We will use fertilization in the bi-ciliated green alga Chlamydomonas as a model system to investigate conserved cellular and molecular mechanisms of ciliary signaling and the gamete membrane fusion reaction. When Chlamydomonas gametes of opposite types are mixed together they adhere to each other by complementary adhesion receptors on their cilia. Interaction between the adhesion receptors (encoded by SAG1 in plus gametes and SAD1 in minus gametes) initiates a signaling pathway within the cilia that is transmitted to the cell body and 1) triggers the gametes rapidly (~ 5 minutes) to undergo a striking redistribution of pre-existing ciliary adhesion polypeptides from the cell body plasma membrane to the base of the cilium, followed by trafficking onto the ciliary membrane; and 2) uncovers and activates apically localized protrusions - - the plus and minus mating structures - - on the cell body of each gamete between the two cilia. Each mating structure bears a gamete-specific adhesion protein, distinct from those on the cilia, that bind the tips of the two mating structures together. Mating structure adhesion is rapidly followed by lipid bilayer merger through the action of the gamete-specific broadly conserved protein, HAP2. Soon after fusion, the mating structure adhesion molecules and the ciliary adhesion receptors receptors are down-regulated. In our ciliary signaling and protein trafficking studies, we have found that ciliary adhesion-induced apical localization of SAG1 polypeptide depends on singlet microtubules in the cytoplasm and action of the retrograde intraflagellar transport (IFT) motor. On the other hand, contrary to current models, dynamic trafficking of SAG1 into cilia does not require the anterograde IFT motor. Finally, we determined that SAG1 movement into cilia is uni-directional. During down-regulation, SAG1 does not return to the cell body, but the entire pre-existing complement of the protein is shed into the medium in the form of ciliary ectosomes. Our findings challenge existing, largely untested models of ciliary membrane protein trafficking and set the stage for new strategies to investigate cellular and molecular mechanisms of the dynamic regulation of the protein composition of cilia. In our studies of the gamete membrane fusion reaction, we have identified the missing member of a receptor pair essential for adhesion between the plus and minus mating structures. And, in what we feel is a major advance in the field, we discovered that the fusion-essential HAP2 protein is a Class II fusion protein in the same family as Dengue and Zika virus fusion proteins. Mutational or immunological interference with the HAP2 ?fusion loop? does not interfere with HAP2 localization to the mating structure, but renders HAP2 inactive in bilayer fusion. Our discoveries make possible a detailed, structure- and function- based molecular dissection of eukaryotic gamete fusion. Furthermore they open the possibility that strategies used to block viral transmission can be used to interfere with transmission of protozoan pathogens, including the HAP2-containing malaria organism, Plasmodium.
This is a proposal to use fertilization in the unicellular, biciliated green alga Chlamydomonas as a model system to study conserved mechanism of ciliary signaling and the gamete membrane fusion reaction. We will determine the cellular and molecular mechanisms that underlie dynamic regulation of the properties of the primary cilium during cilium generated signaling. And, we will use our recent discovery that gamete lipid bilayer merger depends on an ancient Class II fusion protein as the foundation for a more complete molecular understanding of gamete fusion.
| Feng, Juan; Dong, Xianchi; Pinello, Jennifer et al. (2018) Fusion surface structure, function, and dynamics of gamete fusogen HAP2. Elife 7: |
| Angrisano, Fiona; Sala, Katarzyna A; Da, Dari F et al. (2017) Targeting the Conserved Fusion Loop of HAP2 Inhibits the Transmission of Plasmodium berghei and falciparum. Cell Rep 21:2868-2878 |
