An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma.

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. However, how the parasites coordinate exocytosis with host interaction is unknown. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and conserved in Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. Using cutting-edge imaging tools, including expansion microscopy and cryo-electron tomography, we show that, unlike previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associate with the exocytic site-prior to the invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarities with GPCR proteins. Collectively our data imply that the CRMP complex acts as a host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

Diversification of CORVET tethers facilitates transport complexity in Tetrahymena thermophila.

In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate Tetrahymena thermophila possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking proteins, while retaining the related CORVET complex. Here, we show that Tetrahymena encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal versus recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how 'backfilling' following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways.This article has an associated First Person interview with the first author of the paper.

The mitochondrial citrate synthase from Tetrahymena thermophila does not form an intermediate filament.

The mitochondrial citrate synthase (mCS) purified from the ciliate Tetrahymena thermophila has been reported to form intermediate-filament-like structures during conjugation and to self-assemble into fibers when recombinantly expressed. This would represent a rare example of a tractable and recent origin of a novel cytoskeletal element. In an attempt to investigate the evolutionary emergence of this behavior, we re-investigated the ability of Tetrahymena's mCS to form filaments in vivo. Using strep-tagged mCS in Tetrahymena and monoclonal antibodies, we found no evidence of filamentous structures during conjugation or starvation. Extensive biochemical characterization of mCS revealed that the self-assembly of recombinant protein is triggered by a specific chemical moiety shared by MES and HEPES buffers used in previous studies. The absence of indicative phenotypes in fiber-deficient GFP-tagged mutants indicates that Tetrahymena mCS did not evolve a structural role in sexual reproduction or metabolic regulation.

Ciliate Microtubule Diversities: Insights from the EFBTU3 Tubulin in the Antarctic Ciliate Euplotes focardii.

Protozoans of the Phylum Ciliophora (ciliates) assemble many diverse microtubular structures in a single cell throughout the life cycle, a feature that made them useful models to study microtubule complexity and the role of tubulin isotypes. In the Antarctic ciliate Euplotes focardii we identified five ß-tubulin isotypes by genome sequencing, named EFBTU1, EFBTU2, EFBTU3, EFBTU4 and EFBTU5. By using polyclonal antibodies directed against EFBTU2/EFBTU1 and EFBTU3, we show that the former isotypes appear to be involved in the formation of all microtubular structures and are particularly abundant in cilia, whereas the latter specifically localizes at the bases of cilia. By RNA interference (RNAi) technology, we silenced the EFBTU3 gene and provided evidence that this isotype has a relevant role in cilia regeneration upon deciliation and in cell division. These results support the long-standing concept that tubulin isotypes possess functional specificity in building diverse microtubular structures.

Unraveling the Elusive Rhoptry Exocytic Mechanism of Apicomplexa.

Apicomplexan parasites are unicellular eukaryotes that invade the cells in which they proliferate. The development of genetic tools in Toxoplasma, and then in Plasmodium, in the 1990s allowed the first description of the molecular machinery used for motility and invasion, revealing a crucial role for two different secretory organelles, micronemes and rhoptries. Rhoptry proteins are injected directly into the host cytoplasm not only to promote invasion but also to manipulate host functions. Nonetheless, the injection machinery has remained mysterious, a major conundrum in the field. Here we review recent progress in uncovering structural components and proteins implicated in rhoptry exocytosis and explain how revisiting early findings and considering the evolutionary origins of Apicomplexa contributed to some of these discoveries.

An Alveolata secretory machinery adapted to parasite host cell invasion.

Apicomplexa are unicellular eukaryotes and obligate intracellular parasites, including Plasmodium (the causative agent of malaria) and Toxoplasma (one of the most widespread zoonotic pathogens). Rhoptries, one of their specialized secretory organelles, undergo regulated exocytosis during invasion1. Rhoptry proteins are injected directly into the host cell to support invasion and subversion of host immune function2. The mechanism by which they are discharged is unclear and appears distinct from those in bacteria, yeast, animals and plants. Here, we show that rhoptry secretion in Apicomplexa shares structural and genetic elements with the exocytic machinery of ciliates, their free-living relatives. Rhoptry exocytosis depends on intramembranous particles in the shape of a rosette embedded into the plasma membrane of the parasite apex. Formation of this rosette requires multiple non-discharge (Nd) proteins conserved and restricted to Ciliata, Dinoflagellata and Apicomplexa that together constitute the superphylum Alveolata. We identified Nd6 at the site of exocytosis in association with an apical vesicle. Sandwiched between the rosette and the tip of the rhoptry, this vesicle appears as a central element of the rhoptry secretion machine. Our results describe a conserved secretion system that was adapted to provide defence for free-living unicellular eukaryotes and host cell injection in intracellular parasites.

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms.

Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER) and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent secretory cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

N6-methyldeoxyadenosine directs nucleosome positioning in Tetrahymena DNA.

BACKGROUND: N6-methyldeoxyadenosine (6mA or m6dA) was shown more than 40 years ago in simple eukaryotes. Recent studies revealed the presence of 6mA in more prevalent eukaryotes, even in vertebrates. However, functional characterizations have been limited. RESULTS: We use Tetrahymena thermophila as a model organism to examine the effects of 6mA on nucleosome positioning. Independent methods reveal the enrichment of 6mA near and after transcription start sites with a periodic pattern and anti-correlation relationship with the positions of nucleosomes. The distribution pattern can be recapitulated by in vitro nucleosome assembly on native Tetrahymena genomic DNA but not on DNA without 6mA. Model DNA containing artificially installed 6mA resists nucleosome assembling compared to unmodified DNA in vitro. Computational simulation indicates that 6mA increases dsDNA rigidity, which disfavors nucleosome wrapping. Knockout of a potential 6mA methyltransferase leads to a transcriptome-wide change of gene expression. CONCLUSIONS: These findings uncover a mechanism by which DNA 6mA assists to shape the nucleosome positioning and potentially affects gene expression.

Remodeling the Specificity of an Endosomal CORVET Tether Underlies Formation of Regulated Secretory Vesicles in the Ciliate Tetrahymena thermophila.

In the endocytic pathway of animals, two related complexes, called CORVET (class C core vacuole/endosome transport) and HOPS (homotypic fusion and protein sorting), act as both tethers and fusion factors for early and late endosomes, respectively. Mutations in CORVET or HOPS lead to trafficking defects and contribute to human disease, including immune dysfunction. HOPS and CORVET are conserved throughout eukaryotes, but remarkably, in the ciliate Tetrahymena thermophila, the HOPS-specific subunits are absent, while CORVET-specific subunits have proliferated. VPS8 (vacuolar protein sorting), a CORVET subunit, expanded to 6 paralogs in Tetrahymena. This expansion correlated with loss of HOPS within a ciliate subgroup, including the Oligohymenophorea, which contains Tetrahymena. As uncovered via forward genetics, a single VPS8 paralog in Tetrahymena (VPS8A) is required to synthesize prominent secretory granules called mucocysts. More specifically, Δvps8a cells fail to deliver a subset of cargo proteins to developing mucocysts, instead accumulating that cargo in vesicles also bearing the mucocyst-sorting receptor Sor4p. Surprisingly, although this transport step relies on CORVET, it does not appear to involve early endosomes. Instead, Vps8a associates with the late endosomal/lysosomal marker Rab7, indicating that target specificity switching occurred in CORVET subunits during the evolution of ciliates. Mucocysts belong to a markedly diverse and understudied class of protist secretory organelles called extrusomes. Our results underscore that biogenesis of mucocysts depends on endolysosomal trafficking, revealing parallels with invasive organelles in apicomplexan parasites and suggesting that a wide array of secretory adaptations in protists, like in animals, depend on mechanisms related to lysosome biogenesis.

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila.

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1-knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment.

A telomerase-associated RecQ protein-like helicase resolves telomeric G-quadruplex structures during replication.

It is well established that G-quadruplex DNA structures form at ciliate telomeres and their formation throughout the cell-cycle by telomere-end-binding proteins (TEBPs) has been analyzed. During replication telomeric G-quadruplex structure has to be resolved to allow telomere replication by telomerase. It was shown that both phosphorylation of TEBPß and binding of telomerase are prerequisites for this process, but probably not sufficient to unfold G-quadruplex structure in timely manner to allow replication to proceed. Here we describe a RecQ-like helicase required for unfolding of G-quadruplex structures in vivo. This helicase is highly reminiscent of human RecQ protein-like 4 helicase as well as other RecQ-like helicase found in various eukaryotes and E. coli. In situ analyses combined with specific silencing of either the telomerase or the helicase by RNAi and co-immunoprecipitation experiments demonstrate that this helicase is associated with telomerase during replication and becomes recruited to telomeres by this enzyme. In vitro assays showed that a nuclear extract prepared from cells in S-phase containing both the telomerase as well as the helicase resolves telomeric G-quadruplex structure. This finding can be incorporated into a mechanistic model about the replication of telomeric G-quadruplex structures during the cell cycle.

Distinct functional roles of ß-tubulin isotypes in microtubule arrays of Tetrahymena thermophila, a model single-celled organism.

BACKGROUND: The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical ß-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and ß-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a ß-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical ß-tubulin, BTU2, and six genes (BLT1-6) yield five divergent ß-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2. METHODOLOGY/PRINCIPAL FINDINGS: With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins in vivo, we transformed Tetrahymena with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically. CONCLUSION/SIGNIFICANCE: We conclude that Tetrahymena uses a family of distinct ß-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.