The centrosome, the primary microtubule organizing center of the cell, serves to nucleate and organize the interphase microtubule array and the asters at the spindle poles during mitosis (Kellogg et al., 1994
; Pickett-Heaps, 1969
). Since the purpose of mitosis is the equal segregation of the duplicated genome at mitosis, the cell can contain two and only two centrosomes at the onset of mitosis. If the centrosome fails to duplicate, the next mitosis will either be monopolar or bipolar with one anastral pole. This means that the cell will fail to divide or one daughter will not inherit a centrosome. Over-duplication of the centrosome raises the chances that the cell will assemble a multipolar spindle at mitosis, which inevitably leads to unequal chromosome segregation and aneuploid daughter cells.
To ensure that mitosis will be strictly bipolar, the cell must coordinate centrosome doubling with nuclear DNA synthesis. The link between centrosome duplication and DNA synthesis is ensured by the rise in cyclin E/cdk2 and/or cyclin A/cdk2 kinase activities which participate in determining when both events begin (Hinchcliffe et al., 1999
; Lacey et al., 1999
; Matsumoto et al., 1999
; reviewed in Hinchcliffe and Sluder, 2002
). Importantly, the cell must also have a means to ensure that the centrioles do not reduplicate during S phase. A centrosome intrinsic block to reduplication exists, at least in normal human cells, which ensures that once the centrosome has duplicated it will not do so again even though the cellular conditions are permissive for duplication (Wong and Stearns, 2003
; Tsou and Stearns 2006
To better understand the mechanisms that limit centrosome duplication, we have investigated a phenomenon described by Hinchcliffe et al. (1998)
. When protein synthesis in sea urchin zygotes is completely blocked from before fertilization, the zygotes arrest in first S phase and the paternal centrosome repeatedly reduplicates (also see Gard et al., 1990
; Sluder et al., 1990
). This is consistent with observations of centrosome reduplication in zygotes arrested in S phase of the first or second cell cycle by inhibition of DNA synthesis (Hinchcliffe et al., 1998
). However, a different pattern of centrosome reproduction was observed when protein synthesis was completely blocked beginning at prophase of first mitosis. The zygotes went through mitosis, because they had completed all preparations for first division before protein synthesis was inhibited. The two blastomeres reformed nuclei and arrested in a G1-like state and each blastomere contained two complete centrosomes for up to 8 hours or the equivalent of seven division cycles. The centrosome inherited at the end of mitosis had duplicated just once in a normal fashion, but no more. The difference in centrosome reproduction at the two arrest points is not based in differing levels of cyclin B/cdk1 activity; H1 kinase activity was at the prefertilization level for both cell cycle arrest points due to the complete inhibition of protein synthesis (Hinchcliffe et al., 1998
). Why centrosomes reduplicate during S phase arrest but are limited to duplicating only once during a G1 arrest has been a mystery.
In the sea urchin zygote, translation of cyclin E mRNA is activated after fertilization and the level of this protein remains constant during early development, suggesting that there is continuous turnover of cyclin E during early development (Sumerel et al., 2001
). Perhaps arrest in S and G1 by blocking protein synthesis might bring about a differential decline in cyclin E protein levels under the two arrest conditions and hence a change in its associated kinase activity. To test this possibility, we repeated the experiments of Hinchcliffe et al. (1998)
and characterized the levels of cyclin E protein and its associated kinase activity when the zygotes were arrested in first S phase and in the G1-like state under conditions in which protein synthesis was completely blocked.