Timing of calcium and protein synthesis requirements for the first mitotic cell cycle in fertilised Xenopus eggs

Abstract

Mitosis is governed by the activity of the M-phase promoting factor (MPF). In some systems, particularly early embryos, transient increases in calcium concentration have been shown to be necessary for mitosis and regulate its timing. By microinjection of the calcium buffer, dibromoBAPTA, into fertilised Xenopus eggs, we have assessed whether calcium events are required to initiate MPF activation and inactivation. Since initial experiments showed that this buffer inhibited protein synthesis, we measured when mitosis and cleavage became independent of translation. We found that, after a period of protein synthesis essential for cleavage, there was a phase during which continued translation affected the timing of cleavage, but was not essential for its occurrence. Measurement of MPF activity in single embryos injected with calcium buffer at different times in the first cell cycle, showed that there were two sensitive periods. The first period of sensitivity blocked MPF activation and coincided with the time at which cleavage became completely independent of protein synthesis. The second sensitive period occurred just before histone kinase activity peaked, and was necessary for kinase inactivation. Preventing inactivation in this way arrested egg extracts in mitosis. These results support the view that transient increases in free calcium concentration contribute to mitotic progression by first triggering MPF activation and subsequently, with elevated MPF activity, inducing its inactivation.