Evidence for involvement of metalloendoproteases in a step in sea urchin gamete fusion

Membrane fusion events are required in three steps in sea urchin fertilization: the acrosome reaction in sperm, fusion of the plasma membrane of acrosome-reacted sperm with the plasma membrane of the egg, and exocytosis of the contents of the egg cortical granules. We recently reported the involvement of a Zn2+-dependent metalloendoprotease in the acrosome reaction (Farach, H. C., D. I. Mundy, W. J. Strittmatter, and W. J. Lennarz. 1987. J. Biol. Chem. 262:5483-5487). In the current study, we investigated the possible involvement of metalloendoproteases in the two other fusion events of fertilization. The use of inhibitors of metalloendoproteases provided evidence that at least one of the fusion events subsequent to the acrosome reaction requires such enzymes. These inhibitors did not block the binding of sperm to egg or the process of cortical granule exocytosis. However, sperm-egg fusion, assayed by the ability of the bound sperm to establish cytoplasmic continuity with the egg, was inhibited by metalloendoprotease substrate. Thus, in addition to the acrosome reaction, an event in the gamete fusion process requires a metalloendoprotease.     

Globin genes are present in Ciona intestinalis

The key position of the Ciona intestinalis basal to the vertebrate phylogenetic tree brings up the question of which respiratory proteins are used by the tunicate to facilitate oxygen transport and storage. The publication of the Ciona draft genome sequence suggests that globin genes are completely missing and that-like some molluscs and arthropods-the sea squirt uses hemocyanin instead of hemoglobin for respiration. However, we report here the presence and expression of at least four distinct globin gene/protein sequences in Ciona. This finding is in agreement with the ancestral phylogeny of the vertebrate globins. Moreover, it seems likely that the Ciona hemocyanin-like sequences have enzymatic instead of respiratory functions.     

Isolation and characterization of genes that are expressed during Ciona intestinalis metamorphosis

In ascidians, the events of metamorphosis transform the non-feeding, mobile tadpole larva into a filter-feeding, fixed juvenile, and the process involves rearrangements of cells, two organs and physiological changes. Differential screening was used to isolate two genes that are not expressed in swimming larvae but are expressed immediately after the initiation of metamorphosis in Ciona intestinalis. One of the genes, Ci-meta1, encodes a polypeptide with a putative secretion signal sequence, 6 epidermal growth factor (EGF)-like repeats and 13 calcium-binding EGF-like repeats. The gene begins to be expressed immediately after the beginning of metamorphosis in the adhesive organ and is likely to be associated with the signal response for metamorphosis. Another gene named Ci-meta2 encodes a protein with a putative secretion signal and three thrombospondin type-1 repeats. Ci-meta2 gene expression begins at the larval stage and is upregulated in the metamorphosing juveniles. Ci-meta2 expression is found in three regions; the adhesive organ which is also associated with settlement, the neck region between the trunk and the tail of the larva which is associated with tail resorption, and dorsal regions of the trunk which correspond to the location of the siphon primordium. This gene may be involved in the dynamic arrangement of cells during ascidian metamorphosis.     

Evidence that talin alternative splice variants from Ciona intestinalis have different roles in cell adhesion

Background  

Talins are large, modular cytoskeletal proteins found in animals and amoebozoans such as Dictyostelium discoideum. Since the identification of a second talin gene in vertebrates, it has become increasingly clear that vertebrate Talin1 and Talin2 have non-redundant roles as essential links between integrins and the actin cytoskeleton in distinct plasma membrane-associated adhesion complexes. The conserved C-terminal I/LWEQ module is important for talin function. This structural element mediates the interaction of talins with F-actin. The I/LWEQ module also targets mammalian Talin1 to focal adhesion complexes, which are dynamic multicomponent assemblies required for cell adhesion and cell motility. Although Talin1 is essential for focal adhesion function, Talin2 is not targeted to focal adhesions. The nonvertebrate chordate Ciona intestinalis has only one talin gene, but alternative splicing of the talin mRNA produces two proteins with different C-terminal I/LWEQ modules. Thus, C. intestinalis contains two talins, Talin-a and Talin-b, with potentially different activities, despite having only one talin gene.     

Evidence for dynamic and multiple roles for huntingtin in Ciona intestinalis

Although mutations in the huntingtin gene (HTT) due to poly-Q expansion cause neuropathology in humans (Huntington’s disease; HD), the normal function(s) of the gene and its protein (HTT) remain obscure. With new information from recently sequenced invertebrate genomes, the study of new animal models opens the possibility of a better understanding of HTT function and its evolution. To these ends, we studied huntingtin expression pattern and dynamics in the invertebrate chordate Ciona intestinalis. Ciona huntingtin (Ci-HTT) shows a biphasic expression pattern during larval development and prior to metamorphosis. A single form of huntingtin protein is present until the early larval stages, at which time two different mass proteins become evident in the metamorphically competent larva. An antibody against Ci-HTT labeled 50 cells in the trunk mesenchyme regions in pre-hatching and hatched larvae and probably represents the distribution of the light form of the protein. Dual labeling with anti-Ci-HTT and anti-aldoketoreductase confirmed the presence of Ci-HTT in mesenchyme cells. Suppression of Ci-HTT RNA by a morpholino oligonucleotide reduced the number and apparent mobility of Ci-HTT positive cells. In Ciona, HTT expression has a dynamic temporal and spatial expression pattern that in ontogeny precedes metamorphosis. Although our results may reflect a derived function for the protein in pre- and post-metamorphic events in Ciona, we also note that as in vertebrates, there is evidence for multiple differential temporal expression, indicating that this protein probably has multiple roles in ontogeny and cell migration.     

A genome-wide survey of genes for enzymes involved in pigment synthesis in an ascidian, Ciona intestinalis

The draft genome sequence and a large quantity of EST and cDNA information are now available for the ascidian Ciona intestinalis. In the present study, genes involved in pigment synthesis pathways were identified in the decoded genome of Ciona, and information about these genes was obtained from available EST and cDNA sequences. It was found that the Ciona genome contains orthologous genes for each enzyme of the melanin, pteridine, ommochrome, papiliochrome, and heme synthesis pathways. Several appear as independent duplications in the Ciona genome. Because cDNA clones for all but two of these genes have already been isolated by the cDNA project, C. intestinalis will provide an experimental system to explore molecular mechanisms underlying color patterns, through future genome-wide studies.     

Mechanisms of chromosome banding. II. Evidence that histones are not involved

C-and G-banding in mouse metaphase chromosomes is unaffected by exposure of the chromosomes to 0.2 N HCl for 4 h. Electrophoretic studies indicate that this is adequate to completely remove the histones from fixed and dried chromatin thus indicating that histones are not involved in C- or G-banding.     

Inducible galectins are expressed in the inflamed pharynx of the ascidian Ciona intestinalis

Although ascidians belong to a key group in chordate phylogenesis, amino acid sequences of Ciona intestinalis galectin-CRDs (CiLgals-a and -b) have been retained too divergent from vertebrate galectins. In the present paper, to contribute in disclosing Bi-CRD galectin evolution a novel attempt was carried out on CiLgals-a and -b CRDs phylogenetic analysis, and their involvement in ascidian inflammatory responses was shown. CiLgals resulted aligned with Bi-CRD galectins from vertebrates (Xenopus tropicalis, Gallus gallus, Mus musculus, Homo sapiens), cephalochordates (Branchiostoma floridae), echinoderms (Strongylocentrotus purpuratus) and a mono-CRD galectin from the ascidian Clavelina picta. The CiLgals-a N-terminal and C-terminal CRDs contain the signature sequence involved in carbohydrate binding, whereas the CiLgals-b C-CRD presents only three out of seven key aminoacids and it could not be suitable as sugar binding motif. Sequence similarity between clusters suggests an evolutionary model based on CRD domain gene duplication and sequence diversification. In particular CiLgals-b N-CRD and C-CRD were similar to each other and both grouped with the ascidian C. picta mono-CRD. Homology modeling process shows a CiLgals molecular structure superimposed to chicken and mouse galectins. The CiLgals-a and CiLgals-b genes were upregulated by LPS inoculation suggesting that they are inducible and expressed in the inflamed pharynx as revealed by real-time PCR analysis. Finally, in situ hybridization and immunohistochemical assays showed their localization in the inflamed tissues, while immunoblotting analysis indicated that CiLgals can form oligomers.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

Novel genes involved in canonical Wnt/beta-catenin signaling pathway in early Ciona intestinalis embryos

We report here characterization of five genes for novel components of the canonical Wnt/ β -catenin signaling pathway. These genes were identified in the ascidian Ciona intestinalis through a loss-of-function screening for genes required for embryogenesis with morpholinos, and four of them have counterparts in vertebrates. The five genes we studied are as follows: Ci-PGAP1 , a Ciona orthologue of human PGAP1 , which encodes GPI (glycosylphosphatidylinositol) inositol-deacylase, Ci-ZF278 , a gene encoding a C2H2 zinc-finger protein, Ci-C10orf11 , a Ciona orthologue of human C10orf11 that encodes a protein with leucine-rich repeats, Ci-Spatial/C4orf17 , a single counterpart for two human genes Spatial and C4orf17 , and Ci-FLJ10634 , a Ciona orthologue of human FLJ10634 that encodes a member of the J-protein family. Knockdown of each of the genes mimicked β -catenin knockdown and resulted in suppression of the expression of β -catenin downstream genes ( Ci-FoxD , Ci-Lhx3 , Ci-Otx and Ci-Fgf9/16/20 ) and subsequent endoderm formation. For every gene, defects in knockdown embryos were rescued by overexpression of a constitutively active form, but not wild-type, of Ci- β -catenin. Dosage-sensitive interactions were found between Ci-β-catenin and each of the genes. These results suggest that these five genes act upstream of or parallel to Ci- β -catenin in the Wnt/ β -catenin signaling pathway in early Ciona embryos.     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

Characterisation of cellulose-binding proteins that are involved in the adhesion mechanism of Fibrobacter intestinalis DR7

Cellulose-binding proteins (CBP) isolated from cell envelopes of the cellulolytic bacterium Fibrobacter intestinalis strain DR7 were studied in order to investigate the adhesion mechanism. The proteins were examined for their reaction with antibodies that specifically block bacterial adhesion, response to glycosylation staining and monosaccharide composition. To this end, the effect of some monosaccharides (CBP components) on blocking of DR7 adhesion to cellulose was determined. Previous study had shown the occurrence of 16 CBP in the outer membrane and periplasm of DR7, of which 6 had endoglucanase activity (Miron and Forsberg 1998). Data from the present study show that most of the 16 CBP of DR7, except for the 38-, 90- and 180-kDa proteins, are glycosylated. Rabbit antibodies that specifically block DR7 adhesion were prepared by affinity preabsorption of antiserum against wild-type DR7 with bacterial cells of its adherence-defective mutant (DR7-M). The preabsorbed antibodies reacted positively in Western blotting with glycosylated CBP of 225, 200, 150, 70, 45 and <38 kDa from the DR7 outer membrane, and reacted weakly with CBP of DR7-M. Modification of glycosidic residues attached to the CBP of DR7 by periodate oxidation prevented any reaction with the preabsorbed antibodies. Monosaccharide analysis by HPLC of isolated CBP from the outer membrane and periplasm of DR7 cells, showed that galactosamine, glucosamine, galacturonic acid, and glucuronic acid were the predominant monosaccharide components of CBP that can block the adhesion of DR7 cells to cellulose. It is suggested that some glycosylated residues of CBP may have a predominant role in the adhesion of DR7 to cellulose. Received: 10 July 1998 / Received revision: 12 November 1998 / Accepted: 14 November 1998