Fertilization triggers release from meiotic arrest and initiates events that prepare for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phospho-regulation is still unknown. We examined absolute protein and phospho-site dynamics after fertilization by mass spectrometry-based proteomics. To do this, we developed a new approach for calculating the stoichiometry of phospho-sites from multiplexed proteomics that is compatible with dynamic, stable and multi-site phosphorylation. Overall, the data suggest that degradation is limited to a few low abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium activated kinases. We identify putative degradation targets as well as new components of the block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.
Welcome to the Wühr Lab!
The Wühr Lab is located in the Lewis-Sigler Institute at Princeton University. We aim to elucidate principles by which molecules self-organize into much larger organelles and cells. Specifically, we study how proteins partition between nucleus and cytoplasm and how different nuclear composition affects cellular function. To address these questions, we develop and employ quantitative proteomics in combination with computational, biochemical and imaging approaches.
The Wühr Lab (from left to right): Eyan Yeung, Matt Sonnett, Martin Wühr, Lillia Ryazanova, and Elizabeth Van Itallie