Extensive gain and loss of photosystem I subunits in chromerid algae, photosynthetic relatives of apicomplexans.

In oxygenic photosynthesis the initial photochemical processes are carried out by photosystem I (PSI) and II (PSII). Although subunit composition varies between cyanobacterial and plastid photosystems, the core structures of PSI and PSII are conserved throughout photosynthetic eukaryotes. So far, the photosynthetic complexes have been characterised in only a small number of organisms. We performed in silico and biochemical studies to explore the organization and evolution of the photosynthetic apparatus in the chromerids Chromera velia and Vitrella brassicaformis, autotrophic relatives of apicomplexans. We catalogued the presence and location of genes coding for conserved subunits of the photosystems as well as cytochrome b6f and ATP synthase in chromerids and other phototrophs and performed a phylogenetic analysis. We then characterised the photosynthetic complexes of Chromera and Vitrella using 2D gels combined with mass-spectrometry and further analysed the purified Chromera PSI. Our data suggest that the photosynthetic apparatus of chromerids underwent unique structural changes. Both photosystems (as well as cytochrome b6f and ATP synthase) lost several canonical subunits, while PSI gained one superoxide dismutase (Vitrella) or two superoxide dismutases and several unknown proteins (Chromera) as new regular subunits. We discuss these results in light of the extraordinarily efficient photosynthetic processes described in Chromera.

Budding of the Alveolate Alga Vitrella brassicaformis Resembles Sexual and Asexual Processes in Apicomplexan Parasites.

Ease of cultivation and availability of genomic data promoted intensive research of free-living phototrophic relatives of apicomplexans, i.e. Chromera velia and Vitrella brassicaformis. Chromera and Vitrella differ significantly in their physiology, morphology, phylogenetic position and genomic features, but Vitrella has not gained as much attention. Here we describe two types of Vitrella zoosporangia. One contains zoospores surrounded by roughly structured matter, with an intracytoplasmic axoneme predicted to develop into a mature flagellum upon spore release, similarly to Plasmodium microgametes; in the second type, cells concurrently bud off the center of the sporangium, surrounded by smooth matter, and flagella develop extracellularly. This process of budding is reminiscent of microsporogenesis as seen in Toxoplasma. We suggest one (or both) of these processes generates gamete-like flagellate progeny. Based on live staining, fusion of zoospores does occur in cultures of V. brassicaformis. We failed to find an apical structure similar to the pseudoconoid in any life stage. V. brassicaformis may therefore either represent an ancestral state lacking an apical complex or has lost the apical complex secondarily. We propose that the common ancestor of Apicomplexa and "chrompodellids" exhibited a complex life cycle, which was reduced in chromerids and colpodellids as dictated by their environment.

Assembly mechanism of Trypanosoma brucei BILBO1, a multidomain cytoskeletal protein.

Trypanosoma brucei BILBO1 (TbBILBO1) is an essential component of the flagellar pocket collar of trypanosomes. We recently reported the high resolution structure of the N-terminal domain of TbBILBO1. Here, we provide further structural dissections of its other three constituent domains: EF-hand, coiled coil, and leucine zipper. We found that the EF-hand changes its conformation upon calcium binding, the central coiled coil forms an antiparallel dimer, and the C-terminal leucine zipper appears to contain targeting information. Furthermore, interdimer interactions between adjacent leucine zippers allow TbBILBO1 to form extended filaments in vitro. These filaments were additionally found to condense into fibers through lateral interactions. Based on these experimental data, we propose a mechanism for TbBILBO1 assembly at the flagellar pocket collar.

Morphology of the trypanosome bilobe, a novel cytoskeletal structure.

The trypanosome bilobe is a cytoskeletal structure of unclear function. To date, four proteins have been shown to localize stably to it: TbMORN1, TbLRRP1, TbCentrin2, and TbCentrin4. In this study, a combination of immunofluorescence microscopy and electron microscopy was used to explore the morphology of the bilobe and its relationship to other nearby cytoskeletal structures in the African trypanosome procyclic trypomastigote. The use of detergent/salt-extracted flagellum preparations was found to be an effective way of discerning features of the cytoskeletal ultrastructure that are normally obscured. TbMORN1 and TbCentrin4 together define a hairpin structure comprising an arm of TbCentrin4 and a fishhook of TbMORN1. The two arms flank a specialized microtubule quartet and the flagellum attachment zone filament, with TbMORN1 running alongside the former and TbCentrin4 alongside the latter. The hooked part of TbMORN1 sits atop the flagellar pocket collar marked by TbBILBO1. The TbMORN1 bilobe occasionally exhibits tendrillar extensions that seem to be connected to the basal and probasal bodies. The TbMORN1 molecules present on these tendrils undergo higher rates of turnover than those for molecules on the main bilobe structure. These observations have been integrated with previous detailed descriptions of the cytoskeletal elements in trypanosome cells.

Evolution of distorted pellicle patterns in rigid photosynthetic euglenids (phacus dujardin).

Members of the euglenid genus Phacus are morphologically differentiated from other photosynthetic species by the presence of a rigid cytoskeleton (pellicle) and predominantly dorsoventrally flattened, leaf-shaped cells. In order to better understand the evolutionary history of this lineage, we used scanning electron microscopy to examine patterns of pellicle strips in Phacus acuminatus, Phacus longicauda var. tortus, Phacus triqueter, Phacus segretii, Phacus pleuronectes, Phacus similis, Phacus pusillus, Phacus orbicularis, Phacus warszewiczii, and Discoplastis spathirhyncha, a putative close relative of Phacus and Lepocinclis. Our observations showed that while the earliest diverging species in our analyses, namely P. warszewiczii, has three whorls of exponential reduction, most members of Phacus have clustered patterns of posterior strip reduction that are bilaterally symmetrical distortions of the radially symmetrical "whorled" patterns found in other photosynthetic euglenids. Comparative morphology, interpreted within the context of molecular phylogenetic analyses of combined nuclear small subunit rDNA and partial nuclear large subunit rDNA sequences, demonstrates that clustered patterns of posterior strip reduction arose after the divergence of Phacus from other photosynthetic euglenids and are the result of developmental processes that govern individual strip length. Clustered patterns of pellicle strips in Phacus do not appear to be adaptively significant themselves; they evolved in association with the origin of cell flattening and cell rigidity, which may be adaptations to a planktonic lifestyle.

Morphology and molecular phylogeny of a marine interstitial tetraflagellate with putative endosymbionts: Auranticordis quadriverberis n. gen. et sp. (Cercozoa).

BACKGROUND: Comparative morphological studies and environmental sequencing surveys indicate that marine benthic environments contain a diverse assortment of microorganisms that are just beginning to be explored and characterized. The most conspicuous predatory flagellates in these habitats range from about 20-150 mum in size and fall into three major groups of eukaryotes that are very distantly related to one another: dinoflagellates, euglenids and cercozoans. The Cercozoa is a diverse group of amoeboflagellates that cluster together in molecular phylogenies inferred mainly from ribosomal gene sequences. These molecular phylogenetic studies have demonstrated that several enigmatic taxa, previously treated as Eukaryota insertae sedis, fall within the Cercozoa, and suggest that the actual diversity of this group is largely unknown. Improved knowledge of cercozoan diversity is expected to help resolve major branches in the tree of eukaryotes and demonstrate important cellular innovations for understanding eukaryote evolution. RESULTS: A rare tetraflagellate, Auranticordis quadriverberis n. gen. et sp., was isolated from marine sand samples. Uncultured cells were in low abundance and were individually prepared for electron microscopy and DNA sequencing. These flagellates possessed several novel features, such as (1) gliding motility associated with four bundled recurrent flagella, (2) heart-shaped cells about 35-75 microm in diam., and (3) bright orange coloration caused by linear arrays of muciferous bodies. Each cell also possessed about 2-30 pale orange bodies (usually 4-5 microm in diam.) that were enveloped by two membranes and sac-like vesicles. The innermost membrane invaginated to form unstacked thylakoids that extended towards a central pyrenoid containing tailed viral particles. Although to our knowledge, these bodies have never been described in any other eukaryote, the ultrastructure was most consistent with photosynthetic endosymbionts of cyanobacterial origin. This combination of morphological features did not allow us to assign A. quadriverberis to any known eukaryotic supergroup. Thus, we sequenced the small subunit rDNA sequence from two different isolates and demonstrated that this lineage evolved from within the Cercozoa. CONCLUSION: Our discovery and characterization of A. quadriverberis underscores how poorly we understand the diversity of cercozoans and, potentially, represents one of the few independent cases of primary endosymbiosis within the Cercozoa and beyond.

NOVEL PELLICLE SURFACE PATTERNS ON EUGLENA OBTUSA (EUGLENOPHYTA) FROM THE MARINE BENTHIC ENVIRONMENT: IMPLICATIONS FOR PELLICLE DEVELOPMENT AND EVOLUTION(1).

Euglena obtusa F. Schmitz possesses novel pellicle surface patterns, including the greatest number of strips (120) and the most posterior subwhorls of strip reduction in any euglenid described so far. Although the subwhorls form a mathematically linear pattern of strip reduction, the pattern observed here differs from the linear pattern described for Euglena mutabilis F. Schmitz in that it contains seven linear subwhorls, rather than three, and is developmentally equivalent to three whorls of exponential reduction, rather than two. These properties imply that the seven-subwhorled linear pattern observed in E. obtusa is evolutionarily derived from an ancestral bilinear pattern, rather than from a linear pattern, of strip reduction. Furthermore, analysis of the relative lateral positions of the strips forming the subwhorls in E. obtusa indicates that (1) the identity (relative length, lateral position, and maturity) of each strip in any mother cell specifies that strip's identity in one of the daughter cells following pellicle duplication and cell division, (2) the relative length of any given pellicle strip regulates the length of the nascent strip it will produce during pellicle duplication, and (3) pellicle pores develop within the heels of the most mature pellicle strips. These observations suggest that continued research on pellicle development could eventually establish an ideal system for understanding mechanisms associated with the morphogenesis and evolution of related eukaryotic cells.

Macroevolution of complex cytoskeletal systems in euglenids.

Euglenids comprise a group of single-celled eukaryotes with diverse modes of nutrition, including phagotrophy and photosynthesis. The level of morphological diversity present in this group provides an excellent system for demonstrating evolutionary transformations in morphological characters. This diversity also provides compelling evidence for major events in eukaryote evolution, such as the punctuated effects of secondary endosymbiosis and mutations in underlying developmental mechanisms. In this essay, we synthesize evidence for the origin, adaptive significance and diversification of the euglenid cytoskeleton, especially pellicle ultrastructure, pellicle surface patterns, pellicle strip number and the feeding apparatus. We also highlight holes in our knowledge that must be filled before we are able to confidently describe euglenid cell biology and infer the earliest stages in euglenid evolution. Nonetheless, by possessing combinations of characters resulting from adaptive change and morphostasis, euglenids have retained key pieces of evidence necessary for reconstructing the early evolution and diversification of eukaryotic life.