Centrin Is Necessary for the Formation of the Motile Apparatus in Spermatids ofMarsilea

Abstract

During spermiogenesis in the water fern, Marsilea vestita, basal bodies are synthesized de novo in cells that lack preexisting centrioles, in a particle known as a blepharoplast. We have focused on basal body assembly in this organism, asking what components are required for blepharoplast formation. Spermiogenesis is a rapid process that is activated by placing dry microspores into water. Dry microspores contain large quantities of stored protein and stored mRNA, and inhibitors reveal that certain proteins are translated from stored transcripts at specific times during development. Centrin translation accompanies blepharoplast appearance, while ␤-tubulin translation occurs later, during axonemal formation. In asking whether centrin is an essential component of the blepharoplast, we used antisense, sense, and double-stranded RNA probes made from the Marsilea centrin cDNA, MvCen1, to block centrin translation. We employed a novel method to introduce these RNAs directly into the cells. Antisense and sense both arrest spermiogenesis when blepharoplasts should appear, and dsRNA made from the same cDNA is an effective inhibitor at concentrations at least 10 times lower than either of the single-stranded RNA used in these experiments. Blepharoplasts are undetectable and basal bodies fail to form. Antisense, sense, and dsRNA probes made from Marsilea ␤-tubulin permitted normal development until axonemes form. In controls, antisense, sense, and dsRNA, made from a segment of HIV, had no effect on spermiogenesis. Immunoblots suggest that translational blocks induced by centrin-based RNA are gene specific and concentration dependent, since neither ␤-tubulin-nor HIV-derived RNAs affects centrin translation. The disruption of centrin translation affects microtubule distributions in spermatids, since centrin appears to control formation of the cytoskeleton and motile apparatus. These results show that centrin plays an essential role in the formation of a motile apparatus during spermiogenesis of M. vestita. INTRODUCTIONThroughout most of the eukaryotic realm, the formation of male gametes involves the synthesis and assembly of a motile apparatus. The motile apparatus comprises flagellar or ciliary axonemes, whose rapid beat frequency and precise beat shape are the consequence of mechanochemical interactions between scores of proteins that are arranged in a remarkably complex cylindrical array (Dutcher, 1995). A controlling template for axonemal formation is the basal body (Mizukami and Gall, 1966), which resides in the cortical cytoplasm at the base of the axoneme (Ringo, 1967;Fulton, 1971; Dibbayawan et al., 1995). Basal bodies closely resemble centrioles (Gould, 1975;Kuriyama and Borisy, 1981;Kochanski and Borisy, 1990), and they are usually formed from or in close association with centrioles (Dirksen, 1991;Marshall and Rosenbaum, 2000), (however, see Fulton [1971]; Fulton and Dingle [1971]; Gould [1975]). The ubiquity of this association has led to the perception that all basal bodies arise from these cytoplasmic organell...

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The Nuclear RNA-binding Protein Sam68 Translocates to the Cytoplasm and Associates with the Polysomes in Mouse Spermatocytes

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Rapid and Efficient Suppression of Gene Expression in a Single-Cell Model System, Ceratopteris richardii

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