Midwestern University

Identification of Signaling Molecules That Function at Fertilization - Project Summary/abstract During Fertilization, the Previously Quiescent Egg Is Activated to Begin Embryonic Development. One of the Roles of This Activation Is to Initiate the Pathways That Lead to Cell Cycle Progression and the Eventual Activation of the Zygotic Genome. However, the Molecular Mechanisms Through Which Sperm- Egg Interaction Leads to These Events Are Not Completely Understood. This Proposal Will Assemble the Components of the Signaling Pathways That Link Egg Activation to the Initiating Signal, Whether It Is a Soluble Molecule From the Sperm or Egg Cytoplasm or Cell Surface Receptor in the Egg (or Sperm) Plasma Membrane. Molecular Tools Based Upon Signaling Molecules Previously Cloned From a Sea Star Cdna Library Will Be Used for the In-depth Study of the Signaling Pathways. the Sea Star Has Been Chosen as the Model System for Several Reasons, Including the Availability of High Quality Molecular Reagents, the Ability to Obtain Large Quantities of Gametes, the Ease With Which Cells Can Be Microinjected and Assayed for Signaling Events, and the Optical Clarity of the Egg for Microscopy. Specific Domains From Known Signaling Molecules (SRC1, SRC3, and PLC Are in Hand) Will Be Expressed as Fusion Proteins That Will Be Used to Identify Interacting Proteins Following Secondary Selection Using Anti-phosphotyrosine Antibodies. the Identity of the Proteins That Interact With SRC1, SRC3, or PLC Will Be Determined by Tandem Mass Spectrometry Amino Acid Sequencing Followed by Database Identification Using Our Newly Published Sea Star Egg Transcriptome (PRJNA398668). We Have Developed Methods for Identification of Tyrosine-phosphorylated Proteins From Zygote Samples, Particularly Those That Interact With the SH2 Domains of SRC and PLC. the Identified Cdna for Each Signaling Molecule Will Be Cloned by Rt-pcr to Produce a Library of Molecules for Further Study. This Proposal Explores the Functional Aspects of Each Identified Molecule at Using a Combination of Rna Depletion Strategies and Direct Protein Interference With Function-blocking Antibodies And/or Fusion Protein Constructs. Real-time Interaction Between Designed Protein Constructs With Fertilized Eggs Lysates Will Be Monitored Using Bio-layer Interferometry. the Result Will Be the Elucidation of a Network of Proteins That Interact in the Echinoderm Egg to Facilitate Egg Activation During Fertilization. This Project Will Engage Biomedical Science Graduate Students Along With Medical and Veterinary Students in the Laboratory. All Students Will Be Trained to Work With Gametes, and in Bioinformatics and Selected Molecular Biology Methods by the Pi and by Peer-peer Teaching. Students Will Earn Co- Authorship on Manuscripts and Project Funds Will Also Support Attendance at Regional and National Research Conferences to Allow the Students to Present Their Work.