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.