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

2017 ◽  
Author(s):  
Simona Amodeo ◽  
Martin Jakob ◽  
Torsten Ochsenreiter
Keyword(s):  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
Super Resolution ◽  
Genome Replication ◽  
The Novel ◽  
Replication Factor ◽  
Summary Statement ◽  
Super Resolution Microscopy ◽  
Novel Protein

AbstractThe 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.Summary StatementMiRF172 is a novel protein involved in the reattachment of replicated minicircles in Trypanosoma brucei, which requires the mitochondrial segregation machinery for proper localization.

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

2020 ◽  
Author(s):  
Simona Amodeo ◽  
Ana Kalichava ◽  
Albert Fradera-Sola ◽  
Eloïse Bertiaux-Lequoy ◽  
Paul Guichard ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Eukaryotic Cell ◽  
The Novel ◽  
Detergent Treatment ◽  
Summary Statement ◽  
First Time

AbstractProper 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.

scholarly journals Characterization of the novel mitochondrial genome replication factor MiRF172 inTrypanosoma brucei

2018 ◽  
Vol 131 (8) ◽  
pp. jcs211730 ◽  
Author(s):  
Simona Amodeo ◽  
Martin Jakob ◽  
Torsten Ochsenreiter
Keyword(s):  
Mitochondrial Genome ◽  
Genome Replication ◽  
The Novel ◽  
Replication Factor

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

2021 ◽  
pp. jcs.254300
Author(s):  
Simona Amodeo ◽  
Ana Kalichava ◽  
Albert Fradera-Sola ◽  
Eloïse Bertiaux-Lequoy ◽  
Paul Guichard ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
Proteome Analysis ◽  
Protozoan Parasite ◽  
Eukaryotic Cell ◽  
The Novel ◽  
Precise Position ◽  
Associated Proteins ◽  
First Time

Proper mitochondrial genome inheritance is important for eukaryotic cell survival. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome, the kDNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described. However, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC associated protein TAP110. Depletion as well as overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA associated proteins including a TEX-like protein that dually localizes to the nucleus and the kDNA potentially linking replication/segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself, however once TAP110 has been assembled it also interacts with the kDNA. Finally, for the first time we use ultrastructure expansion microscopy in trypanosomes to reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.

scholarly journals A molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei

2017 ◽  
Author(s):  
Anneliese Hoffmann ◽  
Sandro Käser ◽  
Martin Jakob ◽  
Simona Amodeo ◽  
Camille Peitsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
De Novo ◽  
Molecular Model ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Kinetoplast Dna ◽  
Exclusion Zone ◽  
Super Resolution Microscopy

AbstractIn 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.Significance 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.Abbreviation(EZF)Exclusion zone filaments(ULF)Unilateral filament(TAC)tripartite attachment complex(OM)outer mitochondrial(IM)inner mitochondrial(BSF)bloodstream form(PCF)procyclic form(kDNA)kinetoplast DNA(gRNA)guide RNA(SBFSEM)Serial block face-scanning electron microscopy(Tet)tetracyclin(STED)Stimulated Emission Depletion

scholarly journals Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy

2017 ◽  
Vol 56 (22) ◽  
pp. 6131-6135 ◽  
Author(s):  
Anne Burgert ◽  
Jan Schlegel ◽  
Jérôme Bécam ◽  
Sören Doose ◽  
Erhard Bieberich ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Regulated protein stabilization underpins the functional interplay among basal body components in Trypanosoma brucei

2019 ◽  
Vol 295 (3) ◽  
pp. 729-742
Author(s):  
Kieu T. M. Pham ◽  
Ziyin Li
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Super Resolution ◽  
Specific Protein ◽  
Protein Stabilization ◽  
Structured Illumination ◽  
Human Parasite ◽  
Evolutionarily Conserved ◽  
Body Components ◽  
Super Resolution Microscopy

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei. Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.

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