The crystal structures of semi-synthetic aequorins.
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The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca(2+). Semi-synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca(2+). To understand the structural basis of the Ca(2+)-sensitivity, we determined the crystal structures of four semi-synthetic aequorins (cp-, i-, br- and n-aequorins) at resolutions of 1.6-1.8 A. In general, the protein structures of these semi-synthetic aequorins are almost identical to native aequorin. Of the four EF-hand domains in the molecule, EF-hand II does not bind Ca(2+), and the loop of EF-hand IV is clearly deformed. It is most likely that the binding of Ca(2+) with EF-hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i-, br-, and n-aequorins have bulky 2-substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca(2+) to aequorin. In cp-aequorin, the cyclopentylmethyl group that substitutes for the original 8-benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light-emitting reaction. The differences of various semi-synthetic aequorins in Ca(2+)-sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca(2+)-binding. (+info)
Redox signaling in colonial hydroids: many pathways for peroxide.
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Studies of mitochondrial redox signaling predict that the colonial hydroids Eirene viridula and Podocoryna carnea should respond to manipulations of reactive oxygen species (ROS). Both species encrust surfaces with feeding polyps connected by networks of stolons; P. carnea is more 'sheet-like' with closely spaced polyps and short stolons, while E. viridula is more 'runner-like' with widely spaced polyps and long stolons. Treatment with the chemical antioxidant vitamin C diminishes ROS in mitochondrion-rich epitheliomuscular cells (EMCs) and produces phenotypic effects (sheet-like growth) similar to uncouplers of oxidative phosphorylation. In peripheral stolon tips, treatment with vitamin C triggers a dramatic increase of ROS that is followed by tissue death and stolon regression. The enzymatic anti-oxidant catalase is probably not taken up by the colony but, rather, converts hydrogen peroxide in the medium to water and oxygen. Exogenous catalase does not affect ROS in mitochondrion-rich EMCs, but does increase the amounts of ROS emitted from peripheral stolons, resulting in rapid, runner-like growth. Treatment with exogenous hydrogen peroxide increases ROS levels in stolon tips and results in somewhat faster colony growth. Finally, untreated colonies of E. viridula exhibit higher levels of ROS in stolon tips than untreated colonies of P. carnea. ROS may participate in a number of putative signaling pathways: (1) high levels of ROS may trigger cell and tissue death in peripheral stolon tips; (2) more moderate levels of ROS in stolon tips may trigger outward growth, inhibit branching and, possibly, mediate the redox signaling of mitochondrion-rich EMCs; and (3) ROS may have an extra-colony function, perhaps in suppressing the growth of bacteria. (+info)
Spatiotemporal characteristics and mechanisms of intracellular Ca(2+) increases at fertilization in eggs of jellyfish (Phylum Cnidaria, Class Hydrozoa).
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We have clarified, for the first time, the spatiotemporal patterns of intracellular Ca(2+) increases at fertilization and the Ca(2+)-mobilizing mechanisms in eggs of hydrozoan jellyfish, which belong to the evolutionarily old diploblastic phylum, Cnidaria. An initial Ca(2+) increase just after fertilization took the form of a Ca(2+) wave starting from one cortical region of the egg and propagating to its antipode in all of four hydrozoan species tested: Cytaeis uchidae, Cladonema pacificum, Clytia sp., and Gonionema vertens. The initiation site of the Ca(2+) wave was restricted to the animal pole, which is known to be the only area of sperm-egg fusion in hydrozoan eggs, and the wave propagating velocity was estimated to be 4.2-5.9 mum/s. After a Ca(2+) peak had been attained by the initial Ca(2+) wave, the elevated Ca(2+) gradually declined and returned nearly to the resting value at 7-10 min following fertilization. Injection of inositol 1,4,5-trisphosphate (IP(3)), an agonist of IP(3) receptors (IP(3)R), was highly effective in inducing a Ca(2+) increase in unfertilized eggs; IP(3) at a final intracellular concentration of 12-60 nM produced a fully propagating Ca(2+) wave equivalent to that observed at fertilization. In contrast, a higher concentration of cyclic ADP-ribose (cADPR), an agonist of ryanodine receptors (RyR), only generated a localized Ca(2+) increase that did not propagate in the egg. In addition, caffeine, another stimulator of RyR, was completely without effect. Sperm-induced Ca(2+) increases in Gonionema eggs were severely affected by preinjection of heparin, an inhibitor of Ca(2+) release from IP(3)R. These results strongly suggest that there is a well-developed IP(3)R-, but not RyR-mediated Ca(2+) release mechanism in hydrozoan eggs and that the former system primarily functions at fertilization. Our present data also demonstrate that the spatial characteristics and mechanisms of Ca(2+) increases at fertilization in hydrozoan eggs resemble those reported in higher triploblastic animals. (+info)
Mitochondria as integrators of information in an early-evolving animal: insights from a triterpenoid metabolite.
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Mitochondria have the capacity to integrate environmental signals and, in animals with active stem cell populations, trigger responses in terms of growth and growth form. Colonial hydroids, which consists of feeding polyps connected by tube-like stolons, were treated with avicis, triterpenoid electrophiles whose anti-cancer properties in human cells are mediated in part by mitochondria. In treated hydroids, both oxygen uptake and mitochondrial reactive oxygen species were diminished relative to controls, similar to that observed in human cells exposed to avicins. While untreated colonies exhibit more stolon branches and connections in the centre of the colony than at the periphery, treated colonies exhibit the opposite: fewer stolon branches in the centre of the colony than at the periphery. The resulting growth form suggest an inversion of the normal pattern of colony development mediated by mitochondrial and redox-related perturbations. An as-yet-uncharacterized gradient within the colony may determine the ultimate phenotypic effects of avicin perturbation. (+info)
Complex colony-level organization of the deep-sea siphonophore Bargmannia elongata (Cnidaria, Hydrozoa) is directionally asymmetric and arises by the subdivision of pro-buds.
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Siphonophores are free-swimming colonial hydrozoans (Cnidaria) composed of asexually produced multicellular zooids. These zooids, which are homologous to solitary animals, are functionally specialized and arranged in complex species-specific patterns. The coloniality of siphonophores provides an opportunity to study the major transitions in evolution that give rise to new levels of biological organization, but siphonophores are poorly known because they are fragile and live in the open ocean. The organization and development of the deep-sea siphonophore Bargmannia elongata is described here using specimens collected with a remotely operated underwater vehicle. Each bud gives rise to a precise, directionally asymmetric sequence of zooids through a stereotypical series of subdivisions, rather than to a single zooid as in most other hydrozoans. This initial description of development in a deep-sea siphonophore provides an example of how precise colony-level organization can arise, and illustrates that the morphological complexity of cnidarians is greater than is often assumed. (+info)
Bioluminescent and red-fluorescent lures in a deep-sea siphonophore.
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Bioluminescence (light production) and fluorescence (re-emission of absorbed radiation as light) are found in an unaccountably diverse array of marine organisms, where their functions are largely unknown. Here we report a deep-sea siphonophore that twitches glowing lures to attract fish. This is rare evidence of bioluminescence used for prey attraction among nonvisual marine organisms. The lures also contain red fluorescent material that shifts the wavelength of emitted light. The existence of a red-luminescent invertebrate suggests that long-wavelength light plays a greater role in marine interactions than previously suspected. (+info)
The modulus of elasticity of fibrillin-containing elastic fibres in the mesoglea of the hydromedusa Polyorchis penicillatus.
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Hydromedusan jellyfish swim by rhythmic pulsation of their mesogleal bells. A single swimming muscle contracts to create thrust by ejecting water from the subumbrellar cavity. At the end of the contraction, energy stored in the deformation of the mesogleal bell powers the refilling stage, during which water is sucked back into the subumbrellar cavity. The mesoglea is a mucopolysaccharide gel reinforced with radially oriented fibres made primarily of a protein homologous to mammalian fibrillin. Most of the energy required to power the refill stroke is thought to be stored by stretching these fibres. The elastic modulus of similar fibrillin-rich fibres has been measured in other systems and found to be in the range of 0.2 to 1.1 MPa. In this paper, we measured the diameters of the fibres, their density throughout the bell, and the mechanical behaviour of the mesoglea, both in isolated samples and in an intact bell preparation. Using this information, we calculated the stiffness of the fibres of the hydromedusa Polyorchis penicillatus, which we found to be approximately 0.9 MPa, similar in magnitude to other species. This value is two orders of magnitude more compliant than the stiffness of the component fibrillin microfibrils previously reported. We show that the structure of the radial fibres can be modelled as a parallel fibre-reinforced composite and reconcile the stiffness difference by reinterpreting the previously reported data. We separate the contributions to the bell elasticity of the fibres and mesogleal matrix and calculate the energy storage capacity of the fibres using the calculated value of their stiffness and measured densities and diameters. We conclude that there is enough energy potential in the fibres alone to account for the energy required to refill the subumbrellar cavity. (+info)
An evolutionary conserved role of Wnt signaling in stem cell fate decision.
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Wnt/Frizzled/ss-catenin-based signaling systems play diverse roles in metazoan development, being involved not only in the establishment of body axes in embryogenesis but also in regulating stem cell fate in mammalian post-embryonic development. We have studied the role the canonical Wnt cascade plays in stem cell fate determination in Hydractinia, a member of the ancient metazoan phylum Cnidaria, by analyzing two key molecules in this pathway, frizzled and ss-catenin, and blocking GSK-3. Generally, frizzled was expressed in cells able to divide but absent in post-mitotic, terminally differentiated cells such as nerve cells and nematocytes. Transcripts of frizzled were identified in all embryonic stages beginning with maternal transcripts in the oocyte. Following gastrulation and in the planula larva, frizzled expression concentrated in the central endodermal mass from which the first interstitial stem cells and their derivatives arise. In post-metamorphic development, high levels of frizzled transcripts were detected in interstitial stem cells. Activating downstream events of the Wnt-cascade in the post-metamorphic life phase by blocking GSK-3 with paullones induced recruitment of nematocytes and nerve cells from the pool of interstitial stem cells. Terminal differentiation was preceded by an initial burst of proliferation of frizzled-positive i-cells. In activated i-cells, ss-catenin appeared in the cytoplasm, later in the nucleus. It was subsequently again observed in the cytoplasm and eventually faded out during terminal differentiation. Our results suggest an ancient role of Wnt signaling in stem cell fate determination. (+info)