Molecular characterization of radial spoke subcomplex containing radial spoke protein 3 and heat shock protein 40 in sperm flagella of the ascidian Ciona intestinalis

Members of the heat-shock protein (HSP)40 regulate the protein folding activity of HSP70 proteins and help the functional specialization of this molecular chaperone system in various types of cellular events. We have recently identified Hsp40 as a component of flagellar axoneme in the ascidian Ciona intestinalis, suggesting a correlation between Hsp40 related chaperone system and flagellar function. In this study, we have found that Ciona 37-kDa Hsp40 is extracted from KCl-treated axonemes with 0.5 M KI solution and comigrates with radial spoke protein (RSP)3 along with several proteins as a complex through gel filtration and ion exchange columns. Peptide mass fingerprinting with matrix-assisted laser desorption ionization/time of flight/mass spectrometry revealed that other proteins in the complex include a homolog of sea urchin spokehead protein (homolog of RSP4/6), a membrane occupation and recognition nexus repeat protein with sequence similarity with meichroacidin, and a functionally unknown 33-kDa protein. A spoke head protein, LRR37, is not included in the complex, suggesting that the complex constructs the stalk of radial spoke. Immunoelectron microscopy indicates that Hsp40 is localized in the distal portion of spoke stalk, possibly at the junction between spoke head and the stalk.     

Building a radial spoke: flagellar radial spoke protein 3 (RSP3) is a dimer

Radial spokes are critical multisubunit structures required for normal ciliary and eukaryotic flagellar motility. Experimental evidence indicates the radial spokes are mechanochemical transducers that transmit signals from the central pair apparatus to the outer doublet microtubules for local control of dynein activity. Recently, progress has been made in identifying individual components of the radial spoke, yet little is known about how the radial spoke is assembled or how it performs in signal transduction. Here we focus on radial spoke protein 3 (RSP3), a highly conserved AKAP located at the base of the radial spoke stalk and required for radial spoke assembly on the doublet microtubules. Biochemical approaches were taken to further explore the functional role of RSP3 within the radial spoke structure and for control of motility. Chemical crosslinking, native gel electrophoresis, and epitope-tagged RSP3 proteins established that RSP3 forms a dimer. Analysis of truncated RSP3 proteins indicates the dimerization domain coincides with the previously characterized axoneme binding domain in the N-terminus. We propose a model in which each radial spoke structure is built on an RSP3 dimer, and indicating that each radial spoke can potentially localize multiple PKAs or AKAP-binding proteins in position to control dynein activity and flagellar motility.     

Insulin-Like Immunoreactivity in the Neural Ganglion of the Ascidian Ciona intestinalis

Antisera raised against salmon insulin and bovine insulin were used to assess the distribution of insulin-like immunoreactive neurons in the neural ganglion of Ciona intestinalis. Cell bodies of two sizes were found in cortical and medullary positions, together with an extensive network of immunoreactive fibres. These observations indicate that an insulin-like material is an important component of the central nervous system in Ciona and gives weight to the hypothesis that protochordates utilize insulin-like factors as neurotransmitters or modulators, as do their invertebrate ancestors.     

Assembly of flagellar radial spoke proteins in Chlamydomonas: identification of the axoneme binding domain of radial spoke protein 3

Radial spokes of the eukaryotic flagellum extend from the A tubule of each outer doublet microtubule toward the central pair microtubules. In the paralyzed flagella mutant of Chlamydomonas pf14, a mutation in the gene for one of 17 polypeptides that comprise the radial spokes results in flagella that lack all 17 spoke components. The defective gene product, radial spoke protein 3 (RSP3), is, therefore, pivotal to the assembly of the entire spoke and may attach the spoke to the axoneme. We have synthesized RSP3 in vitro and assayed its binding to axonemes from pf14 cells to determine if RSP3 can attach to spokeless axonemes. In vitro, RSP3 binds to pf14 axonemes, but not to wild-type axonemes or microtubules polymerized from purified chick brain tubulin. The sole axoneme binding domain of RSP3 is located within amino acids 1-85 of the 516 amino acid protein; deletion of these amino acids abolishes binding by RSP3. Fusion of amino acids 1-85 or 42-85 to an unrelated protein confers complete or partial binding activity, respectively, to the fusion protein. Transformation of pf14 cells with mutagenized RSP3 genes indicates that amino acids 18-87 of RSP3 are important to its function, but that the carboxy-terminal 140 amino acids can be deleted with little effect on radial spoke assembly or flagellar motility.     

Concanavalin A Modifies Allo-specific Sperm-Egg Interactions in the Ascidian, Ciona intestinalis

In the self sterile ascidian, Ciona intestinalis , the spermatozoa rarely bind to the vitelline coat of autologous eggs and never penetrate it. We report here that concanavalin A (ConA), a lectin recognizing mannose or glucose residues of carbohydrates, can modify these self- and nonself-specific sperm-egg interactions. When eggs were pretreated with 0.1–0.5 mg/ml of ConA, about two thousand spermatozoa became attached to the autologous vitelline coat within five minutes of insemination. The effect of ConA was not modified by the addition of D-mannose or pretreatment of spermatozoa with ConA, showing that ConA does not function merely as a ligand bridging the sperm and vitelline coat. In contrast to the marked enhancement of sperm-egg binding, ConA did not facilitate the penetration of spermatozoa through the autologous vitelline coat. Even in non-autologous insemination, it blocked the sperm penetration and, consequently, fertilization did not occur, as shown by Rosati et al. (1978). D-Mannose, when mixed with ConA in advance, completely abolished this inhibitory effect of ConA. Lotus agglutinin, a fucose-binding lectin, was less effective and wheat germ agglutinin and soy bean agglutinin had no effect on sperm entry in the perivitelline space. The results of this study are discussed in relation to the possible involvement of mannosyl and/or glucosyl glycoconjugates in allo-specific sperm-egg interactions.     

Cytological Characterization of Self Incompatibility in Gametes of the Ascidian, Ciona intestinalis

We have determined how many elements are involved in the regulation of self-fertilization in the solitary ascidian, Ciona intestinalis that is an incompletely self-sterile species. Animals collected in the field were repeatedly induced to spawn in order to examine their selfing ratios. About 20% of them were self-fertile, although the ratios fluctuated considerably among respective spawnings. Naturally or acid-induced self-fertile gametes required much longer time for selfing than that for crossing. Egg-suspending seawater (egg water) as such activated sperm motility, but it lowered conspicuously the self-fertilization ratio. Self-sterile spermatozoa could scarcely bind to the vitelline coat (VC) of glycerinated autologous eggs. or in case they bound well to it the sperm flagella ceased to beat within five min of the 'insemination'. The staining of sectioned gametes with DAPI, a fluorescent dye of DNA, showed that in selfing the spermatozoa could hardly penetrate the VC even though they bound well to it. The results of this study show that the block of self-fertilization can be classified into four elements from a phenomenal viewpoint, such as egg water, low affinity of sperm-VC binding, inactivation of bound sperm and difficulty in sperm penetration through the VC.     

The leucine-rich repeat as a protein recognition motif

Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with diverse functions, including GTPase-activating protein rna1p from the ribonuclease-inhibitor-like subfamily; spliceosomal protein U2A', Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily; Skp2 from the cysteine-containing subfamily; and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.     

Self-Nonself Recognition Activity Extracted from Self-Sterile Eggs of the Ascidian, Ciona intestinalis

Eggs of the hermaphrodite, self-sterile ascidian, Ciona intestinalis , were washed with acid seawater (pH 3.2), and the washing solution was then adjusted to pH 8.2. This solution was found to inhibit only the binding of non-autologous sperm to the vitelline coat (VC) of eggs, indicating that it contained self-nonself recognition activity. This activity was heat-stable and insensitive to trypsin, but was destroyed by V-8 protease and α-glucosidase. Both the hydrophobic and hydrophilic components of a lyophilized powder of the extract showed allo-recognizing activity. On TLC, the hydrophobic components gave a major spot of glucose (Glc) and a peptide spot(s) containing mainly glutamic acid and/or glutamine (Glx). The glucosyl conjugate was purified by HPLC and shown to block sperm-egg binding to various extents. Individual peptide subfractions had no inhibitory activity, but in combination they showed inhibitory activity. These findings suggest that the acid extract of Ciona eggs contains a Glc-enriched nonspecific inhibitor of sperm-egg binding, which could be the primary effector of self-incompatibility, and Glx-enriched modulators, which serve as acceptors of allo-sperm. The cooperative interactions of these components may be responsible for the diversity of allo-recognition in Ciona gametes.     

Helper Function of Follicle Cells in Sperm-Egg Interactions of the Ascidian, Ciona intestinalis

Studies were made on the involvement in sperm-egg interactions of follicle cells of Ciona intestinalis , which are tall, vacuolated cells attached to the outer surface of the egg vitelline coat. The basal surface of the follicle cells is polygonal. The borders between cells could easily be observed by the binding of fluorescent SBA (soy bean agglutinin), a lectin recognizing N-acetylgalactosamine (GaINAc) residues. At fertilization many spermatozoa aggregate along these polygonal borders of cells on the vitelline coat, through which they entered the perivitelline space. The removal of follicle cells was sometimes associated with loss of SBA-binding sites, and in such cases the sperm did not show a hexagonal pattern of aggregation, but became dispersed all over the vitelline coat. Removal of the follicle sometimes delayed fertilization. Examination of sections of gametes stained with DAPI, a fluorescent dye staining DNA, showed that removal of the follicle reduced the number of spermatozoa bound to the vitelline coat and, more especially, the number of spermatozoa penetrating through the vitelline coat. The blockage of GalNAc residues on the vitelline coat with SBA did not appreciably affect the time course of fertilization or the number of sperm associated with eggs. These findings are discussed in relation to the role of follicle cells in facilitating sperm aggregation on the vitelline coat and their penetration through it.     

Identification of neuron-specific promoters in Ciona intestinalis

We isolated 5' flanking regions of four genes, Ci-Galphai1, Ci-arr, Ci-vAChTP, and Ci-vGAT, each of which is expressed in distinct sets of neurons in the central nervous system of the ascidian Ciona intestinalis, and we examined their function by introducing green fluorescent protein (GFP)-fusion constructs into Ciona embryos. The reporter gene driven by the 5' flanking region of Ci-Galphai1, Ci-arr, and Ci-vAChTP recapitulated the endogenous gene expression patterns, while that of Ci-vGAT can drive GFP expression in particular subsets of neurons expressing the endogenous gene. Deletion analysis revealed that the Ci-Galphai1 promoter consists of multiple regulatory modules controlling the expression in different types of cells. The GFP fluorescence enabled visualization of cell bodies and axons of different sets of neurons in ascidian larvae. These promoters can be a powerful tool for studying molecular mechanisms of neuronal development as well as neuron networks and functions in ascidians.     

A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex

A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs.     

Disruption of the A-kinase anchoring domain in flagellar radial spoke protein 3 results in unregulated axonemal cAMP-dependent protein kinase activity and abnormal flagellar motility

Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility.     

Identification of a novel testis-specific leucine-rich protein in humans and mice

A novel testis-specific protein, termed LRTP, was identified by screening both human and mouse testis and mouse pachytene spermatocyte cDNA libraries. Sequence analyses (GenBank accession number: AF092208) revealed that LRTP contains an amino terminus leucine-rich repeat domain. There are several acidic regions rich in glutamic acid in the C-terminus. The sequence, by GenBank search, shows similarities to LANP and SDS22+, leucine-rich repeat proteins localized to the nucleus and involved in the regulation of protein phosphatases. In mouse, the mRNA is first detected at about Day 14 postpartum, presumably when mid-pachytene spermatocytes are first seen. In situ hybridization confirmed the expression of the LRTP mRNA at this stage of spermatogenesis. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of pachytene and diplotene cells, corresponding to late prophase of meiosis I. Immunohistochemical localization is markedly reduced in secondary spermatocytes, suggesting a functional association of LRTP with meiosis. An LRTP cDNA probe did not bind to mouse ovary RNA in a dot blot assay.     

An Ultrastructural Study of the Hemocytes of the Pericardial Body in the Ascidian Ciona intestinalis (L.)

Abstract The hemocytes of the pericardial body of Ciona intestinalis were studied by electron microscopy. Our findings showed that stem cells, clear vesicular granulocytes, microgranulocytes, unilocular granulocytes and globular granulocytes are present at the periphery of the smaller-sized pericardial bodies. The stem cells are small round cells with a large nucleus, with or without nucleolus, and homogeneous cytoplasm containing numerous ribosomes. The clear vesicular granulocytes are characterized by an ameboid shape and cytoplasm containing several large electron-lucent vacuoles and small electron-dense granules. The microgranulocytes are variable in shape and contain numerous large electron-dense granules. The unilocular granulocytes show a single large vacuole with an electron-dense or electron-lucent content and a thin layer of peripheral cytoplasm that contains the flattened nucleus. The globular granulocytes are characterized by the presence of large vacuoles containing either fibrogranular material or electron-dense aggregates.     

Identification of a unique TLR2-interacting peptide motif in a microbial leucine-rich repeat protein

Pathogenesis of many bacterially-induced inflammatory diseases is driven by Toll-like receptor (TLR) mediated immune responses following recognition of bacterial factors by different TLRs. Periodontitis is a chronic inflammation of the tooth supporting apparatus often leading to tooth loss, and is caused by a Gram-negative bacterial consortium that includes Tannerella forsythia. This bacterium expresses a virulence factor, the BspA, which drives periodontal inflammation by activating TLR2. The N-terminal portion of the BspA protein comprises a leucine-rich repeat (LRR) domain previously shown to be involved in the binding and activation of TLR2. The objective of the current study was to identify specific epitopes in the LRR domain of BspA that interact with TLR2. Our results demonstrate that a sequence motif GC(S/T)GLXSIT is involved in mediating the interaction of BspA with TLR2. Thus, our study has identified a peptide motif that mediates the binding of a bacterial protein to TLR2 and highlights the promiscuous nature of TLR2 with respect to ligand binding. This work could provide a structural basis for designing peptidomimetics to modulate the activity of TLR2 in order to block bacterially-induced inflammation.     

Synleurin,a novel leucine-rich repeat protein that increases the intensity of pleiotropic cytokine responses

We have identified and characterized a novel single span transmembrane leucine-rich repeat protein, synleurin, that renders cells highly sensitive to the activation by cytokines and lipopolysaccharide (LPS). The major part of the extracellular domain consists of a leucine-rich repeats (LRR) cassette. The LRR central core has 12 analogous LRR repeating modules arranged in a seamless tandem array. The LRRs are most homologous to that of chondroadherin, insulin-like growth factor binding proteins, platelet glycoprotein V, slits, and toll-like receptors. Synleurin expression was detected at low levels in many tissues, including smooth muscle, brain, uterus, pancreas, cartilage, adipose, spleen, and testis. When synleurin is ecotopically expressed in transfected cells, the cells exhibit amplified responses to bFGF, EGF, PDGF-B, IGF-1, IGF-2, and LPS. Synleurin gene (slrn) maps to human chromosome at 5q12. The name synleurin reflects its synergistic effect on cytokine stimulation and its prominent leucine-rich repeats.