Changes in the abundance and distribution of conserved centrosomal, cytoskeletal and ciliary proteins during spermiogenesis in Marsilea vestita.
Spermiogenesis in the male gametophytes of the water fern Marsilea vestita is a precise and rapid process resulting in the production of ciliated gametes. Development begins from a single cell within the microspore wall that undergoes nine rapid cell division cycles in distinct planes to produce 32 spermatids that are surrounded by 7 sterile cells. Thereafter, the de novo formation of basal bodies occurs in a discrete cytoplasmic particle known as a blepharoplast, with the subsequent formation of a complex ciliary apparatus in elongating spermatids. The rate and extent of development appear to be controlled at a post-transcriptional level, where the sudden translation of specific stored mRNAs (e.g., centrin) results in the formation of particular structures in the cells (e.g., blepharoplasts). We show here that additional centrosomal and cytoskeletal antigens known as SF assemblin, p95 kDa protein, delta tubulin, gamma tubulin, Xgrip109, Aik, CTR453, RanBPM, BX63, RSP6, and alpha tubulin each exhibit specific localization patterns both on immunoblots of gametophyte protein isolates and in fixed cells. BAp90, PP4, and RLC exhibit specific localization patterns in fixed cells. We show that the antigens exhibit complex patterns of abundance during spermiogenesis. In an attempt to identify regulatory agents involved in spermiogenesis, we employed a RNAi-based screen of 41 randomly selected gametophyte cDNAs on developing populations of synchronously growing gametophytes. The gametophytes treated with each of the RNAi probes exhibited arrest at a specific stage of development. A consequence of anomalous development was the block to assembly of the ciliary apparatus, an effect highlighted by altered staining with anti-centrin, anti-beta-tubulin, and anti-RSP6 antibodies. Our results show that complex, integrated processes of translation and protein partitioning apparently underlie the assembly of the ciliary apparatus during spermiogenesis in male gametophytes of M. vestita.
Klink
Vincent P
VP
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
Wolniak
Stephen M
SM
D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D018385:Centrosome / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D007150:Immunohistochemistry; D043964:Marsileaceae / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D012330:RNA, Double-Stranded / Q000235:genetics / Q000494:pharmacology; D012098:Reproduction / Q000276:immunology / Q000502:physiology; D016415:Sequence Alignment; D013170:Spores / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*
D000906:Antibodies; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D010940:Plant Proteins; D012330:RNA, Double-Stranded