Mitochondrial genome maintenance - the kinetoplast story.

Mitochondrial DNA replication is an essential process in most eukaryotes. Similar to the diversity in mitochondrial genome size and organization in the different eukaryotic supergroups, there is considerable diversity in the replication process of the mitochondrial DNA. In this review, we summarize the current knowledge of mitochondrial DNA replication and the associated factors in trypanosomes with a focus on Trypanosoma brucei, and provide a new model of minicircle replication for this protozoan parasite. The model assumes the mitochondrial DNA (kinetoplast DNA, kDNA) of T. brucei to be loosely diploid in nature and the replication of the genome to occur at two replication centers at the opposing ends of the kDNA disc (also known as antipodal sites, APS). The new model is consistent with the localization of most replication factors and in contrast to the current model, it does not require the assumption of an unknown sorting and transport complex moving freshly replicated DNA to the antipodal sites. In combination with the previously proposed sexual stages of the parasite in the insect vector, the new model provides a mechanism for maintenance of the mitochondrial genetic diversity

Characterization of two novel proteins involved in mitochondrial DNA anchoring in Trypanosoma brucei.

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC

Distinct 3′ UTRs regulate the life-cycle-specific expression of two TCTP paralogs in Trypanosoma brucei

The translationally controlled tumor protein (TCTP; also known as TPT1 in mammals) is highly conserved and ubiquitously expressed in eukaryotes. It is 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 human and animal pathogen, Trypanosoma brucei. We identified two paralogs (TCTP1 and TCTP2) that are differentially expressed in the life cycle of the parasite. The genes have identical 5′ untranslated regions (UTRs) and almost identical open-reading frames. The 3′UTRs differ substantially in sequence and length, and are sufficient for the exclusive expression of TCTP1 in procyclic- and TCTP2 in bloodstream-form parasites. Furthermore, we characterize which parts of the 3′UTR are needed for TCTP2 mRNA stability. RNAi experiments demonstrate that TCTP1 and TCTP2 expression is essential for normal cell growth in procyclic- and bloodstream-form parasites, respectively. Depletion of TCTP1 in the procyclic form cells leads to aberrant cell and mitochondrial organelle morphology, as well as enlarged, and a reduced number of, acidocalcisomes

Cryo-electron tomography sheds light on the elastic nature 2 of the Trypanosoma brucei tripartite attachment complex

In contrast to many eukaryotic organisms, 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). Segregation of the replicated kDNA is coordinated by the basal body of the cell's single flagellum. The tripartite attachment complex (TAC) forms a physical connection between the proximal end of the basal body and the kDNA. This allows anchoring of the kDNA throughout the cell cycle and couples kDNA segregation with the separation of the basal bodies prior to cell division. Over the past years, several components of the TAC have been identified. To shed light on the structure of the cytoplasmic part of the TAC (known as the exclusion zone), we performed cryo-electron tomography on whole cells. This allowed us to acquire three-dimensional high-resolution images of the exclusion zone in situ . We observed that the exclusion zone filaments offer great mechanical flexibility for basal body movement. We measured the dimensions of the individual structural elements of the area, as well as the overall orientation and positioning of the basal bodies towards the mitochondrial kDNA pocket. Using a combination of experimental data and modelling, we generated a structural model of the exclusion zone protein p197. Our findings suggest that the majority of p197 consists of a string of spectrin-like repeats. We propose that these structural units provide the architecture of a molecular spring and that they are required in the TAC to withstand the mechanical forces generated through basal body repositioning events during kDNA segregation and motility of the organism

Combination of RNAi and proteomics targeting TAC102, TbmtHMG44 and TbKAP68 in BSF cells.

A) Schematic overview of flagellar extraction from wild type cells and cells with TAC102, TbmtHMG44 or TbKAP68 RNAi in combination with quantitative mass spectrometry. B) Volcano plot showing proteins depleted in flagella extracted from cells after three days of TAC102 RNAi versus flagella extracts from wild type cells. The threshold was set as follows: p-value 1 or S7, S8, S9 and S10 Figs and S2 Table).</p

Protein purifications, additional replicates and further experiments supporting the interaction data of TbKAP68 and TbmtHMG44.

Protein purifications, additional replicates and further experiments supporting the interaction data of TbKAP68 and TbmtHMG44.</p

Candidates of the YFP-TAC102 immunoprecipitation.

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.</div

Mitochondrial prediction for the proteins depleted upon A) TAC102, B) TbmtHMG44 or C) TbKAP68 RNAi.

Mitochondrial prediction for the proteins depleted upon A) TAC102, B) TbmtHMG44 or C) TbKAP68 RNAi.</p

Numerical basis of graphs shown in the manuscript.

Numerical basis of graphs shown in the manuscript.</p

TAC and kDNA are required for localization of TbmtHMG44 and TbKAP68 to the interface between TAC102 and the kDNA.

A) 2D-STED immunofluorescence microscopy of TbmtHMG44-HA relative to TAC102 (monoclonal anti-TAC102 antibody) and the kDNA (DAPI, acquired using confocal microscopy). Scale bar 500 nm. B) 2D-STED immunofluorescence microscopy of TbKAP68-PTP relative to TAC102 and the kDNA. Localization dynamics of TbKAP68 across the cell cycle are depicted in S2 Fig. Scale bar 500 nm. C) Quantitative analysis of the fluorescent signals for TAC102 (top) and TbmtHMG44-HA (bottom) relative to the basal body marker YL1/2, over the course of depletion and reassembly of the TAC. (exemplary images from widefield fluorescence microscopy in S3A Fig; quantification of kDNA loss in S3C Fig) n ≥ 100 cells D) Quantitative analysis of the fluorescent signals for TAC102 (top) and TbKAP68-PTP (bottom) relative to the basal body marker YL1/2, over the course of depletion and reassembly of the TAC (exemplary images from widefield fluorescence microscopy in S3B Fig; quantification of kDNA loss in S3D Fig) n ≥ 100 cells. p.i., post induction; p.r.; post recovery. E) Microscopic analysis of TbmtHMG44 (top) or TbKAP68 (bottom) on isolated flagella (widefield fluorescence microscopy; immunodetection of HA or PTP epitope tags and TAC102, detection of DNA with DAPI, visualization of flagella with phase contrast (PH)). Flagella are either DNase I treated or untreated Scale bars 5 μm.</p