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Architecture of the mitochondrial genome segregation machinery in Trypanosoma brucei

Architecture of the mitochondrial genome segregation machinery in Trypanosoma brucei

Session

Stream 5: Late Breaking

Authors

Irina Bregy (3, 4, 5), Ana Kalichava (4, 5), Dr. Julika Radecke (2), Salome Aeschlimann (1), Dr. Alistair Siebert (2), Prof. André Schneider (1), Prof. Benoît Zuber (3), Prof. Torsten Ochsenreiter (4)

Affiliations

1. Department of Chemistry and Biochemistry, University of Bern
2. Diamond Light Source
3. Institute of Anatomy, Universtiy of Bern
4. Institute of Cell Biology, University of Bern
5. Graduate School for Cellular and Biomedical Sciences

Keywords

cryo-electron tomography, transmission electron microscopy, ultrastructure expansion microscopy, confocal microscopy, Trypanosoma brucei, mitochondria

Abstract text

Summary

Being a human and animal parasite, Trypanosoma brucei has been studied heavily in the past decades. Nevertheless, many studies focus on trypanosomes as a model organism for the various unique properties of the parasite. Our research is focusing on T. brucei mitochondrial DNA segregation. Because trypanosomes only have a single mitochondrion per cell, with a single mitochondrial DNA network, the process of mitochondrial DNA segregation must be precisely arranged, to avoid loss of the mitochondrial genome or parts thereof. The structure responsible for mitochondrial DNA segregation has been termed the Tripartite Attachment Complex (= TAC). Over the last decades, many components of the TAC have been identified. We here report the ongoing journey of solving the TAC architecture on a structural level. With the combined power of cryo electron tomography, and ultrastructure expansion microscopy, we were able to visualize the TAC protein p197 within the cell. This represents a first step in building an accurate three dimensional model of the TAC structure.

The study in detail

Besides its role as a disease causing agent, the protist parasite Trypanosoma brucei is also of great importance in the study of mitochondrial evolution. Trypanosomes are early branching eukaryotes of the order of the kinetoplastida. Unlike most eukaryotes, kinetoplastid species harbor one single mitochondrion per cell. Some essential genes are encoded in the singular mitochondrial genome of these organisms. Correct mitochondrial genome replication and segregation is thus a crucial cell cycle step that has to be precisely organized by the cell. The TAC as a genome segregation machinery is an example of a unique structure based on common strategies. While being unique to kinetoplastid species, the TAC is anchored at, and organized by centrioles, a very universal structure among eukarya. Furthermore, even though the TAC is structurally unique, it can be understood as analogous to the kinetochore structure that is known from chromosome segregation during mitosis. Because of these parallels to universal features of the eukarya, unraveling the TAC structure is of great interest from an evolutionary point of view.

Using cryo electron tomography, we are able to visualize the TAC region in situ. This allows us to observe the cytosolic part of the TAC in a close to native situation, within the intact cell. We observe flexible filaments connecting the proximal end of the basal body to the outer mitochondrial membrane. From previous studies, it is known that p197 is a TAC protein that has been localized to this area. We therefore used ultrastructure expansion microscopy (U-ExM) to further analyze p197 localization. U-ExM enables superresolution imaging by improving axial and lateral effective resolution due to an isotropic increase in distance between the individual molecules to be analysed. In U-ExM, we apply series of chemical steps to introduce a polymer matrix into T. brucei in order to expand the cell up to 4.5X. Applying U-ExM to double-tagged p197 cell line, we were able to visualize both termini of the protein within the same expanded cell. The experiment clearly showed that the localizations of the N- and C-terminus of the protein differ significantly. While the C-terminus is localized at the basal body, the N-terminus is localized much closer to the kDNA, about where we would expect the mitochondrial membrane to be. 

Taken together, these results suggest that the TAC protein p197 is a filamentous protein of high structural flexibility, that is C-terminally anchored to the basal body and the pro basal body, while it is anchored to the outer mitochondrial membrane at its N-terminus. Whether p197 binds the respective structures itself, or via short linker proteins, remains to be investigated. It is however unlikely, that p197 inserts into the mitochondrial membrane, as it is not predicted to have a transmembrane domain.