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.

Axonemes are highly organized microtubule-based structures conserved in many eukaryotes. In an attempt to study axonemes by a proteomics approach, we selectively cloned cDNAs of axonemal proteins by immunoscreening the testis cDNA library from the ascidian Ciona intestinalis by using an antiserum against whole axonemes. We report here a 37-kDa protein of which cDNA occurred most frequently among total positive clones. This protein, named LRR37, belongs to the class of SDS22ϩ leucine-rich repeat (LRR) family. LRR37 is different from the LRR outer arm dynein light chain reported in Chlamydomonas and sea urchin flagella, and thus represents a novel axonemal LRR protein. Immunoelectron microscopy by using a polyclonal antibody against LRR37 showed that it is localized on the tip of the radial spoke, most likely on the spoke head. The LRR37 protein in fact seems to form a complex together with radial spoke protein 3 in a KI extract of the axonemes. These results suggest that LRR37 is a component of the radial spoke head and is involved in the interaction with other radial spoke components or proteins in the central pair projection.

Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca 2+ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca 2+ -binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized.Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca 2+ -binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF-hand Ca 2+ -binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross-linking experiment showed that calaxin binds to β-tubulin in situ. Overlay experiments further indicated that calaxin binds the β-dynein heavy chain of outer arm dynein in the presence of Ca 2+ .Conclusions. These results suggest that calaxin is a potential Ca 2+ -dependent modulator of outer arm dynein in metazoan cilia and flagella.

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Spermatozoa undergo dramatic physiological changes at fertilization. In the ascidian Ciona intestinalis, an egg-derived substance named SAAF induces both sperm activation and chemotaxis to the egg. To elucidate the molecular mechanism underlying these phenomena, whole sperm proteins before and after SAAF-treatment were analyzed by two-dimensional gel electrophoresis. By comparison of spot patterns before and after activation, we found twelve proteins that changed the isoelectric points. Seven proteins were shown to be axonemal proteins and others were suggested to be non-axonemal components. Analysis of these proteins by MS-based proteomic system revealed that components of several substructures of the axonemes underwent the changes in isoelectric point at sperm activation, including WD-repeat intermediate chains of outer and inner arm dyneins and a radial spoke protein LRR37, as well as novel axonemal proteins with armadillo repeats or SMC domain. Molecules for cell signaling such as 14-3-3 proteins, Skp1 and VCP/p97 also showed isoelectric changes at sperm activation. These results show a comprehensive feature for signaling mechanism of the activation of spermatozoa at fertilization and also shed new lights on the regulation of ciliary and flagellar movements.