Cryo-electron tomography sheds light on the largest protein in the Trypanosoma brucei tripartite attachment complex

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). The replicated kDNA is segregated during cell division by the tripartite attachment complex (TAC), a multi-protein bridge that physically links each daughter kDNA to a basal body (BB). BB movements drive kDNA segregation prior to cell division. How the TAC accommodates constant BB movements while maintaining a stable kDNA anchor throughout the cell cycle has remained unclear. Here we used cryo-electron tomography to image the cytoplasmic part of the TAC in its native context. We resolved the BB, the mitochondrial membranes, and the exclusion zone filaments (EZFs) connecting the BB and pro-BB to the outer mitochondrial membrane (OMM) and quantified the geometry of the region across many cells. EZF lengths spanned to nm in zoid cells and up to nm in NP-treated cells, and the BB occupied a wide range of positions and orientations relative to the OMM, while the pro-BB sat closer and more constrained. Building on these observations and prior evidence that p alone defines the BB-OMM distance, we propose that p is a length-variable connector: the length of a tandem array of $\alpha$-helical repeats depends on perpetuity. It is made available under a CC-BY-NC-ND International license. preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in bioRxiv preprint doi: [https://doi.org/1.0/./](https://www.google.com/search?q=https://doi.org/1.0/./).; this version posted September , . The copyright holder for this their relative orientation, and filaments of different lengths coexist at one basal body. How that orientation is set remains open, but such a connector reconciles stable kDNA anchoring with the mechanical demands of BB movement during the cell cycle.