An advanced fast method for the evaluation of multiple immunolabelling using gold nanoparticles based on low-energy STEM.

We present a powerful method for the simultaneous detection of Au nanoparticles located on both sides of ultrathin sections. The method employs a high-resolution scanning electron microscope (HRSEM) operating in scanning transmission electron microscopy (STEM) mode in combination with the detection of backscattered electrons (BSE). The images are recorded simultaneously during STEM and BSE imaging at the precisely selected accelerating voltage. Under proper imaging conditions, the positions of Au nanoparticles on the top or bottom sides can be clearly differentiated, hence showing this method to be suitable for multiple immunolabelling using Au nanoparticles (NPs) as markers. The difference between the upper and lower Au NPs is so large that it is possible to apply common software tools (such as ImageJ) to enable their automatic differentiation. The effects of the section thickness, detector settings and accelerating voltage on the resulting image are shown. Our experimental results correspond to the results modelled in silico by Monte Carlo (MC) simulations.

Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure.

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.

Ultrastructure and 3D reconstruction of a diplonemid protist (Diplonemea) and its novel membranous organelle.

IMPORTANCE: The knowledge of cell biology of a eukaryotic group is essential for correct interpretation of ecological and molecular data. Although diplonemid protists are one of the most species-rich lineages of marine eukaryotes, only very fragmentary information is available about the cellular architecture of this taxonomically diverse group. Here, a large serial block-face scanning electron microscopy data set complemented with light and fluorescence microscopy allowed the first detailed three-dimensional reconstruction of a diplonemid species. We describe numerous previously unknown peculiarities of the cellular architecture and cell division characteristic for diplonemid flagellates, and illustrate the obtained results with multiple three-dimensional models, comprehensible for non-specialists in protist ultrastructure.

Minimal resin embedding of SBF-SEM samples reduces charging and facilitates finding a surface-linked region of interest.

BACKGROUND: For decoding the mechanism of how cells and organs function information on their ultrastructure is essential. High-resolution 3D imaging has revolutionized morphology. Serial block face scanning electron microscopy (SBF-SEM) offers non-laborious, automated imaging in 3D of up to ~ 1 mm3 large biological objects at nanometer-scale resolution. For many samples there are obstacles. Quality imaging is often hampered by charging effects, which originate in the nonconductive resin used for embedding. Especially, if the imaged region of interest (ROI) includes the surface of the sample and neighbours the empty resin, which insulates the object. This extra resin also obscures the sample's morphology, thus making navigation to the ROI difficult. RESULTS: Using the example of small arthropods and a fish roe we describe a workflow to prepare samples for SBF-SEM using the minimal resin (MR) embedding method. We show that for imaging of surface structures this simple approach conveniently tackles and solves both of the two major problems-charging and ROI localization-that complicate imaging of SBF-SEM samples embedded in an excess of overlying resin. As the surface ROI is not masked by the resin, samples can be precisely trimmed before they are placed into the imaging chamber. The initial approaching step is fast and easy. No extra trimming inside the microscope is necessary. Importantly, charging is absent or greatly reduced meaning that imaging can be accomplished under good vacuum conditions, typically at the optimal high vacuum. This leads to better resolution, better signal to noise ratio, and faster image acquisition. CONCLUSIONS: In MR embedded samples charging is minimized and ROI easily targeted. MR embedding does not require any special equipment or skills. It saves effort, microscope time and eventually leads to high quality data. Studies on surface-linked ROIs, or any samples normally surrounded by the excess of resin, would benefit from adopting the technique.

A Novel Group of Dynamin-Related Proteins Shared by Eukaryotes and Giant Viruses Is Able to Remodel Mitochondria From Within the Matrix.

The diverse GTPases of the dynamin superfamily play various roles in the cell, as exemplified by the dynamin-related proteins (DRPs) Mgm1 and Opa1, which remodel the mitochondrial inner membrane in fungi and metazoans, respectively. Via an exhaustive search of genomic and metagenomic databases, we found previously unknown DRP types occurring in diverse eukaryotes and giant viruses (phylum Nucleocytoviricota). One novel DRP clade, termed MidX, combined hitherto uncharacterized proteins from giant viruses and six distantly related eukaryote taxa (Stramenopiles, Telonemia, Picozoa, Amoebozoa, Apusomonadida, and Choanoflagellata). MidX stood out because it was not only predicted to be mitochondria-targeted but also to assume a tertiary structure not observed in other DRPs before. To understand how MidX affects mitochondria, we exogenously expressed MidX from Hyperionvirus in the kinetoplastid Trypanosoma brucei, which lacks Mgm1 or Opa1 orthologs. MidX massively affected mitochondrial morphology from inside the matrix, where it closely associates with the inner membrane. This unprecedented mode of action contrasts to those of Mgm1 and Opa1, which mediate inner membrane remodeling in the intermembrane space. We speculate that MidX was acquired in Nucleocytoviricota evolution by horizontal gene transfer from eukaryotes and is used by giant viruses to remodel host mitochondria during infection. MidX's unique structure may be an adaptation for reshaping mitochondria from the inside. Finally, Mgm1 forms a sister group to MidX and not Opa1 in our phylogenetic analysis, throwing into question the long-presumed homology of these DRPs with similar roles in sister lineages.

A neo-functionalized homolog of host transmembrane protein controls localization of bacterial endosymbionts in the trypanosomatid Novymonas esmeraldas.

The stability of endosymbiotic associations between eukaryotes and bacteria depends on a reliable mechanism ensuring vertical inheritance of the latter. Here, we demonstrate that a host-encoded protein, located at the interface between the endoplasmic reticulum of the trypanosomatid Novymonas esmeraldas and its endosymbiotic bacterium Ca. Pandoraea novymonadis, regulates such a process. This protein, named TMP18e, is a product of duplication and neo-functionalization of the ubiquitous transmembrane protein 18 (TMEM18). Its expression level is increased at the proliferative stage of the host life cycle correlating with the confinement of bacteria to the nuclear vicinity. This is important for the proper segregation of bacteria into the daughter host cells as evidenced from the TMP18e ablation, which disrupts the nucleus-endosymbiont association and leads to greater variability of bacterial cell numbers, including an elevated proportion of aposymbiotic cells. Thus, we conclude that TMP18e is necessary for the reliable vertical inheritance of endosymbionts.

Massive Accumulation of Strontium and Barium in Diplonemid Protists.

Barium and strontium are often used as proxies of marine productivity in palaeoceanographic reconstructions of global climate. However, long-searched biological drivers for such correlations remain unknown. Here, we report that taxa within one of the most abundant groups of marine planktonic protists, diplonemids (Euglenozoa), are potent accumulators of intracellular barite (BaSO4), celestite (SrSO4), and strontiobarite (Ba,Sr)SO4. In culture, Namystinia karyoxenos accumulates Ba2+ and Sr2+ 42,000 and 10,000 times higher than the surrounding medium, forming barite and celestite representing 90% of the dry weight, the greatest concentration in biomass known to date. As heterotrophs, diplonemids are not restricted to the photic zone, and they are widespread in the oceans in astonishing abundance and diversity, as their distribution correlates with environmental particulate barite and celestite, prevailing in the mesopelagic zone. We found diplonemid predators, the filter-feeding zooplankton that produces fecal pellets containing the undigested celestite from diplonemids, facilitating its deposition on the seafloor. To the best of our knowledge, evidence for diplonemid biomineralization presents the strongest explanation for the occurrence of particulate barite and celestite in the marine environment. Both structures of the crystals and their variable chemical compositions found in diplonemids fit the properties of environmentally sampled particulate barite and celestite. Finally, we propose that diplonemids, which emerged during the Neoproterozoic era, qualify as impactful players in Ba2+/Sr2+ cycling in the ocean that has possibly contributed to sedimentary rock formation over long geological periods. IMPORTANCE We have identified that diplonemids, an abundant group of marine planktonic protists, accumulate conspicuous amounts of Sr2+ and Ba2+ in the form of intracellular barite and celestite crystals, in concentrations that greatly exceed those of the most efficient Ba/Sr-accumulating organisms known to date. We propose that diplonemids are potential players in Ba2+/Sr2+ cycling in the ocean and have possibly contributed to sedimentary rock formation over long geological periods. These organisms emerged during the Neoproterozoic era (590 to 900 million years ago), prior to known coccolithophore carbonate biomineralization (~200 million years ago). Based on reported data, the distribution of diplonemids in the oceans is correlated with the occurrence of particulate barite and celestite. Finally, diplonemids may provide new insights into the long-questioned biogenic origin of particulate barite and celestite and bring more understanding of the observed spatial-temporal correlation of the minerals with marine productivity used in reconstructions of past global climate.

CEP164C regulates flagellum length in stable flagella.

Cilia and flagella are required for cell motility and sensing the external environment and can vary in both length and stability. Stable flagella maintain their length without shortening and lengthening and are proposed to "lock" at the end of growth, but molecular mechanisms for this lock are unknown. We show that CEP164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei. CEP164C localizes to mature basal bodies of fully assembled old flagella, but not to growing new flagella, and basal bodies only acquire CEP164C in the third cell cycle after initial assembly. Depletion of CEP164C leads to dysregulation of flagellum growth, with continued growth of the old flagellum, consistent with defects in a flagellum locking mechanism. Inhibiting cytokinesis results in CEP164C acquisition on the new flagellum once it reaches the old flagellum length. These results provide the first insight into the molecular mechanisms regulating flagella growth in cells that must maintain existing flagella while growing new flagella.

Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei.

In almost all eukaryotes, mitochondria maintain their own genome. Despite the discovery more than 50 y ago, still very little is known about how the genome is correctly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome, the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure correct segregation of the mitochondrial genome via the basal bodies movement, during the cell cycle. Using superresolution microscopy, we precisely localize each of the currently known TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum toward the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on the biochemical analysis, the TAC consists of several nonoverlapping subcomplexes, suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC is required for correct mitochondrial organelle positioning but not for organelle biogenesis or segregation.

RSM22, mtYsxC and PNKD-like proteins are required for mitochondrial translation in Trypanosoma brucei.

Mitochondrial ribosomes evolved from prokaryotic ribosomes, with which they therefore share more common features than with their counterparts in the cytosol. Yet, mitochondrial ribosomes are highly diverse in structure and composition, having undergone considerable changes, including reduction of their RNA component and varying degree of acquisition of novel proteins in various phylogenetic lineages. Here, we present functional analysis of three putative mitochondrial ribosome-associated proteins (RSM22, mtYsxC and PNKD-like) in Trypanosoma brucei, originally identified by database mining. While in other systems the homologs of RSM22 are known as components of mitochondrial ribosomes, YsxC was linked with ribosomes only in bacteria. The PNKD-like protein shows similarity to a human protein, the defects of which cause PNKD (paroxysmal non-kinesigenic dyskinesia). Here we show that all three proteins are important for the growth of T. brucei. They play an important function in mitochondrial translation, as their ablation by RNAi rapidly and severely affected the de novo synthesis of mitochondrial proteins. Moreover, following the RNAi-mediated depletion of RSM22, structure of the small subunit of mitochondrial ribosome becomes severely compromised, suggesting a role of RSM22 in ribosomal assembly and/or stability.

Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria.

Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance.

Aerobic mitochondria of parasitic protists: Diverse genomes and complex functions.

In this review the main features of the mitochondria of aerobic parasitic protists are discussed. While the best characterized organelles are by far those of kinetoplastid flagellates and Plasmodium, we also consider amoebae Naegleria and Acanthamoeba, a ciliate Ichthyophthirius and related lineages. The simplistic view of the mitochondrion as just a power house of the cell has already been abandoned in multicellular organisms and available data indicate that this also does not apply for protists. We discuss in more details the following mitochondrial features: genomes, post-transcriptional processing, translation, biogenesis of iron-sulfur complexes, heme metabolism and the electron transport chain. Substantial differences in all these core mitochondrial features between lineages are compatible with the view that aerobic protists harbor organelles that are more complex and flexible than previously appreciated.

Malleable mitochondrion of Trypanosoma brucei.

The importance of mitochondria for a typical aerobic eukaryotic cell is undeniable, as the list of necessary mitochondrial processes is steadily growing. Here, we summarize the current knowledge of mitochondrial biology of an early-branching parasitic protist, Trypanosoma brucei, a causative agent of serious human and cattle diseases. We present a comprehensive survey of its mitochondrial pathways including kinetoplast DNA replication and maintenance, gene expression, protein and metabolite import, major metabolic pathways, Fe-S cluster synthesis, ion homeostasis, organellar dynamics, and other processes. As we describe in this chapter, the single mitochondrion of T. brucei is everything but simple and as such rivals mitochondria of multicellular organisms.

Mitochondrial heat shock protein machinery hsp70/hsp40 is indispensable for proper mitochondrial DNA maintenance and replication.

UNLABELLED: Mitochondrial chaperones have multiple functions that are essential for proper functioning of mitochondria. In the human-pathogenic protist Trypanosoma brucei, we demonstrate a novel function of the highly conserved machinery composed of mitochondrial heat shock proteins 70 and 40 (mtHsp70/mtHsp40) and the ATP exchange factor Mge1. The mitochondrial DNA of T. brucei, also known as kinetoplast DNA (kDNA), is represented by a single catenated network composed of thousands of minicircles and dozens of maxicircles packed into an electron-dense kDNA disk. The chaperones mtHsp70 and mtHsp40 and their cofactor Mge1 are uniformly distributed throughout the single mitochondrial network and are all essential for the parasite. Following RNA interference (RNAi)-mediated depletion of each of these proteins, the kDNA network shrinks and eventually disappears. Ultrastructural analysis of cells depleted for mtHsp70 or mtHsp40 revealed that the otherwise compact kDNA network becomes severely compromised, a consequence of decreased maxicircle and minicircle copy numbers. Moreover, we show that the replication of minicircles is impaired, although the lack of these proteins has a bigger impact on the less abundant maxicircles. We provide additional evidence that these chaperones are indispensable for the maintenance and replication of kDNA, in addition to their already known functions in Fe-S cluster synthesis and protein import. IMPORTANCE: Impairment or loss of mitochondrial DNA is associated with mitochondrial dysfunction and a wide range of neural, muscular, and other diseases. We present the first evidence showing that the entire mtHsp70/mtHsp40 machinery plays an important role in mitochondrial DNA replication and maintenance, a function likely retained from prokaryotes. These abundant, ubiquitous, and multifunctional chaperones share phenotypes with enzymes engaged in the initial stages of replication of the mitochondrial DNA in T. brucei.

Evolution of parasitism in kinetoplastid flagellates.

Kinetoplastid protists offer a unique opportunity for studying the evolution of parasitism. While all their close relatives are either photo- or phagotrophic, a number of kinetoplastid species are facultative or obligatory parasites, supporting a hypothesis that parasitism has emerged within this group of flagellates. In this review we discuss origin and evolution of parasitism in bodonids and trypanosomatids and specific adaptations allowing these protozoa to co-exist with their hosts. We also explore the limits of biodiversity of monoxenous (one host) trypanosomatids and some features distinguishing them from their dixenous (two hosts) relatives.

Growing diversity of trypanosomatid parasites of flies (Diptera: Brachycera): frequent cosmopolitism and moderate host specificity.

Widely distributed, highly prevalent and speciose, trypanosomatid flagellates represent a convenient model to address topics such as host specificity, diversity and distribution of parasitic protists. Recent studies dealing with insect parasites of the class Kinetoplastea have been focused mainly on trypanosomatids from true bugs (Heteroptera), even though flies (Diptera, Brachycera) are also known as their frequent hosts. Phylogenetic position, host specificity and geographic distribution of trypanosomatids parasitizing dipteran hosts collected in nine countries on four continents (Bulgaria, Czech Republic, Ecuador, Ghana, Kenya, Madagascar, Mongolia, Papua New Guinea and Turkey) are presented. Spliced leader (SL) RNA gene repeats and small subunit (SSU) rRNA genes were PCR amplified from trypanosomatids infecting the gut of a total of forty fly specimens belonging to nine families. While SL RNA was mainly used for barcoding, SSU rRNA was utilized in phylogenetic analyses. Thirty-six different typing units (TUs) were revealed, of which 24 are described for the first time and represent potential new species. Multiple infections with several TUs are more common among brachyceran hosts than in true bugs, reaching one third of cases. When compared to trypanosomatids from heteropteran bugs, brachyceran flagellates are more host specific on the genus level. From seven previously recognized branches of monoxenous trypanosomatids, the Blastocrithidia and "jaculum" clades accommodate almost solely parasites of Heteroptera; two other clades (Herpetomonas and Angomonas) are formed primarily by flagellates found in dipteran hosts, with the most species-rich Leishmaniinae and the small Strigomonas and "collosoma" clades remaining promiscuous. Furthermore, two new clades of trypanosomatids from brachyceran flies emerged in this study. While flagellates from brachyceran hosts have moderate to higher host specificity, geographic distribution of at least some of them seems to be cosmopolitan. Moreover, the genus Angomonas, so far known only from South America, is present on other continents as well.

YCF45 protein, usually associated with plastids, is targeted into the mitochondrion of Trypanosoma brucei.

YCF45 belongs to a family of proteins of unknown function usually located in the chloroplast of plants. Its highly conserved homologues were found in the genomes of several Trypanosoma and Leishmania species. HA(3)-tagging of the YCF45 protein with the start codon as annotated in the Gene(DB) revealed its cytosolic localization in the cultured procyclic stage of Trypanosoma brucei. However, when a more upstream located start codon was used in another HA(3)-tagged construct, the resulting protein was targeted to the mitochondrion. We propose that YCF45 was acquired by an ancestral trypanosomatid by horizontal gene transfer and in the absence of a plastid was re-targeted to the mitochondrion.

Probing for primary functions of prohibitin in Trypanosoma brucei.

Prohibitins (PHBs) 1 and 2 are small conserved proteins implicated in a number of functions in the mitochondrion, as well as in the nucleus of eukaryotic cells. The current understanding of PHB functions comes from studies of model organisms such as yeast, worm and mouse, but considerable debate remains with regard to the primary functions of these ubiquitous proteins. We exploit the tractable reverse genetics of Trypanosoma brucei, the causative agent of African sleeping sickness, in order to specifically analyse the function of PHB in this highly divergent eukaryote. Using inducible RNA interference (RNAi) we show that PHB1 is essential in T. brucei, where it is confined to the cell's single mitochondrion forming a high molecular weight complex. PHB1 and PHB2 appear to be indispensible for mitochondrial translation. Their ablation leads to a decrease in mitochondrial membrane potential, however no effect on the level of reactive oxygen species was observed. Flagellates lacking either PHB1 or both PHB1 and PHB2 exhibit significant morphological changes of their organelle, most notably its inflation. Even long after the loss of the PHB proteins, mtDNA was unaltered and mitochondrial cristae remained present, albeit displaced to the periphery of the mitochondrion, which is in contrast to other eukaryotes.