Catarina Gadelha

Discovery of novel intermediate forms redefines the fungal tree of life

Fungi are the principal degraders of biomass in terrestrial ecosystems and establish important interactions with plants and animals. However, our current understanding of fungal evolutionary diversity is incomplete and is based upon species amenable to growth in culture. These culturable fungi are typically yeast or filamentous forms, bound by a rigid cell wall rich in chitin. Evolution of this body plan was thought critical for the success of the Fungi, enabling them to adapt to heterogeneous habitats and live by osmotrophy: extracellular digestion followed by nutrient uptake. Here we investigate the ecology and cell biology of a previously undescribed and highly diverse form of eukaryotic life that branches with the Fungi, using environmental DNA analyses combined with fluorescent detection via DNA probes. This clade is present in numerous ecosystems including soil, freshwater and aquatic sediments. Phylogenetic analyses using multiple ribosomal RNA genes place this clade with Rozella, the putative primary branch of the fungal kingdom. Tyramide signal amplification coupled with group-specific fluorescence in situ hybridization reveals that the target cells are small eukaryotes of 3–5 μm in length, capable of forming a microtubule-based flagellum. Co-staining with cell wall markers demonstrates that representatives from the clade do not produce a chitin-rich cell wall during any of the life cycle stages observed and therefore do not conform to the standard fungal body plan. We name this highly diverse clade the cryptomycota in anticipation of formal classification.

Basal body movements orchestrate membrane organelle division and cell morphogenesis in Trypanosoma brucei

The defined shape and single-copy organelles of Trypanosoma brucei mean that it provides an excellent model in which to study how duplication and segregation of organelles is interfaced with morphogenesis of overall cell shape and form. The centriole or basal body of eukaryotic cells is often seen to be at the centre of such processes. We have used a combination of electron microscopy and electron tomography techniques to provide a detailed three-dimensional view of duplication of the basal body in trypanosomes. We show that the basal body duplication and maturation cycle exerts an influence on the intimately associated flagellar pocket membrane system that is the portal for secretion and uptake from this cell. At the start of the cell cycle, a probasal body is positioned anterior to the basal body of the existing flagellum. At the G1–S transition, the probasal body matures, elongates and invades the pre-existing flagellar pocket to form the new flagellar axoneme. The new basal body undergoes a spectacular anti-clockwise rotation around the old flagellum, while its short new axoneme is associated with the pre-existing flagellar pocket. This rotation and subsequent posterior movements results in division of the flagellar pocket and ultimately sets parameters for subsequent daughter cell morphogenesis.

Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes

A key feature of immune evasion for African trypanosomes is the functional specialization of their surface membrane in an invagination known as the flagellar pocket (FP), the cells sole site of endocytosis and exocytosis. The FP membrane is biochemically distinct yet continuous with those of the cell body and the flagellum. The structural features maintaining this individuality are not known, and we lack a clear understanding of how extracellular components gain access to the FP. Here, we have defined domains and boundaries on these surface membranes and identified their association with internal cytoskeletal features. The FP membrane appears largely homogeneous and uniformly involved in endocytosis. However, when endocytosis is blocked, receptor-mediated and fluid-phase endocytic markers accumulate specifically on membrane associated with four specialized microtubules in the FP region. These microtubules traverse a distinct boundary and associate with a channel that connects the FP lumen to the extracellular space, suggesting that the channel is the major transport route into the FP.

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa.

ABSTRACT Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.

Basal body and flagellum mutants reveal a rotational constraint of the central pair microtubules in the axonemes of trypanosomes

Productive beating of eukaryotic flagella and cilia requires a strict regulation of axonemal dynein activation. Fundamental to any description of axonemal beating is an understanding of the significance of the central pair microtubules and the degree to which central pair rotation has a role. However, for the majority of organisms, it is unclear whether the central pair actually rotates. Using an extra-axonemal structure as a fixed reference, we analysed the orientation of the central pair in African trypanosomes and other kinetoplastid protozoa. A geometric correction allowed the superposition of data from many cross-sections, demonstrating that the axis of the central pair is invariant and that there is no central pair rotation in these organisms. Analysis of mutants depleted in particular flagellar and basal body proteins [γ-tubulin, δ-tubulin, Parkin co-regulated gene product (PACRG) or the paraflagellar rod protein PFR2] allowed a dissection of the mechanisms for central pair constraint. This demonstrated that orientation is independent of flagellum attachment and beating, but is influenced by constraints along its length and is entirely dependent on correct positioning at the basal plate.

Improvement on the visualization of cytoskeletal structures of protozoan parasites using high-resolution field emission scanning electron microscopy (FESEM)

The association of high resolution field emission scanning electron microscopy (FESEM), with a more efficient system of secondary electron (SE) collection and in-lens specimen position, provided a great improvement in the specimen’s topographical contrast and in the generation of high-resolution images. In addition, images obtained with the use of the high-resolution backscattered electrons (BSE) detector provided a powerful tool for immunocytochemical analysis of biological material. In this work, we show the contribution of the FESEM to the detailed description of cytoskeletal structures of the protozoan parasites Herpetomonas megaseliae, Trypanosoma brucei and Giardia lamblia. High-resolution images of detergent extracted H. megaseliae and T. brucei showed the profile of the cortical microtubules, also known as sub-pellicular microtubules (SPMT), and protein bridges cross-linking them. Also, it was possible to visualize fine details of the filaments that form the lattice-like structure of the paraflagellar rod (PFR) and its connection with the axoneme. In G. lamblia, it was possible to observe the intricate structure of the adhesive disk, funis (a microtubular array) and other cytoskeletal structures poorly described previously. Since most of the stable cytoskeletal structures of this protozoan rely on tubulin, we used the BSE images to accurately map immunolabeled tubulin in its cytoskeleton. Our results suggest that the observation of detergent extracted parasites using FESEM associated to backscattered analysis of immunolabeled specimens represents a new approach for the study of parasite cytoskeletal elements and their protein associations.

A novel role for BRCA1 in regulating breast cancer cell spreading and motility.

BRCA1 interacts with ERM proteins at leading edges and focal adhesion sites and modulates motility via its ubiquitin ligase activity.

Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture

DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.

Proteomic Analysis of Clathrin Interactions in Trypanosomes Reveals Dynamic Evolution of Endocytosis

Endocytosis is a vital cellular process maintaining the cell surface, modulating signal transduction and facilitating nutrient acquisition. In metazoa, multiple endocytic modes are recognized, but for many unicellular organisms the process is likely dominated by the ancient clathrin‐mediated pathway. The endocytic system of the highly divergent trypanosomatid Trypanosoma brucei exhibits many unusual features, including a restricted site of internalization, dominance of the plasma membrane by GPI‐anchored proteins, absence of the AP2 complex and an exceptionally high rate. Here we asked if the proteins subtending clathrin trafficking in trypanosomes are exclusively related to those of higher eukaryotes or if novel, potentially taxon‐specific proteins operate. Co‐immunoprecipitation identified twelve T. brucei clathrin‐associating proteins (TbCAPs), which partially colocalized with clathrin. Critically, eight TbCAPs are restricted to trypanosomatid genomes and all of these are required for robust cell proliferation. A subset, TbCAP100, TbCAP116, TbCAP161 and TbCAP334, were implicated in distinct endocytic steps by detailed analysis of knockdown cells. Coupled with the absence of orthologs for many metazoan and fungal endocytic factors, these data suggest that clathrin interactions in trypanosomes are highly lineage‐specific, and indicate substantial evolutionary diversity within clathrin‐mediated endocytosis mechanisms across the eukaryotes.

Specializations in a successful parasite: What makes the bloodstream-form African trypanosome so deadly?

Most trypanosomatid parasites have both arthropod and mammalian or plant hosts, and the ability to survive and complete a developmental program in each of these very different environments is essential for life cycle progression and hence being a successful pathogen. For African trypanosomes, where the mammalian stage is exclusively extracellular, this presents specific challenges and requires evasion of both the acquired and innate immune systems, together with adaptation to a specific nutritional environment and resistance to mechanical and biochemical stresses. Here we consider the basis for these adaptations, the specific features of the mammalian infective trypanosome that are required to meet these challenges, and how these processes both inform on basic parasite biology and present potential therapeutic targets.