Signal transduction of Ca2+ oscillations in eggs at fertilization (360G-Wellcome-090063_Z_09_Z)

Egg activation and early development in animals is controlled by intracellular factors, such as Ca2+ increases, cell cycle proteins, and protein kinases. The downstream effects of Ca2+ are mediated by protein kinases and most studies on eggs have focussed on protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaMKII). The technology to monitor Ca2+ in single eggs with fluorescent and luminescence probes has existed for some time. Fluorescent fusion proteins have also allowed us t o follow the concentration changes of proteins such as cyclin B in living eggs. However, to analyse the activity of, or phosphorylation due to, protein kinases, we still rely on cell-destructive biochemical assays. In this project, we will use FRET-based indicators for measuring the activity of PKC and CaMKII in oocytes, eggs and early embryos. We shall first establish how a range of different Ca2+ stimuli are transduced by these protein kinases in mouse eggs. Physiological changes in PKC and C aMKII will also be determined in real time during oogenesis and during early development in sea urchins and ascidians. We shall also generate a new range of luciferase-based probes and test their potential to monitor the dynamics of protein phosphorylation in intact eggs.