Conserved mechanisms of ciliary signaling and cell-cell fusion

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We use the bi-ciliated, unicellular green alga Chlamydomonas as a model system to study conserved mechanisms of ciliary signaling and cell-cell fusion during fertilization. Ciliary adhesion between plus and minus gametes induces a rapid, ~15-fold increase in intracellular cAMP in the cell body that leads to activation of the cells in preparation for the gamete membrane fusion reaction. In our studies of ciliary signaling, we are examining the functions of the protein kinase GSPK, the adenylyl cyclase IMP4, and the protein phosphatase PP2A3 in the ciliary adhesion-induced increase in cAMP. One key assay in these studies is measurement of ciliary adhesion-induced cAMP production in gametes expressing wild type and mutant forms of the proteins. New fluorescent reagents are now being used in other systems to rapidly quantify cellular cAMP levels in live cells and we anticipate that they can be used with flow cytometry to quantify cAMP concentration in our cells. In studies of the mechanisms of gamete fusion, we showed that interaction between the membrane protein FUS1 on the mating structure of activated plus gametes with the membrane protein MAR1 on the mating structure of minus gametes leads to attachment of the two gametes at their mating structures. We have demonstrated that MAR1 is bifunctional. Not only does MAR1 interact with FUS1 during mating structure adhesion, but in resting minus gametes, MAR1 is biochemically and functionally associated on the minus mating structure with the conserved class II membrane fusion protein, HAP2. Unlike viral class II fusion proteins, however, whose fusogenic reconfiguration is triggered by low pH, the fusogenic reconfiguration of HAP2 is triggered by FUS1-MAR1 interactions. We and our collaborators have just determined the X-ray structure of the FUS1-MAR1 complex, showing that its is a heterotetramer composed of two MAR1 monomers and two FUS1 monomers. We will use structure-guided mutagenesis in combination with phenotypic analyses to identify the structural domains and specific residues in FUS1, MAR1, and HAP2 important both to maintain them in their prefusion forms and to regulate their function during the fusion reaction. We have just determined that a flow cytometry-based assay for cell-cell fusion is 20 times faster than our current microscopy-based assay and allows us to assess 30,000 cells/per sample compared to 300 cells/sample in our microscopic assay. The currently available flow cytometer is well past its prime, is unreliable, and lacks the laser technology needed for newly available fluorescent probes. Here, we are requesting funds towards the purchase of a new Becton Dickinson Symphony A1 Flow Cytometry system that we believe will substantively enhance the efficiency and thorougness of our research.