Stephen Jones and Adam Hernandez worked as a team on their UBM research project in Dr. Carroll’s laboratory. The main focus of their project this year is to develop the biological basis to allow for the development of a Neural Network for the study of two signaling pathways. We are using the starfish oocyte and egg as a model system to understand two important processes: 1) the regulation of the phosphorylation of Mitogen Activated Protein kinase (MAPK) during oocyte maturation; and 2) the signaling network leading to activation of phospholipase Cgamma during fertilization. The activities so far on these projects: 1) MAPK project – Adam Hernandez has been the leader of this project. He is using biological data produced by two undergraduate scientists (Britney Pennington and Ana Mantilla) and two graduate students (Leia Shuhaibar and Ioana Policeanu) to begin to develop a Neural Network model so that we can begin to test mechanisms for the phosphorylation of MAPK during oocyte maturation and the dephosphorylation of MAPK during fertilization. Adam has made significant progress in learning the language of the Neural Net program and he has develop a simple working model for MAPK signaling in oocytes; 2) PLCgamma project – Stephen Jones has been the leader of this project with support from Adam Hernandez and Dr. Carroll. Stephen has been working on methods to identify proteins that interact with PLCgamma during fertilization in the starfish. He has produced a recombinant GST-PLCgamma fusion protein that interacts with other tyrosine-phosphorylated egg proteins during fertilization. He is optimizing methods to attempt to identify these proteins by either mass spectrometry or screening western blots using antibodies. At the same time, Stephen and Adam are working together to develop the strategy for a neural net model of this system. Research Findings:
- MAPK findings: Using a phosphorylation-state specific antibody, we defined the timeframe over which MAPK becomes phosphorylated during starfish oocyte maturation and then dephosphorylated following fertilization. Since the ultimate goal is to understand how MAPK is regulated during these transitions, the MAPK phosphorylation changes in single starfish oocytes were compared to those observed in oocyte populations. Changes in MAPK phosphorylation are more abrupt when visualized in single oocytes than when oocyte populations are assayed. Oocytes incubated with farnesyl transferase inhibitor III (FPT III) do not undergo germinal vesicle breakdown or MAPK phosphorylation in response to 1-MA, suggesting that Ras is involved in this process. The single cell system in starfish is novel and will allow examination of the variability of individual oocytes in response to differing stimuli. This is something that cannot be observed when working with protein samples prepared from populations of cells. We are also excited about the possibility of coupling microinjection of individual cells with the analysis of protein phosphorylation state from the same exact cells. The initial report of the development of the single cell assay is currently being written up for publication.
- PLCgamma project: Using an affinity interaction strategy, we have discovered that numerous tyrosine phosphorylated proteins interact with the SH2 domains of PLCgamma both in the unfertilized mature egg and during fertilization. We have discovered that some of these interacting proteins are entering the egg from the sperm cytoplasm during fertilization. At this time, we do not know the identity of any of these proteins. An initial attempt to identify three of these proteins by affinity interaction with the PLCgamma SH2 domains followed by gel electrophoresis and mass spectrometry did not identify any proteins other than the GST-PLCgamma fusion protein. We are currently trying different experiments to isolate the interacting proteins from the fusion protein.