The long-term goals of my research are to understand the cellular and molecular mechanisms of fertilization in eukaryotic cells. Our laboratory uses fertilization of the biflagellated alga Chlamydomonas as a model system. Like sperm and eggs in multicellular organisms, Chlamydomonas gametes of opposite sexes (mt+ and mt-) are endowed with adhesion and signal transduction molecules in their flagella that enable them to bind to each other to initiate a signal transduction cascade. When gametes are mixed together they adhere to each other by their flagella, thereby activating protein kinases and an adenylyl cyclase. The consequent increase in intracellular cAMP triggers several biochemical and morphological events (collectively termed gamete activation) that, within minutes after flagellar contact, render the gametes able to fuse to form a zygote (the equivalent of a fertilized egg in multicellular organisms). While sperm-egg interactions in multicellular organisms are widely studied, we know little about the cellular and molecular mechanisms that underlie gamete adhesion, signaling or gamete fusion in mouse or humans.
In addition to being an excellent model system for studying fertilization, Chlamydomonas stands out for its contributions to our understanding of the assembly and function of cilia and flagella. Recent studies in Chlamydomonas revealed the existence of a novel transport system composed of motor proteins (kinesin-II and cytoplasmic dynein) and intraflagellar transport particles (composed of 17 proteins) that carry flagellar proteins from the cell body to the flagella and back. Subsequent examination of cilia in mouse embryos, the mouse eye, C. elegans sensory cilia, and human kidney tubule cells, among others, showed that each of these organelles contain the same set of motor proteins and intraflagellar transport particle proteins originally discovered in Chlamydomonas.
At present, three research problems are under investigation in my laboratory:
- Recently we determined that the first Chlamydomonas member of the Ipl1/aurora family of protein kinases (a molecule we named CALK) is an effector in a pathway for regulated flagellar disassembly. When cells receive a signal to remove their flagella, CALK is phosphorylated and activated. Currently, we are testing the model that CALK regulates the movement of flagellar components into flagella by controlling the intraflagellar transport (IFT) machinery.
- In studies aimed at identifying the molecular components of the signal transduction pathway stimulated by cell-cell adhesion, we have discovered that a novel flagellar protein tyrosine kinase is activated during adhesion of mt+ and mt- gametes. To our surprise, we found that adhesion-induced activation of this protein kinase requires the activity of the intraflagellar transport motor kinesin-II. We are using biochemical and molecular biological methods to identify and characterize this new soluble protein tyrosine kinase and its substrate to determine its role in activation of the flagellar adenylyl cyclase, and we intend to learn more about the role of kinesin-II in coupling flagellar adhesion to activation of the signal transduction pathway.
- In related studies we are using Chlamydomonas to identify and characterize proteins essential for cell-cell fusion. To date the only gamete-specific molecule shown by gene disruption evidence to be essential for gamete fusion in any eukaryote is the Chlamydomonas gene fus1. We have just shown that the Fus1 protein is localized at the specialized cell surface site at which gamete fusion occurs in Chlamydomonas, a microvillous-like, actin-filled organelle called the fertilization tubule. Using other sterile mutants, we also determined that the Fus1 protein is essential for the first stage of gamete fusion, which is docking of mt+ and mt- gametes. Currently, we are investigating the domains of the Fus1 protein essential for adhesion and fusion and we are searching for the protein on mt- gametes that interacts with Fus1 to consummate fusion.