ASGSB 1999 Annual Meeting Abstracts

MACROMOLECULAR ORDER FROM DUST AND CLOUDS: RATE-LIMITING STEPS IN THE ASSEMBLY OF THE MOTILE APPARATUS DURING SPERMIOGENESIS IN MARSILEA. S.M. Wolniak, V.P. Klink and C.W. Tsai. Dept. of Cell Biol. and Molec. Genetics, Univ. of Maryland, College Park

Marsilea vestita is a water fern that forms 32 motile male gametes within each of its microspores. Mature spermatozoids emerge 11 h after the dry spore is placed into an aqueous medium. Each spermatozoid has an elongated nucleus, a complex microtubule cytoskeleton and ~140 cilia situated distally along a coiled cell body. The formation of the motile apparatus requires the de novo synthesis of basal bodies, a process that begins in spermatocytes, 4 h after imbibition, in a particle known as a blepharoplast. The blepharoplast functions as a centrosome during the last mitotic division of the spermatocytes. We have focused on basal body assembly, asking what components must be made for blepharoplast maturation. We have shown that dry microspores contain large quantities of stored protein and mRNA. Inhibitor and radiolabeling experiments reveal that certain proteins are translated from stored mRNA at specific times during development. In contrast to -, - and -tubulin, which are abundant as proteins in the dry spores, the translation of centrin, RanBPM and Xgrip109 from stored mRNA accompanies blepharoplast appearance. Antisense centrin RNA probes were designed to test whether centrin translation is necessary for blepharoplast formation. Antisense centrin RNA blocks development at a stage when the blepharoplast should be present, but blepharoplasts are not detectable in these cells. Sense centrin-RNA inhibits development, because of RNAi effects. Antisense RNA probes for cyclin A or cyclin B block mitosis in the gametophyte, similar to treatments with olomoucine or hydroxyurea. None of these treatments forestalls or limits centrin translation, but in the absence of mitosis, we have been unable to detect blepharoplasts. Thus, centrin translation appears to be necessary but not sufficient for blepharoplast formation. Cytoplasmic partitioning appears important for organizing components that assemble into the blepharoplast as these cells acquire an organized centrosome and initiate de novo basal body assembly. 

(Supported by NSF: MCB 9904435)

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