William J. Snell

The Molecules of Mammalian Fertilization

We thank Eduardo Almeida for figures, Nedra Wilson for comments on the text, and members of the fertilization community for helpful discussions. Work in our laboratories was supported by grants from the National Institutes of Health (GM48739, J. W.; GM25661, W. S.) and the National Science Foundation (IBN-9318708, W. S.).

Cilium-generated signaling and cilia-related disorders

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.

The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes

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.

Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas.

Primary cilia are widely used for signal transduction during development and in homeostasis and are assembled and maintained by intraflagellar transport (IFT). Here, we have dissected the role of IFT in signaling within the flagella (structural and functional counterparts of cilia) of the biflagellated green alga Chlamydomonas. Using a conditional IFT mutant enables us to deplete the IFT machinery from intact, existing flagella. We identify a cGMP-dependent protein kinase (CrPKG) within flagella as the substrate of a protein tyrosine kinase activated by flagellar adhesion during fertilization. We demonstrate that flagellar adhesion stimulates association of CrPKG with a new flagellar compartment. Moreover, formation of the compartment requires IFT, and IFT particles themselves are part of the compartment. Our results lead to a model in which the IFT machinery is required not only for assembling cilia and flagella but also for organizing a signaling pathway within the organelles during cilium-generated signaling.

An Aurora Kinase Is Essential for Flagellar Disassembly in Chlamydomonas

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 revealed: from flagellar assembly in Chlamydomonas to human obesity disorders.

The recent identification in Chlamydomonas of the intraflagellar transport machinery that assembles cilia and flagella has triggered a renaissance of interest in these organelles that transcends studies on their well-characterized ability to move. New studies on several fronts have revealed that the machinery for flagellar assembly/disassembly is regulated by homologs of mitotic proteins, that cilia play essential roles in sensory transduction, and that mutations in cilia/basal body proteins are responsible for cilia-related human disorders from polycystic kidney disease to a syndrome associated with obesity, hypertension, and diabetes.

The ciliary membrane

Cilia and flagella are important organizing centers for signaling in both development and disease. A key to their function is a poorly characterized barrier at their base that allows the protein and lipid composition of the ciliary membrane to be distinct from that of the plasma membrane. We review current models of ciliary membrane biogenesis, highlighting several structures, including the ciliary necklace and ciliary pocket, that appear during biogenesis and that likely contribute to the barrier. The regulated movement of membrane proteins and lipids across this barrier is central to the sensory function of these organelles.

A microtubule depolymerizing kinesin functions during both flagellar disassembly and flagellar assembly in Chlamydomonas

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.

Izumo is part of a multiprotein family whose members form large complexes on mammalian sperm

Izumo, a sperm membrane protein, is essential for gamete fusion in the mouse. It has an Immunoglobulin (Ig) domain and an N‐terminal domain for which neither the functions nor homologous sequences are known. In the present work we identified three novel proteins showing an N‐terminal domain with significant homology to the N‐terminal domain of Izumo. We named this region “Izumo domain,” and the novel proteins “Izumo 2,” “Izumo 3,” and “Izumo 4,” retaining “Izumo 1” for the first described member of the family. Izumo 1–3 are transmembrane proteins expressed specifically in the testis, and Izumo 4 is a soluble protein expressed in the testis and in other tissues. Electrophoresis under mildly denaturing conditions, followed by Western blot analysis, showed that Izumo 1, 3, and 4 formed protein complexes on sperm, Izumo 1 forming several larger complexes and Izumo 3 and 4 forming a single larger complex. Studies using different recombinant Izumo constructs suggested the Izumo domain possesses the ability to form dimers, whereas the transmembrane domain or the cytoplasmic domain or both of Izumo 1 are required for the formation of multimers of higher order. Co‐immunoprecipitation studies showed the presence of other sperm proteins associated with Izumo 1, suggesting Izumo 1 forms a multiprotein membrane complex. Our results raise the possibility that Izumo 1 might be involved in organizing or stabilizing a multiprotein complex essential for the function of the membrane fusion machinery. Mol. Reprod. Dev. 76: 1188–1199, 2009.

Signal transduction during fertilization in the unicellular green alga, Chlamydomonas.

Sexual reproduction in the green alga, Chlamydomonas, is regulated by environmental conditions and by cell-cell interactions. After gametogenesis, flagellar adhesion between gametes triggers gamete activation, leading to cell fusion and zygote formation. Recent studies have identified new molecular events that underlie signal transduction during Chlamydomonas fertilization, including expression of a sex-determining protein, phosphorylation of a homeodomain protein, activity of a kinesin II and regulated translocation of an aurora/Ip11-like protein kinase from the cell body to the flagella.