Biology subjects

Amodeo, S.

Publications and source records attributed to Amodeo, S..

5 recordsLinked to original sources

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↗

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↗