The reproduction of centrosomes: nuclear versus cytoplasmic controls

The tight coordination normally found between nuclear events and the doubling of centrosomes at each cell cycle suggests that nuclear activities may be part of the mechanism that controls the reproduction of centrosomes. To determine if this is the case, we used a micropipette to completely remove the nucleus from eggs of the sea urchin Lytechinus variegatus at prophase of the first mitosis, leaving only one centrosome in the cell. The subsequent behavior of this centrosome was then followed in vivo with the polarization microscope. In all cases the centrosome reproduced in a precise 1:2:4:8 fashion with a periodicity that was slightly slower than the centrosome cycle of control eggs. The cell cycle-related changes in centrosome morphology were identical to those of control eggs in that: (a) the astral birefringence varied cyclically to a normal extent, (b) the astral focus enlarged and then flattened during the telophase equivalent, (c) cleavage furrows were initiated as the astral birefringence faded, and (d) daughter centrosomes separated before the increase in astral birefringence at the onset of each mitosis. To determine if centrioles also reproduced normally, enucleate eggs were followed in vivo until they contained eight centrosomes. They were then individually removed from the preparations, fixed, and embedded. Each egg was serially 0.25-micron sectioned for observation with the high voltage electron microscope. We completely reconstructed 23 centrosomes in four eggs; all centrosomes contained two centrioles apiece. These results demonstrate that the subunits for complete centrosome assembly can be stockpiled ahead of time and that the properly controlled use of these subunits for centrosome reproduction does not require nuclear transcription or nuclear DNA synthesis at each cell cycle.