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...

The utilization of stored RNA is a driving force in rapid development. Here, we show that retention and subsequent removal of introns from pre-mRNAs regulate temporal patterns of translation during rapid and posttranscriptionally controlled spermatogenesis of the fern Marsilea vestita. Analysis of RNAseq-derived transcriptomes revealed a large subset of intron-retaining transcripts (IRTs) that encode proteins essential for gamete development. Genomic and IRT sequence comparisons show that other introns have been previously removed from the IRT pre-mRNAs. Fully spliced isoforms appear at distinct times during development in a spliceosome-dependent and transcription-independent manner. RNA interference knockdowns of 17/17 IRTs produced anomalies after the time points when those transcripts would normally be spliced. Intron retention is a functional mechanism for forestalling precocious translation of transcripts in the male gametophyte of M. vestita. These results have broad implications for plant gene regulation, where intron retention is widespread.

Mitotic PtK1 cells, lysed at anaphase into a carbowax 20 M Brij 58 solution, continue to move chromosomes toward the spindle poles and to move the spindle poles apart at 50% in vivo rates for 10 min. Chromosome movements can be blocked by adding metabolic inhibitors to the lysis medium and inhibition of movement can be reversed by adding ATP to the medium. Vanadate at micromolar levels reversibly inhibits dynein ATPase activity and movement of demembranated flagella and cilia. It does not affect glycerinated myofibril contraction or myosin ATPase activity at less than millimolar concentrations. Vanadate at 10-100/xM reversibly inhibits anaphase movement of chromosomes and spindle elongation. After lysis in vanadate, spindles lose their fusiform appearance and become more barrel shaped. In vitro microtubule polymerization is insensitive to vanadate.

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