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

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

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

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

Functional analysis of prohibitin in \kur{Trypanosoma brucei}

In this study the importance of prohibitin1 and prohibitin2 genes for Trypanosoma brucei was examined. RNA interference showed both of them essential for parasites to survive. Knocking down of these genes resulted in altered morphology of the mitochondrion, changes in membrane potential and shut down of mitochondrial translation. No changes were observed in levels of Reactive Oxygen Species and respiration. Both prohibitines are part of big complex present in the mitochondrion

Functional analysis of the YCF 45 gene in procyclic \kur{Trypanosoma brucei}

In this study was shown by RNA interference that YCF 45 gene of putative chloroplast origin is not essential for surviving of procyclic Trypanosoma brucei. Three different construct bearing HA-tagged YCF 45 protein that differs on their N-terminus were generated to reveal the localization of the protein in the cell. Spliced leader acceptor site of YCF 45 mRNA was identified and in comparison to annotated data new start codon was proposed. Fylogeny tree was made to shed some light on the origin of this gene

Kinetoplastids biology, from the group phylogeny and evolution into the secrets of the mitochondrion of one representative: \kur{Trypanosoma brucei}, the model organism in which new roles of the evolutionary conserved genes can be explored

This thesis is composed of two topics, for which trypanosomatids and evolution are common denominators. First part deals with phylogenetic relationships among monoxenous trypanosomatids, with emphasis on flagellates parasitizing dipteran hosts, analyzed mainly from biogeographical and evolutionary perspectives. Second part focuses on the trypanosomatid Trypanosoma brucei, causative agent of severe diseases, which serves as a model organism for functional studies of evolutionary conserved mitochondrial proteins, in particular those involved in replication, maintenance and expression of the mitochondrial genome, also termed the kinetoplast. This thesis identified the mtHsp70/mtHsp40 chaperone machinery as an essential component of replication and maintenance of the kinetoplast, and also identified numerous conditions under which mtHsp70 has a tendency to aggregate. Moreover, several conserved proteins, previously identified to be part of the mitochondrial ribosome, were shown to be important for translation of the mitochondrial transcripts

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

Abstract 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