Uncovering an ancestral green ménage à trois: Contributions of Chlamydomonas to the discovery of a broadly conserved triad of plant fertilization proteins.

During sexual reproduction in the unicellular green alga Chlamydomonas, gametes undergo the conserved cellular events that define fertilization across the tree of life. After initial ciliary adhesion, plus and minus gametes attach to each other at plasma membrane sites specialized for fusion, their bilayers merge, and cell coalescence into a quadri-ciliated cell signals for nuclear fusion. Recent findings show that these conserved cellular events are driven by 3 conserved protein families, FUS1/GEX2, HAP2/GCS1, and KAR5/GEX1. New results also show that species-specific recognition in Chlamydomonas activates the ancestral, viral-like fusogen HAP2 to drive fusion; that the conserved nuclear envelope fusion protein KAR5/GEX1 is also essential for nuclear fusion in Arabidopsis; and that heterodimerization of BELL-KNOX proteins signals for nuclear fusion in Chlamydomonas through early diverging land plants. This review outlines how Chlamydomonas's Janus-like position in evolution along with the ease of working with its gametes have revealed broadly conserved mechanisms.

A cytoplasmic protein kinase couples engagement of Chlamydomonas ciliary receptors to cAMP-dependent cellular responses.

The primary cilium is a cellular compartment specialized for receipt of extracellular signals that is essential for development and homeostasis. Although intraciliary responses to engagement of ciliary receptors are well studied, fundamental questions remain about the mechanisms and molecules that transduce ciliary signals into responses in the cytoplasm. During fertilization in the bi-ciliated alga Chlamydomonas reinhardtii, ciliary adhesion between plus and minus gametes triggers an immediate ∼10-fold increase in cellular cAMP and consequent responses in the cytoplasm required for cell-cell fusion. Here, we identify a new participant in ciliary signaling, Gamete-Specific Protein Kinase (GSPK). GSPK is essential for the adhesion-induced cAMP increase and for rapid gamete fusion. The protein is in the cytoplasm, and the entire cellular complement responds to a signal from the cilium by becoming phosphorylated within 1 min after ciliary receptor engagement. Unlike all other cytoplasmic events in ciliary signaling, GSPK phosphorylation is not responsive to exogenously added cAMP. Thus, during ciliary signaling in Chlamydomonas, a cytoplasmic protein is required to rapidly interpret a still uncharacterized ciliary signal to generate a cytoplasmic response.

MAR1 links membrane adhesion to membrane merger during cell-cell fusion in Chlamydomonas.

Union of two gametes to form a zygote is a defining event in the life of sexual eukaryotes, yet the mechanisms that underlie cell-cell fusion during fertilization remain poorly characterized. Here, in studies of fertilization in the green alga, Chlamydomonas, we report identification of a membrane protein on minus gametes, Minus Adhesion Receptor 1 (MAR1), that is essential for the membrane attachment with plus gametes that immediately precedes lipid bilayer merger. We show that MAR1 forms a receptor pair with previously identified receptor FUS1 on plus gametes, whose ectodomain architecture we find is identical to a sperm adhesion protein conserved throughout plant lineages. Strikingly, before fusion, MAR1 is biochemically and functionally associated with the ancient, evolutionarily conserved eukaryotic Class II fusion protein HAP2 on minus gametes. Thus, the integral membrane protein MAR1 provides a molecular link between membrane adhesion and bilayer merger during fertilization in Chlamydomonas.

Species-specific gamete recognition initiates fusion-driving trimer formation by conserved fusogen HAP2.

Recognition and fusion between gametes during fertilization is an ancient process. Protein HAP2, recognized as the primordial eukaryotic gamete fusogen, is a structural homolog of viral class II fusion proteins. The mechanisms that regulate HAP2 function, and whether virus-fusion-like conformational changes are involved, however, have not been investigated. We report here that fusion between plus and minus gametes of the green alga Chlamydomonas indeed requires an obligate conformational rearrangement of HAP2 on minus gametes from a labile, prefusion form into the stable homotrimers observed in structural studies. Activation of HAP2 to undergo its fusogenic conformational change occurs only upon species-specific adhesion between the two gamete membranes. Following a molecular mechanism akin to fusion of enveloped viruses, the membrane insertion capacity of the fusion loop is required to couple formation of trimers to gamete fusion. Thus, species-specific membrane attachment is the gateway to fusion-driving HAP2 rearrangement into stable trimers.

Transient Internalization and Microtubule-Dependent Trafficking of a Ciliary Signaling Receptor from the Plasma Membrane to the Cilium.

Cilia are ancient organelles used by unicellular and multicellular organisms not only for motility but also to receive and respond to multiple environmental cues, including light, odorants, morphogens, growth factors, and contact with cilia of other cells. Much is known about the cellular mechanisms that deliver membrane proteins to cilia during ciliogenesis. Execution of a ciliary signaling pathway, however, can critically depend on rapid alterations in the receptor composition of the cilium itself, and our understanding of the mechanisms that underlie these rapid, regulated alterations remains limited [1-6]. In the bi-ciliated, unicellular alga Chlamydomonas reinhardtii, interactions between cilia of mating type plus and mating type minus gametes mediated by adhesion receptors SAG1 and SAD1 activate a ciliary signaling pathway [7]. In response, a large, inactive pool of SAG1 on the plasma membrane of plus gametes rapidly becomes enriched in the peri-ciliary membrane and enters the cilia to become active and maintain and enhance ciliary adhesion and signaling [8-14]. Ciliary entry per se of SAG1 is independent of anterograde intraflagellar transport (IFT) [13], but the rapid apical enrichment requires cytoplasmic microtubules and the retrograde IFT motor, dynein 1b [14]. Whether the receptors move laterally within the plasma membrane or transit internally during redistribution is unknown. Here, in coupled immunolocalization/biochemical studies on SAG1, we show that, within minutes after gamete activation is initiated, cell-surface SAG1 is internalized, associates with an apico-basally polarized array of cytoplasmic microtubules, and returns to the cell surface at a peri-ciliary staging area for entry into cilia.

Fusion surface structure, function, and dynamics of gamete fusogen HAP2.

HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of Chlamydomonas HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loops of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the two fusion loops, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on Chlamydomonas gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.

Targeting the Conserved Fusion Loop of HAP2 Inhibits the Transmission of Plasmodium berghei and falciparum.

Inhibiting transmission of Plasmodium is a central strategy in malarial eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. The lack of a structure or known molecular function of current anti-malarial vaccine targets has previously been a hindrance in the development of transmission-blocking vaccines. Structure/function studies have indicated that the conserved gamete membrane fusion protein HAP2 is a class II viral fusion protein. Here, we demonstrate that targeting a function-critical site of the fusion/cd loop with species-specific antibodies reduces Plasmodium berghei transmission in vivo by 58.9% and in vitro fertilization by up to 89.9%. A corresponding reduction in P. falciparum transmission (75.5%/36.4% reductions in intensity/prevalence) is observed in complimentary field studies. These results emphasize conserved mechanisms of fusion in Apicomplexa, while highlighting an approach to design future anti-malarial transmission-blocking vaccines.

The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein.

Sexual 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.

Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols.

Cellular lipids are speculated to act as key intermediates in Hedgehog signal transduction, but their precise identity and function remain enigmatic. In an effort to identify such lipids, we pursued a Hedgehog pathway inhibitory activity that is particularly abundant in flagellar lipids of Chlamydomonas reinhardtii, resulting in the purification and identification of ergosterol endoperoxide, a B-ring oxysterol. A mammalian analog of ergosterol, 7-dehydrocholesterol (7-DHC), accumulates in Smith-Lemli-Opitz syndrome, a human genetic disease that phenocopies deficient Hedgehog signaling and is caused by genetic loss of 7-DHC reductase. We found that depleting endogenous 7-DHC with methyl-ß-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3ß,5α-dihydroxycholest-7-en-6-one, a naturally occurring B-ring oxysterol derived from 7-DHC that also accumulates in Smith-Lemli-Opitz syndrome, blocked Hedgehog signaling by inhibiting activation of the essential transduction component Smoothened, through a mechanism distinct from Smoothened modulation by other lipids.

The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction.

Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction.

Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding.

The role of the primary cilium in key signaling pathways depends on dynamic regulation of ciliary membrane protein composition, yet we know little about the motors or membrane events that regulate ciliary membrane protein trafficking in existing organelles. Recently, we showed that cilium-generated signaling in Chlamydomonas induced rapid, anterograde IFT-independent, cytoplasmic microtubule-dependent redistribution of the membrane polypeptide, SAG1-C65, from the plasma membrane to the periciliary region and the ciliary membrane. Here, we report that the retrograde IFT motor, cytoplasmic dynein 1b, is required in the cytoplasm for this rapid redistribution. Furthermore, signaling-induced trafficking of SAG1-C65 into cilia is unidirectional and the entire complement of cellular SAG1-C65 is shed during signaling and can be recovered in the form of ciliary ectosomes that retain signal-inducing activity. Thus, during signaling, cells regulate ciliary membrane protein composition through cytoplasmic action of the retrograde IFT motor and shedding of ciliary ectosomes.

Characterization, mutagenesis and mechanistic analysis of an ancient algal sterol C24-methyltransferase: Implications for understanding sterol evolution in the green lineage.

Sterol C24-methyltransferases (SMTs) constitute a group of sequence-related proteins that catalyze the pattern of sterol diversity across eukaryotic kingdoms. The only gene for sterol alkylation in green algae was identified and the corresponding catalyst from Chlamydomonas reinhardtii (Cr) was characterized kinetically and for product distributions. The properties of CrSMT were similar to those predicted for an ancient SMT expected to possess broad C3-anchoring requirements for substrate binding and formation of 24ß-methyl/ethyl Δ(25(27))-olefin products typical of primitive organisms. Unnatural Δ(24(25))-sterol substrates, missing a C4ß-angular methyl group involved with binding orientation, convert to product ratios in favor of Δ(24(28))-products. Remodeling the active site to alter the electronics of Try110 (to Leu) results in delayed timing of the hydride migration from methyl attack of the Δ(24)-bond, that thereby produces metabolic switching of product ratios in favor of Δ(25(27))-olefins or impairs the second C1-transfer activity. Incubation of [27-(13)C]lanosterol or [methyl-(2)H3]SAM as co-substrates established the CrSMT catalyzes a sterol methylation pathway by the "algal" Δ(25(27))-olefin route, where methylation proceeds by a conserved SN2 reaction and de-protonation proceeds from the pro-Z methyl group on lanosterol corresponding to C27. This previously unrecognized catalytic competence for an enzyme of sterol biosynthesis, together with phylogenomic analyses, suggest that mutational divergence of a promiscuous SMT produced substrate- and phyla-specific SMT1 (catalyzes first biomethylation) and SMT2 (catalyzes second biomethylation) isoforms in red and green algae, respectively, and in the case of SMT2 selection afforded modification in reaction channeling necessary for the switch in ergosterol (24ß-methyl) biosynthesis to stigmasterol (24α-ethyl) biosynthesis during the course of land plant evolution.

Fertilization: a sticky sperm protein in plants.

During fertilization in eukaryotes, gametes of the opposite sex undergo a complex series of interactions that culminate in cell fusion. A new study on gamete interaction in plants has identified the first protein in multicellular organisms shown by gene disruption to be essential for gamete membrane adhesion.

Organelle size: a cilium length signal regulates IFT cargo loading.

Cilia grow by assembling structural precursors delivered to their tips by intraflagellar transport. New work on ciliary length control indicates that, during ciliary growth, cilia send a length signal to the cytoplasm that regulates cargo loading onto the constitutively trafficking intraflagellar transport machinery.

Activation loop phosphorylation of a protein kinase is a molecular marker of organelle size that dynamically reports flagellar length.

Specification of organelle size is crucial for cell function, yet we know little about the molecular mechanisms that report and regulate organelle growth and steady-state dimensions. The biflagellated green alga Chlamydomonas requires continuous-length feedback to integrate the multiple events that support flagellar assembly and disassembly and at the same time maintain the sensory and motility functions of the organelle. Although several length mutants have been characterized, the requisite molecular reporter of length has not been identified. Previously, we showed that depletion of Chlamydomonas aurora-like protein kinase CALK inhibited flagellar disassembly and that a gel-shift-associated phosphorylation of CALK marked half-length flagella during flagellar assembly. Here, we show that phosphorylation of CALK on T193, a consensus phosphorylation site on the activation loop required for kinase activity, is distinct from the gel-shift-associated phosphorylation and is triggered when flagellar shortening is induced, thereby implicating CALK protein kinase activity in the shortening arm of length control. Moreover, CALK phosphorylation on T193 is dynamically related to flagellar length. It is reduced in cells with short flagella, elevated in the long flagella mutant, lf4, and dynamically tracks length during both flagellar assembly and flagellar disassembly in WT, but not in lf4. Thus, phosphorylation of CALK in its activation loop is implicated in the disassembly arm of a length feedback mechanism and is a continuous and dynamic molecular marker of flagellar length during both assembly and disassembly.

Regulated membrane protein entry into flagella is facilitated by cytoplasmic microtubules and does not require IFT.

The membrane protein composition of the primary cilium, a key sensory organelle, is dynamically regulated during cilium-generated signaling [1, 2]. During ciliogenesis, ciliary membrane proteins, along with structural and signaling proteins, are carried through the multicomponent, intensely studied ciliary diffusion barrier at the base of the organelle [3-8] by intraflagellar transport (IFT) [9-18]. A favored model is that signaling-triggered accumulation of previously excluded membrane proteins in fully formed cilia [19-21] also requires IFT, but direct evidence is lacking. Here, in studies of regulated entry of a membrane protein into the flagellum of Chlamydomonas, we show that cells use an IFT-independent mechanism to breach the diffusion barrier at the flagellar base. In resting cells, a flagellar signaling component [22], the integral membrane polypeptide SAG1-C65, is uniformly distributed over the plasma membrane and excluded from the flagellar membrane. Flagellar adhesion-induced signaling triggers rapid, striking redistribution of the protein to the apical ends of the cells concomitantly with entry into the flagella. Protein polarization and flagellar enrichment are facilitated by cytoplasmic microtubules. Using a conditional anterograde IFT mutant, we demonstrate that the IFT machinery is not required for regulated SAG1-C65 entry into flagella. Thus, integral membrane proteins can negotiate passage through the ciliary diffusion barrier without the need for a motor.

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates.

Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa.

Evolutionarily conserved Delta(25(27))-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii.

Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ(24(28))-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24ß-methyl) and 7-dehydroporiferasterol (C24ß-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ(25(27))-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ(24(28))-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24ß-methyl/ethyl groups and Δ(22(23))E and Δ(25(27))-double bond constructions. When incubated with [methyl-(2)H(3)]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24ß-alkyl sterols, consistent only with a Δ(25(27))-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol.