Centrin is necessary for the formation of the motile apparatus in spermatids of Marsilea.
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 beta-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 beta-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 beta-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.
Klink
V P
VP
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.
Wolniak
S M
SM
D000546:Amanitins / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D063245:Plant Development; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D013081:Sperm Motility; D013091:Spermatogenesis / Q000187:drug effects / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014404:Tubulin / Q000378:metabolism
D000546:Amanitins; D010940:Plant Proteins; D018749:RNA, Plant; D014404:Tubulin; D003513:Cycloheximide