Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control.

Abstract

Abstract. The reproduction, or duplication, of the centrosome is an important event in a cell's preparation for mitosis. We sought to determine if centrosome reproduction is regulated by the synthesis and accumulation of cyclin proteins and/or the synthesis of centrosome-specific proteins at each cell cycle. We continuously treat sea urchin eggs, starting before fertilization, with a combination of emetine and anisomycin, drugs that have separate targets in the protein synthetic pathway. These drugs inhibit the postfertilization incorporation of [35S]methionine into precipitable material by 97.3-100%. Autoradiography of SDS-PAGE gels of drug-treated zygotes reveals that [35S]methionine incorporates exclusively into material that does not enter the gel and material that runs at the dye front; no other labeled bands are detected. Fertilization events and syngamy are normal in drug-treated zygotes, but the cell cycle arrests before first mitosis.The sperm aster doubles once in all zygotes to yield two asters. In a variable but significant percentage of zygotes, the asters continue to double. This continued doubling is slower than normal, asynchronous between zygotes, and sometimes asynchronous within individual zygotes. High voltage electron microscopy of serial semithick sections from drug-treated zygotes reveals that 90% of the daughter centrosomes contain two centrioles of normal appearance. From these results, we conclude that centrosome reproduction in sea urchin zygotes is not controlled by the accumulation of cyclin proteins or the synthesis of centrosomespecific proteins at each cell cycle. New centrosomes are assembled from preexisting pools of ready-to-use subunits. Furthermore, our results indicate that centrosomal and nuclear events are regulated by separate pathways.

Fate of centrosomes following somatic cell nuclear transfer (SCNT) in bovine oocytes

“…Thus, the cell cycle of donor cells influences reproduction of centrosomes following nuclear transfer. However, centrosome duplication does not require de novo protein synthesis, since an inhibitor of protein synthesis, cycloheximide, was present during the first 6 h following nuclear transfer, consistent with previous findings in embryos from other species (Gard et al 1990, Sluder et al 1990). The subunits for complete centrosome assembly can be stockpiled ahead of time, and the properly controlled use of these subunits for centrosome reproduction does not require nuclear transcription or nuclear DNA synthesis at each cell cycle (Sluder et al 1986).…”

Two for two: Cdk2 and its role in centrosome doubling

“…These cyclins are classified as Clb 1 -4 (mitotic B-type cyclins); Clb 5 and 6 (S phase B-type cyclins); and Cln 1, 2 and 3 (the so-called G 1 cyclins). When they deleted the all B-type cyclins (Clb 1 -6), they found that the SPB could reproduce; confirming the work from urchin that centrosome reproduction does not require cyclin B mediated Cdk activity (Sluder et al, 1990;. However, unlike sea urchin centrosomes, yeast SPBs cannot duplicate again in the complete absence of the Clbs.…”

“…The synthesis of cyclin B starting in S phase and the precipitous rise in the activity of this kinase complex in late G2 was known to drive the cell cycle into mitosis (Murray and Kirschner, 1989). Findings that complete inhibition of protein synthesis does not stop repeated centrosome duplication in zygotes provided compelling evidence that the cycle of Cdk1-B activity does not control if and when centrosomes reproduce (Sluder et al, 1990;Gard et al, 1990). Nevertheless, the possibility still remained that the absolute value of Cdk1-B activity might provide conditions that allowed centrosome duplication in early interphase and later, as cyclin B was synthesized, blocked it from late S phase through the end of mitosis.…”

“…Extrinsic cytoplasmic controls determine when the centrosome duplicates in relation to the progression of nuclear events such as DNA synthesis and mitosis. Evidence for cytoplasmic regulation of centrosomal events came from studies on zygotes demonstrating that repeated centrosome duplication occurs in enucleated cells and cells in which protein synthesis is completely blocked (Sluder et al, 1986(Sluder et al, , 1990Gard et al, 1990). These studies indicated that centrosome duplication is not dependent upon the completion of nuclear events such as DNA synthesis per se and can take place in an orderly fashion from pre-existing subunit pools that are not limiting.…”

One to only two: a short history of the centrosome and its duplication

“…For example, early sea urchin and frog zygotes contain complete pools of subunits on hand at fertilization to make many centrioles [68,69]. The same holds true for early Drosophila embryos that are reported to have subunit pools sufficient to assemble 2 Â 10 13 centriole pairs [70].…”