine crystals which enable high reflection with light interference colouration. Platelets shape in body photophores is an unique near-regular hexagonal, probably to allow the homogeneity of reflection angle of the luminescence from photocytes. Focus point of the parabola-like reflector is positioned on the photocytes that ensures the light produced from the photocytes is redirected to the ventral direction. In vitro luminescence reaction using purified luciferase and the substrate coelenterazine showed the light emission at λ_max 454 nm, while reflection spectra of the iridophores exhibit peaks at longer wavelength, which accomplish to alter the luminescence emitted from photocytes to longer wavelength to fit the mesopelagic light environment. Taken together, we revealed multiple mechanistic elaborations in myctophid body photophores to achieve effective control of biochemical luminescence for counterillumination.     

The bioluminescent organs of the deep-sea cephalopod Vampyroteuthis infernalis (Cephalopoda: Vampyromorpha)

Vumpyroteuthis infernalis Chun is the sole member of the cephalopod subclass Vampyromorpha. The three putative types of photophore (fin base, dorsomedial or composite, and skin nodules) are described and it is concluded that the composite organ is not a light-emitting structure. It has structural similarities to an extra-ocular photoreceptor.
The fin-base photophore is capable of being everted from the pigmented pit in which it is seated. Its surface bears chromatophores which may modulate the emission and the photocytes contain an extensive network of microtubular elements and proteoglycan particles. The reflector is formed of a very thick layer of collagen fibrils in different orientations. The skin nodules are widely distributed over the animal. They have a collagenous reflector and core cells with structural similarities to those of the fin-base photocytes.
The photocytes have no morphological links with those of octopods or decapods. The thick collagen reflector, and absence of the iridosomal platelets present in the other subclasses, suggests an early separation of the Vampyromorpha from other coleoids.     

Removal of the polar lobe leads to the formation of functionally deficient photocytes in the annelidChaetopterus variopedatus

Summary The light emitting photocytes ofChaetopterus variopedatus larvae are bilaterally situated within the ectoderm of the post-trochal region. Their histological appearance is similar to that of the adult photocytes. The larval photocytes contain a large quantity of membranous secretory vesicles (photosomes), which probably contain the photoluminescent protein. The two-cellChaetopterus embryo contains a small AB and a large CD blastomere. Previous studies have shown that only the “larvae” resulting from isolated CD blastomeres are able to luminesce. Consistent with these findings, morphologically distinct photocytes are only found in the CD larvae. The removal of the small polar lobe that forms during first cleavage leads to the production of a “larva” that is unable to produce light. All delobed larvae contain morphologically distinct photocytes, which are identical to those in normal larvae except they appear to contain only a small quantity of photosomes. Experimental equalization of first cleavage leads to the production of a double embryo. While photocytes are found in both of the duplicated post-trochal regions, usually only one of these is capable of emitting luminescence. Apparently, the highly localized vagetal material (determinants) responsible for functional light emission is distributed to both halves in only a few cases when first cleavage is experimentally “equalized”. These results indicate that the determinative action of the polar lobe is not required for the formation of the photocytes themselves, but rather for their ability to function as emitters of light. The determinants in the polar lobe ofChaetopterus may control some aspect of the photoluminescence reaction itself, such as the production of the photoprotein.     

The morphology of the bioluminescent tissue of the cephalopod Japetella diaphana (Octopoda: Bolitaenidae)

Maturing females of the octopod Japetella diaphana (Hoyle) develop a luminous oral ring. Studies of specimens of different size show that this structure develops from a muscular ring which undergoes great cellular proliferation, associated with gradual degeneration of the original muscle. The light-producing cells (photocytes) have a relatively uniform cytoplasm whose most characteristic components are small mitochondria, granular aggregates and microtubular or microfibrillar bundles. It is concluded that the original muscle tissue is not transformed directly into luminous tissue.
Possible uses of the luminescence are discussed, based on the postures adopted by live specimens in shipboard aquaria.     

Immunohistochemical and radioautographic evidence of monoamine-containing cells in bioluminescent elytra of the scale-worm Harmothoe imbricata (Polychaeta)

Summary Elytra of the scale-worm Harmothoe imbricata were examined for the presence of monoamine-like immunoreactivities and radioautographic reactions. Serotonin (5-HT)-like immunoreactivity was widely distributed among the cellular constituents of the elytra, being present in epithelial cells including photocytes, in elytral nerves, clear cells and the loose neuronal plexus of the middle compartment. The distribution of [³H]5-HT labelling coincided with that of the immunoreactivity except for an additional reactive band extending through the upper cuticle layer. Tyrosine hydroxylase (TH)-like immunoreactivity was detected in epithelial cells, sensory papillae and elytral ganglion and nerves, with little or no staining in clear cells and plexus neurons of the middle compartment. Radioautographic labelling with [³H]noradrenaline and [³H]adrenaline overlaid many epithelial cells, elytral nerves and sensory papillae, but not the loose neuronal plexus or, apparently, clear cells. It is concluded that monoaminergic systems are widely distributed and that they must play important roles as neuroactive and/or paracrine substances in the elytral neuroectoderm. The distribution of [³H]5-HT label in photocytes also suggests the involvement of serotonergic mechanisms in luminescence control, luminescence being the only known effector activity of elytra.     

The development of bioluminescence in the ctenophore Mnemiopsis leidyi

The photocytes of the ctenophore Mnemiopsis have a discontinuous distribution along the radial canal between the sites where the comb plate cilia cells are located on the side of the canal which contains the testes. They are separated from the lumen of the canal by a population of gastric cells. Cytologically these cells are characterized by a condensed nucleus and cytoplasm which stains lightly with basophilic dyes.The ability of the ctenophore embryo to produce light appears at the developmental stage when the comb plate cilia first begin to grow out. At this stage four light-producing areas are present; each area corresponds to one quadrant of the adult animal. At the sites of light production, a population of cells can be identified that have some of the cytological properties of the photocytes of the adult animal. Within 8–10 hr after light production begins there is a 10-fold increase in the amount of light produced by an embryo and a cytological maturation of its photocytes; during this time period there is no increase in photocyte number. At about the time the embryo begins to feed, each light-producing region splits into two regions, each of which corresponds to a radial canal.During the process of embryogenesis the photocyte cell lineage is first segregated from non-photocytes at the differential division which gives the 8-cell stage embryo. The M macromere lineage goes on to form photocytes, but the E macromere lineage does not. The M macromeres form a micromere at the aboral pole of the embryo at each of the next two cleavages; during these cleavages the potential for photocyte differentiation continues to segregate with the M macromeres. During the division which gives the 64-cell stage the M macromeres divide equally; the potential for photocyte differentiation segregates with the M macromeres nearest the oral-aboral axis. M macromeres which are isolated from the embryo at the 8-, 16-, or 32-cell stage of development will continue to cleave as though they were part of a normal embryo and differentiate to form photocytes.The events that are responsible for the differential division during the formation of the 8-cell stage embryo have been studied by centrifuging eggs to produce fragments of different cytoplasmic composition. Egg fragments which contain only cortical cytoplasm differentiate comb plate cilia cells, but do not produce photocytes. Cortical fragments with a small amount of yolk differentiate comb plate cilia cells and photocytes. Both the M and E macromeres from cortical fragments with no yolk produce comb plate cilia. Only M macromeres containing yolk form photocytes; if an M macromere forms photocytes it does not form comb plate cilia.     

Pharmacomorphological study of denervation induced by 6-hydroxydopamine in Porichthys photophores

The effects of 6-hydroxydopamine (6-OHDA) on the bioluminescent response of Porichthys photophores were investigated as part of a pharmacological study of the neural control of luminescence in this fish. Subcutaneous injections of 6-OHDA induce a luminescent response similar to that of norepinephrine (NE), suggesting a sympathomimetic action. The luminescent response to electrical stimulation is almost completely and irreversibly abolished within 24 hours following low-dose treatment of the photophores with 6-OHDA, while the sensitivity of these organs to exogenous NE is increased significantly over the few days post-treatment. During this period the photophores continuously emitted a steady low-level glow. Electronmicroscopic studies of such photophores revealed progressive destruction of the nerve endings. Photophore luminescent sensitivity to NE subsequently became sub-normal, and at this stage electron microscopy revealed an increasingly larger number of damaged photocytes, supportive cells and, in one case, lens cells. From these results it is suggested that 6-OHDA initially impairs neuro-photocyte transmission by destroying catecholaminergic nerve endings. In turn, the transmitter reuptake mechanism is also impaired, thus accounting for development of supersensitive responses to exogenous NE. Subnormal luminescent responses to NE appear as a result of loss of photocyte competence due to structural deterioration. The latter are interpreted as the consequence of removal of trophic factors supplied by the photophore adrenergic innervation.Suppression of luminescent response to both electrical stimulation and exogenous NE in photophores treated with higher doses of 6-OHDA, may be due to a direct effect of this drug on the receptor sites of the photocytes.     

The fine structure of the light organ of the glowworm Lampyris noctiluca

Summary The four main parts of the glowworm light organ are the cuticle, the hypodermis, the photocyte layer and the reflector cell layer. The hypodermis is one cell thick and it contains hypodermic glands. These glandular cells have a lumen that opens to the outside of the cuticle. Projecting into the lumen are numerous microvilli. Between the hypodermis and photocytes are typical insect tunicated nerve fibres. They pass down between the photocyte and reflector layer cells. They do not appear to innervate the photocytes and they are thought to innervate adjacent muscle fibres or to be sensory. Tracheoles are commonly present between the photocytes but no tracheolar end organs are found. The photocytes contain amorphous granules, mitochondria, photocyte granules and a vesiculated reticulum. All, except the mitochondria, are absent from the reflector layer and so probably have some connection with light production. The reflector layer contains glycogen granules, clear spaces thought to be the sites of urate crystals, and membranous granules. The latter granules are sometimes found in photocytes adjacent to the reflector layer whilst amorphous granules are sometimes absent from these adjacent cells. So a cell layer with some features of the photocyte and reflector layer cells is present. These morphological findings are discussed with regard to the unknown function of the reflector layer and the control of light emission. Acknowledgments. We would like to thank Professor J. Z. Young and Dr. E. G. Gray for their advice and encouragement, Mrs. Jane, Astafiev for drawing fig. 1, Mr. S. Waterman for photographic assistance, Miss Cheryl Martin for secretarial assistance, and many colleagues for help in collecting specimens of glowworms.     

Luminescence of Ca2+-activated photoprotein obelin initiated by NaOCl and MnCl2

The luminescence of obelin is initiated by NaOCl in a reaction mixture containing no calcium. The addition of Mn²⁺ enhances the light emission >300-fold. Sodium azide and histidine, as singlet oxygen quenchers, inhibit NaOCl-activated obelin luminescence in the presence or absence of Mn²⁺. This suggests that the addition of NaOCI to the mixture causes singlet oxygen formation (stimulated by Mn²⁺ ions), and singlet oxygen initiates the light-emitting reaction.     

Ultrastructural organization of lantern shark (Etmopterus spinax Linnaeus, 1758) photophores

Etmopterus spinax Linnaeus, 1758 is a deep-sea lantern shark that emits blue light thanks to thousands of tiny cup-shaped organs made of a pigmented sheath enclosing light-emitting cells topped by an iris-like structure and a lens. In this study, we investigate the ultrastructure of these photophores in order to improve our understanding of the light emission process. The presence of a novel layer, a putative reflector upholstering the pigmented sheath, is highlighted. The intracellular organization of the photocytes is addressed. They appear as regionalized cells: their basal area is occupied by an ovoid nucleus, their medial area is highly vesiculated and their apical area, oriented toward the photophore center, displays small granular inclusions. We hypothesize this granular area to be the intracellular site of photogenesis in E. spinax, as it is also the most fluorescent part of the photocyte.     

Formation of the Ca2+-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils.

1. A method has been developed to incorporate the apoprotein of the Ca2+-activated photoprotein obelin, and mRNA purified from the hydroid Obelia, into the cytoplasm of intact human neutrophils. This was based on internal release from pH-sensitive immunoliposomes taken up initially by phagocytosis. 2. Addition of the prosthetic group of obelin, coelenterazine, to these cells containing apo-obelin or Obelia mRNA resulted in formation of active Ca2+-activated obelin. 3. The obelin formed within the neutrophils responded to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (1 microM) and to the membrane attack complex of complement (C5B6789n). 4. The formation of the apo-obelin from mRNA within neutrophils was inhibited by over 80% in the absence of added amino acids, and by over 90% by the protein-synthesis inhibitor puromycin (100 micrograms/ml). 5. The translation of Obelia mRNA inside cells provides a method for circumventing consumption of Ca2+-activated photoproteins during cell activation or injury, and for monitoring protein synthesis in living cells.     

Uptake and release of [3H]-serotonin in photophores of the midshipman fish, Porichthys notatus

A kinetic analysis of [3H]-5-HT uptake in the photocytes of the photophores of Porichthys notatus revealed a high affinity (Km: 1.71 X 10(-7] and low affinity component (Km: 1.10 X 10(-5) M). The high affinity uptake was sodium- and potassium-dependent but largely insensitive to temperatures between 0 and 20 C. Ouabain (5 X 10(-3) M) and dinitrophenol (10(-3) M) reduced uptake significantly. DMI, imipramine and fluoxetine, in that order of potency, greatly inhibited [3H]-5-HT uptake. Noradrenaline and adrenaline reduced uptake in a non-competitive manner, while dopamine, tryptophan, 5-hydroxytryptophan and Cypridina luciferin had little or not effect on uptake. Non-facilitated luminescent responses to electrical stimulation were accompanied by release of [3H]-5-HT accumulated in the photocytes. Facilitatory luminescence excitation consistently failed to induce the release of [3H]-5-HT. Electrical and adrenaline (10(-5) M) stimulation of photophores after [3H]-5-HT release has occurred, failed to elicit any additional luminescent response. The photophores were responsive to KCN (10(-3) M) under these conditions. The results indicate that a specific carrier-mediated transport system is responsible for photocytic [3H]-5-HT uptake, and that release of photocytic [3H]-5-HT is stringently regulated and followed by inhibition of luminescence excitability.     

Early development of bioluminescence suggests camouflage by counter‐illumination in the velvet belly lantern shark Etmopterus spinax (Squaloidea: Etmopteridae)

The development of luminous structures and the acquisition of luminescence competence during the ontogeny of the velvet belly lantern shark Etmopterus spinax, a deep‐sea squalid species, were investigated. The sequential appearance of nine different luminous zones during shark embryogenesis were established, and a new terminology for them given. These zones form the complex luminous pattern observed in free‐swimming animals. The organogenesis of photophores (photogenic organs) from the different luminous zones was followed, and photophore maturation was marked by the appearance of green fluorescent vesicles inside the photocytes (photogenic cells). Peroxide‐induced light emissions as well as spontaneous luminescence analysis indicated that the ability of E. spinax to produce light was linked to the presence of these fluorescent vesicles and occured prior to birth. The size of photogenic organs, as well as the percentage of ventral body surface area occupied by the luminous pattern and covered by photophores increased sharply during embryogenesis but remained relatively stable in free‐swimming animals. All these results strongly suggest camouflage by counter‐illumination in juvenile E. spinax.     

The role of cleavage in the localization of developmental potential in the Ctenophore Mnemiopsis leidyi

This study defines the time period during which the cellular components that specify comb plates and photocytes become localized in different parts of blastomeres prior to their segregation to separate daughter cells. At the two-cell stage the factors which specify comb plates are localized at the aboral pole of the blastomeres. There is not a significant localization of the factors which specify comb plates and photocytes along the tentacular axis of the embryo. At the four-cell stage, the factors which specify comb plates become localized at one end of the tentacular axis of the blastomeres; however, the factors which specify photocytes have not yet become localized. At the eight-cell stage, the factors which specify these two cell types are segregated to different blastomeres.The role of cleavage in setting up these localized regions of developmental potential has been studied by reversibly inhibiting selected cleavages. After the first division, the pattern of cleavage that follows a period of cleavage inhibition corresponds to the pattern occurring in untreated embryos that began development at the same time. This situation is similar to the “clock” system, which controls many aspects of the pattern of cleavage in sea urchin embryos. The extent to which the factors that specify comb plates and photocytes become localized in a given region of a blastomere is correlated with the kind of cleavage which occurs after a block. Most of the activity involved in localizing developmental potential takes place during cleavage.     

Luminescence Literature

The journal provides comprehensive literature searches on all aspects of luminescence which will be published in most issues. These lists have been produced regularly since 1986—some by year and some by specialized topic. This survey of papers on Obelia, Renilla, Aequorea and Gonyaulax is drawn from the 2000 literature and covers the biology of these organisms and the applications of genes and proteins from these organisms in analysis.     

Cleavage of formate from omega,4-dihydroxyacetophenone. An unusual oxygen-requiring reaction in the bacterial catabolism of 4-hydroxyacetophenone.

The bioluminescence of the ctenophore Mnemiopsis takes place when the photoprotein mnemiopsin in the photocytes reacts with Ca2+. The luminescence is inhibited in sunlight and this photoinhibition is reversible by keeping the live specimens in the dark. The extracts of mnemiopsin are similarly photoinhibited, but the photoinhibition cannot be reversed in the dark. We have found that photo-inhibited mnemiopsin can be re-activated in the dark by incubation with coelenterazine and O2 only in solutions having a pH very close to 9.0. The re-activation in vivo probably takes place in the same manner, using the coelenterazine that is supplied from its abundant storage form. Various lines of experimental evidence suggest that the photoinactivation of mnemiopsin results in the dissociation of coelenterazine and oxygen from the molecule of photoprotein; the dissociated form of the former molecule is an inactive form of coelenterazine, not free coelenterazine.     

A direct demonstration that inositol-trisphosphate induces an increase in intracellular calcium in Limulus photoreceptors

Inositol-trisphosphate was pressure-injected into Limulus ventral photoreceptors; these injections induced electrical responses that mimic several aspects of the electrical responses induced by light. Single cells were also injected with aequorin. Injections of inositol-trisphosphate into such cells induced an increase in luminescence from the intracellular aequorin, even in the absence of extracellular calcium ions. These aequorin responses show directly that inositol-trisphosphate induces an increase in ionized calcium concentration within intact and functioning cells that arises from release of calcium ions from intracellular stores.