Evolution of gap junctions: the missing link?
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Amino Acid Sequence↗
Biomedical subjects
Charles N David
Publications and source records attributed to Charles N David.
The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity.
BACKGROUND: Apoptotic cell death plays an essential part in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss of plasma membrane phospholipid asymmetry and exposure of phosphatidylserine on the outer leaflet. Surface exposure of phosphatidylserine is recognised by a specific receptor (phosphatidylserine receptor, PSR) and is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. RESULTS: We have cloned the PSR receptor from Hydra in order to investigate its function in this early metazoan. Bioinformatic analysis of the Hydra PSR protein structure revealed the presence of three nuclear localisation signals, an AT-hook like DNA binding motif and a putative 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase activity. All of these features are conserved from human PSR to Hydra PSR. Expression of GFP tagged Hydra PSR in hydra cells revealed clear nuclear localisation. Deletion of one of the three NLS sequences strongly diminished nuclear localisation of the protein. Membrane localisation was never detected. CONCLUSIONS: Our results suggest that Hydra PSR is a nuclear 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase. This is in contrast with the proposed function of Hydra PSR as a cell surface receptor involved in the recognition of apoptotic cells displaying phosphatidylserine on their surface. The conservation of the protein from Hydra to human infers that our results also apply to PSR from higher animals.
Amino Acid Motifs↗
Control of planula migration by LWamide and RFamide neuropeptides in Hydractinia echinata.
Planula larvae of Hydractinia echinata (Cnidaria) settled on a substratum migrate toward light. We observed that planula migration is not a continuous process. Instead, it consists of repeating cycles of active migration (about 8 min on average) and inactive resting periods (about 26 min on average). This pattern of periodic migration is regulated by LWamide and RFamide neuropeptides. LWamide (10(-8) mol l(-1)) stimulates migration primarily by making the active periods longer, whereas RFamide (10(-7) mol l(-1)) inhibits migration by blocking the initiation and also shortening the length of the active periods. Since sensory neurons containing LWamides and RFamides are present in planula larvae, it appears likely that planula migration is regulated by the release of endogenous neuropeptides in response to environmental cues.
Animal Migration↗
Replication labeling patterns and chromosome territories typical of mammalian nuclei are conserved in the early metazoan Hydra.
To investigate the evolutionary conservation of higher order nuclear architecture previously described for mammalian cells we have analyzed the nuclear architecture of the simple polyp Hydra. These diploblastic organisms have large nuclei (8-10 microm) containing about 3x10(9) bp of DNA organized in 15 chromosome pairs. They belong to the earliest metazoan phylum and are separated from mammals by at least 600 million years. Single and double pulse labeling with halogenated nucleotides (bromodeoxyuridine, iododeoxyuridine and chlorodeoxyuridine) revealed striking similarities to the known sequence of replication labeling patterns in mammalian nuclei. These patterns reflect a persistent nuclear arrangement of early, mid-, and late replicating chromatin foci that could be identified during all stages of interphase over at least 5-10 cell generations. Segregation of labeled chromatids led after several cell divisions to nuclei with single or a few labeled chromosome territories. In such nuclei distinct clusters of labeled chromatin foci were separated by extended nuclear areas with non-labeled chromatin, which is typical of a territorial arrangement of interphase chromosomes. Our results indicate the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals and suggest the existence of conserved mechanism(s) controlling this architecture.
Animals↗
Inhibition of metamorphosis by RFamide neuropeptides in planula larvae of Hydractinia echinata.
The primitive nervous system in planula larvae of Hydractinia echinata (Cnidaria) has sensory neurons containing LWamide or RFamide neuropeptides. LWamides have been shown to induce metamorphosis of planula larvae into adult polyps. We report here that RFamides act antagonistically to LWamides. RFamides inhibit metamorphosis when applied to planula larvae during metamorphosis induction by treatment with LWamides (or other inducing agents such as CsCl ions, diacylglycerol and bacterial inducers). Our results show further that RFamides act downstream of LWamide release, presumably directly on target cells mediating metamorphosis. These observations support a model in which metamorphosis in H. echinata is regulated by sensory neurons secreting LWamides and RFamides in response to environmental cues.
Animals↗
Molecular cloning and cellular distribution of two 14-3-3 isoforms from Hydra: 14-3-3 proteins respond to starvation and bind to phosphorylated targets.
In the simple metazoan Hydra a clear link between food supply and cell survival has been established. Whilst in plants 14-3-3 proteins are found to be involved in signalling cascades that regulate metabolism, in animals they have been shown to participate in cell survival pathways. In order to explore the possibility that 14-3-3 proteins in Hydra could be involved in regulating metabolism under different conditions of food supply, we have cloned two isoforms of 14-3-3 proteins. We show here that 14-3-3 proteins bind to phosphorylated targets in Hydra and form homo- and heterodimers in vitro. 14-3-3 proteins are localised in the cytoplasm of all cells and also in the nuclei of some epithelial cells. This nuclear localisation becomes more prominent during starvation. Moreover, 14-3-3 protein is present in large amounts in food granules and from this we conclude that it performs functions which are associated with metabolism and food storage in Hydra.
14-3-3 Proteins↗
Cloning and characterisation of PKB and PRK homologs from Hydra and the evolution of the protein kinase family.
Two new serine/threonine protein kinases have been cloned from Hydra cDNA. The first of these kinases belongs to the PKB/Akt family. It is expressed ubiquitously in Hydra at a relatively low level but is upregulated during head regeneration. The second kinase is a member of the PRK/PKN family. It is ubiquitously expressed in Hydra tissue, albeit at a higher level than PKB. Construction of a phylogenetic tree including the Hydra PRK and PKB kinases and two PKC homologs previously cloned by Hassel and comparing them with members of the PKC, PKB and PRK families from porifera, Dictyostelium,yeast, Drosophila, Caenorhabditis and humans provide support for a simple model for the evolution of these kinase families. An ancestral precursor which contained a pleckstrin homology domain in its N-terminus and a C-terminal kinase domain gave rise to PKB in Dictyostelium. From this ancestor the PKB/PRK and PKC families evolved. The pleckstrin homology domain was lost in the PKC and PRK families and kept in the PKB family. PKB homologs have now been found in a variety of multicellular animals with Hydra being the phylogenetically earliest representative. Members of the PRK/PKC family, on the other hand, are also present in fungi. The precursor for these kinases must have contained N-terminal regulatory domains that were retained in fungal PRKs but subsequently partitioned between kinases of the PKC and PRK groups in metazoans.
Amino Acid Sequence↗
Poly-gamma-glutamate synthesis during formation of nematocyst capsules in Hydra.
Nematocysts are explosive organelles found in all Cnidaria. Explosion of nematocyst capsules is driven by the high pressure within the capsule formed by the high concentration of poly-gamma-glutamate in the capsule matrix. Poly-gamma-glutamate is a polyanion that binds cations tightly, including the fluorescent cationic dyes acridine orange and DAPI (4',6-diamidino-2-phenylindole). We have used acridine orange and DAPI staining to localize poly-gamma-glutamate within capsules and to follow the biosynthesis of poly-gamma-glutamate during capsule formation. The results indicate that poly-gamma-glutamate biosynthesis occurs late in capsule formation after invagination of the tubule and that it is accompanied by swelling of the capsule due to increasing osmotic pressure. The matrix in all four capsule types is homogeneously filled with poly-gamma-glutamate. In vivo this poly-gamma-glutamate is complexed with monovalent cations. In addition, poly-gamma-glutamate is formed within the tubule lumen of stenoteles. We argue that this poly-gamma-glutamate is required to drive the two-step explosion process in stenotele nematocysts.
Acridine Orange↗
GFP expression in Hydra: lessons from the particle gun.
The cnidarian Hydra is an important model organism to study pattern formation and tem cell differentiation. In the past, however, it has been difficult to study gene function in Hydra because the animals have hot been accessible to gene transfection studies, we have now developed a method to transiently express GFP-tagged proteins in Hydra using a green fluorescent protein (GFP) expression plasmid under the control of the Hydra actin promoter and a particle gun to introduce it into Hydra cell nuclei. We achieve strong transient GFP expression in a small but reproducible number of epithelial and interstitial cells. Implications for the use of this method to carry out single cell assays with GFP-tagged Hydra proteins are discussed.
Animals↗