Biology subjects

Ochsenreiter, T.

Publications and source records attributed to Ochsenreiter, T..

14 recordsLinked to original sources

bioRxiv2026

A Genome-Wide Genetic Screen Identifies a Novel kDNA Replication Protein in Trypanosomes

Mitochondrial DNA of trypanosomatid parasites is organized into a topologically complex structure, named kinetoplast (kDNA). Replication, segregation and expression of kDNA involve an estimated [~]300 proteins, only a fraction of which have been identified and characterized. Here, we report the development of a genetic screen in Trypanosoma brucei to identify novel kDNA maintenance factors. Of the 20 highest-ranked genes identified, six are known kDNA maintenance factors. We selected one hit, Tb927.8.4240, a gene of previously unknown function, for experimental follow-up. Ultrastructure expansion microscopy using a tagged version of the protein reveals a dynamic localization during the cell cycle. RNAi-mediated ablation of Tb927.8.4240 results in the progressive but incomplete loss of kDNA, with only a minor effect on the tripartite attachment complex, suggesting the protein is involved in kDNA replication but not segregation. The growth phenotype of Tb927.8.4240 ablation is fully rescued in a kDNA-independent genetic background, confirming a specific role in kDNA replication. In summary, we describe a functional genetic screen for the identification of kDNA maintenance factors in trypanosomes, validate one hit as a novel kDNA replication factor, and provide a prioritized hit list as a promising starting point for the future identification of additional factors.

molecular biology↗

bioRxiv2025

Post-translational modifications of microtubules are crucial for malaria parasite transmission

Microtubules, composed of - and {beta}-tubulin polymers, are essential components of the eukaryotic cytoskeleton. They maintain cellular shape and structural integrity and play critical roles in cell division and in intracellular vesicular transport. In Plasmodium, the parasite that causes malaria, nuclear replication during the liver stage is among the fastest known in eukaryotic cells and relies heavily on microtubules for DNA segregation and cytoskeletal organization. Despite their importance, the role of microtubules in liver stage development remains largely unexplored. Here, we investigated microtubule dynamics during liver stage development using a combination of cell and molecular biology techniques, expansion microscopy, and live-cell imaging. By employing antibodies specific for -tubulin post-translational modifications (PTMs), we found that the Plasmodium sporozoites subpellicular microtubules (SSPM) persist during liver infection, giving rise to liver stage parasite microtubule bundles (LSPMB). These LSPMB form multimeric tubulin structures within hepatocytes and are redistributed to the hemi-spindle poles of parasite nuclei during schizogony. Deletion of the C-terminal region encompassing all known Plasmodium -tubulin PTM sites prevented sporozoite migration from the mosquito midgut to the salivary glands, effectively blocking parasite transmission. Using Plasmodium microtubule-specific depolymerisation drugs, we found that while LSPMB are stable in sporozoites, they exhibit dynamic behavior during hepatocyte infection. Given the regulatory role of PTMs in microtubule dynamics, we generated parasite mutants by substituting and deleting key -tubulin C-terminal residues involved in PTMs. Substitution of the polyglutamylation site with alanine and deletion of the C-terminal tyrosination/detyrosination motifs impaired parasite growth during liver infection. Together, our findings reveal extensive microtubule remodeling during liver stage development and establish -tubulin C-terminal modifications as critical regulators of both intracellular development and parasite transmission of Plasmodium parasites.

cell biology↗

bioRxiv2025

mtTF1: A Novel Factor Involved in Mitochondrial Gene Expression in Trypanosoma brucei

Mitochondrial DNA replication and gene expression are essential for cell survival. In Trypanosoma brucei, a protozoan animal and human parasite, the mitochondrial RNA polymerase (mtRNAP) plays roles in both transcription and DNA replication. This study identifies and characterizes the first mitochondrial transcription factor (mtTF1) in the Kinetoplastea. mtRNAP and mtTF1 form a high-molecular-weight complex that localizes to the kinetoplast DNA (kDNA) and is essential for parasite survival in both life cycle stages. Their localization is interdependent, and both proteins influence maxicircle replication, but not minicircle replication. Knockdown of either protein result in altered gene expression, particularly affecting the minor strand of the mitochondrial genome. Since mtTF1 is unique to the Kinetoplastea, it might prove to be a promising drug target.

cell biology↗

bioRxiv2025

Beyond a Linear Structure: The Tubular Organization of the Tripartite Attachment Complex and the Functional Role of TAC53

The Tripartite Attachment Complex (TAC) is essential for mitochondrial DNA (kDNA) segregation in Trypanosoma brucei, providing a physical link between the flagellar basal body and the mitochondrial genome. Although the TACs hierarchical assembly and linear organization have been extensively studied, much remains to be discovered regarding its complete architecture and composition - for instance, our identification of a new TAC component underscores these knowledge gaps. Here, we use a combination of proteomics, RNA interference (RNAi), and Ultrastructure Expansion Microscopy (U-ExM) to characterize the TAC at high resolution and identify a novel component, TAC53 (Tb927.2.6100). Depletion of TAC53 in both procyclic and bloodstream forms results in kDNA missegregation and loss, a characteristic feature of TAC dysfunction. TAC53 localizes to the kDNA in a cell cycle-dependent manner and represents the most kDNA-proximal TAC component identified to date. U-ExM reveals a previously unrecognized tubular architecture of the TAC, with two distinct TAC structures per kDNA disc, suggesting a mechanism for precise kDNA alignment and segregation. Moreover, immunoprecipitation and imaging analyses indicate that TAC53 interacts with known TAC-associated proteins HMG44, KAP68, and KAP3, forming a network at TAC-kDNA the interface. These findings redefine our understanding of TAC architecture and function and identify TAC53 as a key structural component anchoring the mitochondrial genome in T. brucei. Significance StatementThis research identifies a new component (TAC53) of the tripartite attachment complex (TAC), a cellular machinery that anchors the mitochondrial DNA to a cytoskeletal structure, the basal body in Trypanosoma brucei. Using proteomics and high-resolution microscopy, we demonstrate that TAC53 is at the interface of the TAC and the mitochondrial DNA and likely the final piece in this structure. We also describe the overall tubular architecture of the TAC from the basal body to the mitochondrial matrix, and how the presence of two TAC structures per mitochondrial genome can explain the parasites ability to maintain its mitochondrial DNA accurately. In summary, we present a new component and the architecture of the currently best understood mitochondrial DNA segregation mechanism in biology.

cell biology↗

bioRxiv2023

Pre-gelation staining expansion microscopy (PS-ExM) for visualization of the Plasmodium liver stage

Fluorescence and light microscopy are important tools in the history of natural science. However, the resolution of microscopes is limited by the diffraction of light. One possible method to circumvent this physical restriction is the recently developed expansion microscopy (ExM). By physically expanding the sample in a swellable hydrogel, biomolecules are separated in space allowing to image molecules that are otherwise difficult to study due to their size or complexity. Furthermore, the improved resolution is useful to study protein localization and interactions. However, the original ultrastructure ExM (U-ExM) protocol is very time-consuming, and some epitopes are lost during the process. In this study, we developed a shortened pre-gelation staining ExM (PS-ExM) protocol and tested it to investigate the Plasmodium liver stage. The protocol presented in this study allows expanding pre-stained samples, which results in shorter incubation times, better preservation of some epitopes, and the advantage that non-expanded controls can be performed alongside using the same staining protocol. The protocol applicability was accessed throughout the Plasmodium liver stage showing isotropic five-fold expansion. Furthermore, we used PS-ExM to visualize the association of lysosomes to the parasitophorous vacuole membrane (PVM) as an example of visualizing host-pathogen interaction. We are convinced that this new tool will be helpful for a deeper understanding of the biology of the Plasmodium liver stage.

cell biology↗

bioRxiv2023

Cryo-electron tomography sheds light on the elastic nature of the Trypanosoma brucei tripartite attachment complex

Trypanosomes only contain a single mitochondrion per cell. Within that singular mitochondrion, the protist carries a single mitochondrial genome that consists of a complex DNA network, the kinetoplast DNA (kDNA). The replicated kDNA is segregated during cell division by the tripartite attachment complex (TAC), a multi-protein bridge that physically links each daughter kDNA to a basal body (BB). BB movements drive kDNA segregation prior to cell division. How the TAC accommodates constant BB movements while maintaining a stable kDNA anchor throughout the cell cycle has remained unclear. Here we used cryo-electron tomography to image the cytoplasmic part of the TAC in its native context. We resolved the BB, the mitochondrial membranes, and the exclusion zone filaments (EZFs) connecting the BB and pro-BB to the outer mitochondrial membrane (OMM) and quantified the geometry of the region across many cells. EZF lengths spanned 230 to 625 nm in zoid cells and up to 874 nm in NP40-treated cells, and the BB occupied a wide range of positions and orientations relative to the OMM, while the pro-BB sat closer and more constrained. Building on these observations and prior evidence that p197 alone defines the BB-OMM distance, we propose that p197 is a length-variable connector: the length of a tandem array of -helical repeats depends on their relative orientation, and filaments of different lengths coexist at one basal body. How that orientation is set remains open, but such a connector reconciles stable kDNA anchoring with the mechanical demands of BB movement during the cell cycle.

cell biology↗

bioRxiv2022

The mitochondrial genome segregation system of T. brucei: Single p197 molecules connect the basal body with the outer membrane

The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body of the flagellum. Here we studied the architecture of the exclusion zone filament of the TAC that connects the basal body with the mitochondrial outer membrane. The only known component of the exclusion zone filaments is p197. Using genetical, biochemical and microscopical methods we show that p197 has three domains all of which are essential for mitochondrial DNA inheritance. The C-terminus of p197 interacts with the mature and pro-basal body whereas its N-terminus binds to the peripheral outer membrane protein TAC65. The large central region of p197 has a high -helical content and likely acts as a flexible spacer. Replacement of endogenous p197 with a functional version containing N- and C-terminal epitope tags together with expansion microscopy demonstrates that p197 alone can bridge the approximately 170 nm gap between the basal body and the periphery of the outer membrane. This demonstrates the power of expansion microscopy which allows to localize distinct regions within the same molecule and suggests that p197 is the TAC subunit most proximal to the basal body. Significance statementSegregation of the replicated single unit mitochondrial genome of Trypanosoma brucei requires a large hardwired structure that connects the organellar DNA with the flagellar basal body. The cytosolic part of this structure consists of filaments made of single p197 molecules, a protein larger than 600 kDa. p197 has three domains all of which are essential for its function. The N-terminus of p197 is anchored to the peripheral outer membrane protein TAC65 whereas its C-terminus connects to the base of the basal body. The large central domain forms an -helix and consists of at least 26 repeats of 175 aa in length. It provides a flexible linker bridging the approximately 170 nm between the outer membrane and the basal body

cell biology↗

bioRxiv2021

Ultrastructure Expansion Microscopy in Trypanosoma brucei

Abstract-IntroductionThe recently developed ultrastructure expansion microscopy (U-ExM) technique allows to increase the spatial resolution within a cell or tissue for microscopic imaging through the physical expansion of the sample. In this study we validate the use of U-ExM in Trypanosoma brucei by visualizing the nucleus and kDNA as well as proteins of the cytoskeleton, the basal body, the mitochondrion and the ER. T. brucei is a unicellular flagellated protozoan parasite and the causative agent of human African sleeping sickness and Nagana in cattle.The highly polarized parasite cell body is about 25 m in length and is shaped by the subpellicular microtubule corset. Its single flagellum emanates from the posterior part of the cell and is attached along the entire cell body. T. brucei The cell contains all typical organelles of eukaryotic cells including ER, Golgi and mitochondrion. Interestingly, Golgi and mitochondrion are single unit organelles in this protozoan parasite. The signature feature of trypanosomes is the single unit mitochondrial genome, the kinetoplast DNA (kDNA) that is organized in a complex structure of interlocked mini- and maxicircles. The kDNA is segregated during cell division by the tripartite attachment complex (TAC) that connects it via the mitochondrial membranes to the base of the flagellum.

cell biology↗

bioRxiv2020

Characterization of two novel proteins involved in mitochondrial DNA anchoring

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Excavates. T. brucei cells harbor a single mitochondrion with a singular mitochondrial genome, that consists of a unique network of thousands of interwoven circular DNA molecule copies and is termed the kinetoplast DNA (kDNA). To ensure proper inheritance of the kDNA to the daughter cells the genome is linked to the basal body, the master organizer of the cell cycle in trypanosomes. The structure connecting the basal body and kDNA is termed the tripartite attachment complex (TAC). Using a combination of proteomics and RNAi (depletomics) we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and Tb927.11.16120 as novel candidates of a structure that connects the TAC to the kDNA. Both proteins localize in the region of the unilateral filaments between TAC102 and the kDNA and depletion of each leads to a strong kDNA loss phenotype. TbmtHMG44 and Tb927.11.16120 stably associate with extracted flagella, even after DNase treatment however they do require the kDNA for initial assembly. Furthermore we demonstrate that recombinant Tb927.11.16120 is a DNA binding protein and thus a promising candidate to link the TAC to the kDNA.

cell biology↗

bioRxiv2020

Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 in Trypanosoma brucei

Proper mitochondrial genome inheritance is key for eukaryotic cell survival, however little is known about the molecular mechanism controlling this process. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome aka kinetoplast DNA (kDNA). kDNA segregation requires anchoring of the genome to the basal body via the tripartite attachment complex (TAC). Several components of the TAC as well as their assembly have been described, it however remains elusive how the TAC connects to the kDNA. Here, we characterize the TAC associated protein TAP110 and for the first time use ultrastructure expansion microscopy in trypanosomes to reveal that TAP110 is the currently most proximal kDNA segregation factor. The kDNA proximal positioning is also supported by RNAi depletion of TAC102, which leads to loss of TAP110 at the TAC. Overexpression of TAP110 leads to expression level changes of several mitochondrial proteins and a delay in the separation of the replicated kDNA networks. In contrast to other kDNA segregation factors TAP110 remains only partially attached to the flagellum after DNAse and detergent treatment and can only be solubilized in dyskinetoplastic cells, suggesting that interaction with the kDNA might be important for stability of the TAC association. Furthermore, we demonstrate that the TAC, but not the kDNA, is required for correct TAP110 localization in vivo and suggest that TAP110 might interact with other proteins to form a >669 kDa complex. Summary StatementTAP110 is a novel mitochondrial genome segregation factor in Trypanosoma brucei that associates with the previously described TAC component TAC102. Ultrastructure expansion microscopy reveals its proximal position to the kDNA.

cell biology↗

bioRxiv2019

Discovery of new mitochondrial DNA segregation machinery components in Trypanosoma brucei: a comparison of three approaches

Trypanosoma brucei is a single celled eukaryotic parasite and the causative agent of human African sleeping sickness and Nagana in cattle. Aside from its medical relevance T. brucei has also been key to the discovery of several general biological principles including GPI-anchoring, RNA-editing and trans-splicing. The parasite contains a single mitochondrial organelle with a singular genome. Recent studies have identified several molecular components of the mitochondrial genome segregation machinery (tripartite attachment complex, TAC), which connects the basal body of the flagellum to the mitochondrial DNA of T. brucei. The TAC component in closest proximity to the mitochondrial DNA is TAC102. Here we apply and compare three different approaches (proximity labeling, Immunoprecipitation and yeast two-hybrid) to identify novel interactors of TAC102 and subsequently verify their localisation. Furthermore, we establish the direct interaction of TAC102 and p166 in the unilateral filaments of the TAC. Subject areabiochemistry, molecular biology, cellular biology

cell biology↗

bioRxiv2017

Characterization of the Novel Mitochondrial Genome Replication Factor MiRF172 in Trypanosoma brucei

The unicellular parasite Trypanosoma brucei harbors one individual mitochondrial organelle with a singular genome the kinetoplast DNA or kDNA. The kDNA largely consists of concatenated minicircles and a few maxicircles that are also interlocked into the kDNA disc. More than 30 proteins involved in kDNA replication have been described, however several mechanistic questions are only poorly understood. Here, we describe and characterize MiRF172, a novel mitochondrial genome replication factor, which is essential for proper cell growth and kDNA maintenance. Using super-resolution microscopy, we localize MiRF172 to the antipodal sites of the kDNA. We demonstrate that depletion of MiRF172 leads to continuous loss of mini- and maxicircles during the cell division cycle. Detailed analysis suggests that MiRF172 is likely involved in the reattachment of replicated minicircles to the kDNA disc. Furthermore, we provide evidence that the localization of the replication factor MiRF172 not only depends on the kDNA itself, but also on the mitochondrial genome segregation machinery suggesting a tight interaction between the two essential entities.\n\nSummary StatementMiRF172 is a novel protein involved in the reattachment of replicated minicircles in Trypanosoma brucei, which requires the mitochondrial segregation machinery for proper localization.

cell biology↗

bioRxiv2017

A 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 years ago still very little is known about how the genome is properly 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 proper segregation of the mitochondrial genome via the basal bodies movement, during cell cycle. Using super-resolution microscopy we precisely localize each of the currently known unique TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum towards the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on biochemical analysis the TAC consists of several non-overlapping subcomplexes suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC has an impact on mitochondrial organelle positioning however is not required for proper organelle biogenesis or segregation.\n\nSignificance StatementMitochondrial genome replication and segregation are essential processes in most eukaryotic cells. While replication has been studied in some detail much less is known about the molecular machinery required distribute the replicated genomes. Using super-resolution microscopy in combination with molecular biology and biochemistry we show for the first time in which order the segregation machinery is assembled and that it is assembled de novo rather than in a semi conservative fashion in the single celled parasite Trypanosoma brucei. Furthermore, we demonstrate that the mitochondrial genome itself is not required for assembly to occur. It seems that the physical connection of the mitochondrial genome to cytoskeletal elements is a conserved feature in most eukaryotes, however the molecular components are highly diverse.\n\nAbbreviation

cell biology↗

bioRxiv2017

The differentially expressed translationally controlled tumor proteins TCTP1 and TCTP2 in Trypanosoma brucei

1. SummaryIn Trypanosoma brucei we identified two TCTP genes differentially expressed during the parasite life cycle and discovered the mechanism by which this expression is controlled. Furthermore, we demonstrate that TCTP is important for cell growth as well as proper cell and organelle morphology in the insect form of the parasite.\n\n2. AbstractTCTP is a highly conserved protein ubiquitously expressed in eukaryotes. Studies have reported TCTP to be involved in growth and development, cell cycle progression, protection against cellular stresses and apoptosis, indicating the multifunctional role of the protein. Here, for the first time we characterize the expression and function of TCTP in the unicellular parasite Trypanosoma brucei. We identified two paralogue TCTP genes, which we named TbTCTP1 and TbTCTP2. They have identical 5UTRs and only ten single nucleotide polymorphisms in the open reading frames (ORFs). However, the 3UTRs differ dramatically in sequence and length. We found that the two TCTP mRNAs are differentially expressed during T. brucei life cycle. While procyclic form trypanosomes (PCF) express TCTP1, the bloodstream form trypanosomes (BSF), express TCTP2. We link the differential expression to the distinct 3UTRs of the paralogues. In PCF cells, the protein appears to localize in the cytosol. We show that TCTP1 is essential for normal cell growth and has pleiotropic effects on the cells including aberrant cell morphology, enlarged and reduced number of acidocalcisomes and appearance of accumulations in the mitochondria.

microbiology↗