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.

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.

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

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.

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 mm large biological objects at nanometer-scale resolution.

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).

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.

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 (BaSO), celestite (SrSO), and strontiobarite (Ba,Sr)SO.

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.

Molecular model of the mitochondrial genome segregation machinery in .

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 contains a single mitochondrion with a singular genome, the kinetoplast DNA (kDNA).

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.

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.

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.

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.

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.

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.

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.

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.

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.