Microtubule Protein Synthesis Research Articles

Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration

Flagellar regeneration in gametes of Chlamydomonas reinhardi is initiated within 15–20 min after flagellar amputation and proceeds at a rapid but decelerating rate until by 90 min flagellar outgrowth is 80–85% complete. Sufficient flagellar protein reserves exist in the cytoplasm to allow regeneration of flagella 1 3 – 1 2 normal length. Nevertheless, in vivo labeling with 14C-amino acids shows that microtubule protein and other flagellar proteins are synthesized de novo during flagellar regeneration. To determine whether tubulin is synthesized continuously by gametic cells or whether its synthesis is induced as a consequence of deflagellation, we have isolated polyribosomes from deflagellated and control cells, and analyzed the proteins produced by these polyribosomes during in vitro translation. Two proteins of 53,000 and 56,000 molecular weight which co-migrate with flagellar and chick brain tubulin on SDS-polyacrylamide gels and which selectively co-assemble with chick brain tubulin during in vitro microtubule assembly are synthesized by polyribosomes (or polyadenylated mRNA) from deflagellated cells. No microtubule proteins can be detected in the translation products synthesized by polyribosomes (or mRNA) from control cells, clearly indicating that deflagellation results in the induction of tubulin synthesis. Kinetics of tubulin synthesis demonstrate that induction takes place immediately after deflagellation; polyribosomes bearing tubulin mRNA can be detected in the cytoplasm in as little as 15 min after removal of flagella. Maximal rates of tubulin synthesis occur between 45 and 90 min after deflagellation when approximately 14% of the protein being synthesized by the cell is tubulin. This estimate of tubulin synthesis based on in vitro translation data agrees well with in vivo measurements of flagellar tubulin synthesis. While high levels of tubulin production extend well beyond the period of rapid flagellar assembly, synthesis begins to decline after 90 min, and by 180 min after deflagellation only low levels of tubulin mRNA are detectable in polyribosomes.

Regulation of Microtubule Synthesis and Utilization during Early Embryonic Development of the Sea Urchin

Microtubules deployed during early development of the sea urchin embryo are derived both from a preexisting pool of subunits present in the egg and from microtubule protein subunits synthesized in the embryo. Several aspects of microtubule protein synthesis and utilization are reviewed. Microtubule protein synthesis in early development utilizes oogenetic messenger RNA species. Translation of this mRNA is under regulation. Microtubule protein synthesis rises concomitantly with overall protein synthesis at fertilization, but rises at a relatively higher rate later in cleavage stages. Microtubule protein labeled with [3H]-leucine in early development is incorporated into cilia, indicating that newly synthesized protein enters the pool of subunits used in organelle assembly. The microtubule protein pool comprises about 1%of the soluble protein of the egg, and remains constant in size at least until the blastula stage. Direct pool size estimates are consistent with results of experiments on recruitment of microtubule protein subunits into the mitotic apparatus and into regenerating cilia. Soluble and particulate colchicine binding fractions, which have been reported from several systems, appear to be present in sea urchin embryos. The possible role of such fractions are discussed, as are aspects of the regulation of ciliary assembly.

Regulation of microtubule protein synthesis in embryos of the marine snail, Ilyanassa obsoleta

Synthesis of microtubule protein is not detectable in 1-day-old embryos of the snail Ilyanassa obsoleta but is readily detectable after 2 days of development. Microtubule protein synthesis then increases for several days thereafter. Continuous culture of embryos in 25 μg/ml of actinomycin D, an amount sufficient to abolish incorporation of [ 3H]uridine into high molecular weight RNA, blocks ciliation of embryos but has no effect upon microtubule protein synthesis. A mechanism for the selection of microtubule protein mRNA for translation at the appropriate developmental stage appears to exist. The timing of microtubule protein synthesis may be coordinated with ciliogenesis, but it is not coupled to it.

Microtubule protein synthesis during oogenesis and early embryogenesis in Xenopus laevis.

A method is described which permits the preparation of descrete classes of oocytes of different sizes from all stages of oogenesis in Xenopus laevis. This technique is used in the determination of the content of microtubule protein in oocytes during the course of oogenesis. These experiments show that microtubule protein is present in oocytes of all sizes assayed and that the amount is simply related to the volume of the oocyte. In the largest oocytes microtubule protein constitutes 1% of the soluble protein and this amount does not change on maturation and fertilization. These results show that the changes occurring in the oocyte on maturation which allow the cytoplasm to support microtubule polymerization occur as a result of a modification of the pre-existing microtubule protein, not from protein synthesis de novo. These experiments also indicate that the synthesis of microtubule protein either form 'masked' mRNA or from newly synthesized mRNA plays an insignificant role in microtubule protein synthesis at maturation, ovulation and immediately post-fertilization.