Three different calcium wave pacemakers in ascidian eggs

Calcium wave pacemakers in fertilized eggs of ascidians and mouse are assoc iated with accumulations of cortical endoplasmic reticulurn in the vegetal hemisphere. In ascidians, two distinct pacemakers (PM1 and PM2) generate tw o series of calcium waves necessary to drive meiosis I and II. Pacemaker PM 2 is stably localized in a cortical ER accumulation situated in the vegetal contraction pole. We now find that pacemaker PMI is situated in a cortical ER-rich domain that forms around the sperm aster and moves with it during the calcium-dependant cortical contraction triggered by the fertilizing spe rm. Global elevations of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P-3) levels produced by caged Ins(1,4,5)P-3 or caged glycero-myo-PtdIns(4,5)P-2 photoly sis reveal that the cortex of the animal hemisphere, also rich in ER-cluste rs, is the cellular region most sensitive to Ins(1,4,5)P-3 and acts as a th ird type of pacemaker (PM3). Surprisingly, the artificial pacemaker PM3 pre dominates over the natural pacemaker PM2, located at the opposite pole. Mic rotubule depolymerization does not alter the activity nor the location of t he three pacemakers. By contrast, blocking the actomyosin driven cortical c ontraction with cytochalasin B prevents PM1 migration and inhibits PM2 acti vity. PM3, however, is insensitive to cytochalasin B. Our experiments suggest that the three distinct calcium wave pacemakers are probably regulated by different spatiotemporal variations in Ins(1,4,5)P-3 concentration. In particular, the activity of the natural calcium wave pac emakers PM1 and PM2 depends on the apposition of a cortical ER-rich domain to a source of Ins(1,4,5)P-3 production in the cortex.