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Nature And Regulation Of Protein Phosphorylation Changes During Egg Activation In Drosophila Melanogaster
Mature oocytes are held in a developmentally-quiescent, arrested state. For development to occur, these oocytes must transition to a new cellular state that can support the processes of embryogenesis. This transition is achieved by the events of egg activation. My studies focused on protein phosphorylation changes that take place during egg activation in Drosophila. Because there is little or no transcription at this time, egg activation is directed by maternal mRNAs and proteins regulated through posttranscriptional and post-translational mechanisms. Phosphorylation is an abundant post-translational modification with a wide array of regulatory effects. In addition, phosphorylation regulators such as CaMKII and calcineurin are required for egg activation in a variety of organisms. We hypothesize that simultaneously changing the phosphorylation states of a large number of proteins is a key contributor to the cellular changes that encompass the oocyte-to-embryo transition. I applied two different proteomic methods, IMAC and 2D-gel electrophoresis, to identify the proteins that change in phosphorylation state between mature oocytes and unfertilized, activated eggs. This led to the identification of 311 proteins that are phospho-modulated during egg activation. I used RNAi to knock down the genes that encode some of these proteins, testing a total of 71 genes for effects on female fertility. I identified multiple candidates for future study including, mrityu, which is required for progression through the early rounds of embryonic mitosis. I also used the phosphorylation changes of two proteins identified from the proteomics experiments, Spindly and Vap-33-1, as "molecular markers" to examine how the egg activation genes sarah, cortex, and prage relate to the phosphorylation changes that take place at egg activation. I showed that all three genes are upstream of Spindly dephosphorylation, but only sarah and cortex are upstream of Vap-33-1 phosphorylation. These data, along with previous findings in the lab, suggest that sarah and cortex act in a common pathway. Overall, my studies have contributed to our understanding of the roles of protein phosphorylation during egg activation. My results show that phosphorylation is an important area of study if we are to discover the proteins and pathways that regulate the oocyte-to-embryo transition.
Egg activation; Phosphorylation; Proteomics
Wolfner, Mariana Federica
Goldberg, Michael Lewis; Clark, Andrew
Molecular & Cell Biology
Ph.D. of Molecular & Cell Biology
Doctor of Philosophy
dissertation or thesis