The cellular and molecular mechanisms that underlie species-specific membrane fusion between male and female gametes remain largely unknown. Here, by use of gene discovery methods in the green alga Chlamydomonas, gene disruption in the rodent malaria parasite Plasmodium berghei, and distinctive features of fertilization in both organisms, we report discovery of a mechanism that accounts for a conserved protein required for gamete fusion. A screen for fusion mutants in Chlamydomonas identified a homolog of HAP2, an Arabidopsis sterility gene. Moreover, HAP2 disruption in Plasmodium blocked fertilization and thereby mosquito transmission of malaria. HAP2 localizes at the fusion site of Chlamydomonas minus gametes, yet Chlamydomonas minus and Plasmodium hap2 male gametes retain the ability, using other, species-limited proteins, to form tight prefusion membrane attachments with their respective gamete partners. Membrane dye experiments show that HAP2 is essential for membrane merger. Thus, in two distantly related eukaryotes, species-limited proteins govern access to a conserved protein essential for membrane fusion.[Keywords: Gamete fusion; cell-cell fusion; malaria; HAP2; Chlamydomonas, Plasmodium] Supplemental material is available at http://www.genesdev.org. Received January 28, 2008; revised version accepted February 22, 2008. Fusion of gametes of opposite sex (or mating type) to form a zygote is the defining moment in the life of a eukaryote. In the first phase of gamete interactions, cell adhesion molecules displayed on the surfaces of the gametes bring the two cells together. In animals, the sperm plasma membrane binds to the extracellular matrix of the egg (the zona pellucida in mammals and the jelly coat in many invertebrates). The interacting gametes use this first-phase adhesion step not only to bind to each other, but also to initiate a signal transduction cascade that activates the sperm and exposes new, fusogenic regions of the sperm plasma membrane. In the second phase of fertilization, the membrane fusion reaction, the plasma membranes of the two gametes come into intimate contact and then fuse, bringing about cytoplasmic continuity (Primakoff and Myles 2002;Rubinstein et al. 2006). Although these two steps-prefusion attachment of the plasma membranes of gametes and merger of their lipid bilayers-have been experimentally separated using in vitro bioassays, gene disruption studies to date have failed to distinguish the two, and no genes have been identified whose disruption allows prefusion attachment and disallows membrane merger. In mice, several proteins involved in gamete membrane interactions have been described, including ADAMS family members and CRISP proteins on sperm and integrins and tetraspanin family members CD9 and CD81 on eggs (for review, see Ellerman et al. 2006;Inoue et al. 2007;Primakoff and Myles 2007). Izumo, an immunoglobulin superfamily sperm protein that appears to be limited to mammals, is gamete-specific and shown by gene disruption to be essential at a late step in ferti...

SummarySexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa—animals, plants, and protists (including important human pathogens like Plasmodium)—suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.

Biologists have long known that humans experience their environment through cilia. Light, odorant, and sound perception depend on these microtubule-filled, complex organelles present on cells in primary sensory tissues. Recently, discoveries on the mechanism of assembly of cilia (flagella) in the lowly, biflagellated, eucaryotic green alga Chlamydomonas have triggered a renaissance of interest in the organelles along with a recognition of their key sensory roles in nonsensory tissues. Chlamydomonas researchers uncovered an entirely new set of cellular machinery essential for transporting the protein components of cilia and flagella in all ciliated/ flagellated eukaryotic cells between their site of synthesis in the cell body and their site of assembly at the tip of the flagellum (intraflagellar transport: IFT). Prompted by the surprising observations that disruption of IFT genes in mice led to polycystic kidney disease (PKD) and that PKD proteins are present on the sensory cilia of Caenorhabditis elegans, researchers have made a direct connection between PKD and cilia. At least five (and possibly all) of the seven identified human genes disrupted in PKD and a related disorder nephronophthisis encode proteins expressed in the primary cilia that project into the lumen from the epithelial cells that line renal tubules. Moreover, the renal cilia are flow sensors and at least two of the PKD genes encode ciliary transmembrane proteins essential for mechanosensation. Although their roles have not yet been as clearly identified, cilia also are at the center of a rare human disorder, Bardet-Biedl syndrome (BBS), in which patients exhibit phenotypes of common human diseases, including obesity and increased incidence of hypertension and diabetes. Five of the eight known BBS genes encode basal body or cilia proteins in mice or humans, and homologues of two of the remaining genes are present in basal bodies/cilia of model organisms. Here we briefly describe the biology of cilia and flagella, we outline how studies on model organisms have led to our current understanding of the roles of these organelles and their proteins in health and disease, and we highlight the notion that the primary cilia present on cells throughout the body, even those on brain neurons, may be essential for as yet undiscovered cilium-generated signaling functions.

Cilia and flagella play key roles in development and sensory transduction, and several human disorders, including polycystic kidney disease, are associated with the failure to assemble cilia. Here, we show that the aurora protein kinase CALK in the biflagellated alga Chlamydomonas has a central role in two pathways for eliminating flagella. Cells rendered deficient in CALK were defective in regulated flagellar excision and regulated flagellar disassembly. Exposure of cells to altered ionic conditions, the absence of a centriole/basal body for nucleating flagellar assembly, cessation of delivery of flagellar components to their tip assembly site, and formation of zygotes all led to activation of the regulated disassembly pathway as indicated by phosphorylation of CALK and the absence of flagella. We propose that cells have a sensory pathway that detects conditions that are inappropriate for possession of a flagellum, and that CALK is a key effector of flagellar disassembly in that pathway.

Cilia and flagella are dynamic organelles that are assembled and disassembled during cell differentiation, during stress, and during the cell cycle. Although intraflagellar transport (IFT) is well documented to be responsible for transport of ciliary/flagellar precursors from the cell body to the flagella, little is known about the molecular mechanisms for mobilizing the cell body-localized precursors to make them available for transport during organelle assembly or for disassembling the microtubule-based axoneme during shortening. Here, we show that Chlamydomonas kinesin-13 (CrKinesin-13), a member of the kinesin-13 family of microtubule depolymerizing kinesins best known for their roles in the cell cycle, functions in flagellar disassembly and flagellar assembly. Activation of a cell to generate new flagella induces rapid phosphorylation of CrKinesin-13, and activation of flagellar shortening induces the immediate transport of CrKinesin-13 via intraflagellar transport from the cell body into the flagella. Cells depleted of CrKinesin-13 by RNAi assemble flagella after cell division but are incapable of the rapid assembly of flagella that normally occurs after flagellar detachment. Furthermore, they are inhibited in flagellar shortening. Thus, CrKinesin-13 is dynamically regulated during flagellar assembly and disassembly in Chlamydomonas and functions in each.cilia ͉ flagella ͉ intraflagellar transport ͉ Kinesin-13