Cryo-electron tomography sheds light on the largest protein in the Trypanosoma brucei tripartite attachment complex
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eLife Assessment
This study provides valuable insights into the architecture of the tripartite attachment complex in Trypanosoma brucei and how it may preserve mitochondrial genome anchoring during basal body movement. Cryo-electron tomography and structural modelling provide solid support for the observed variability in filament length and basal body positioning, although the proposed mechanism involving p197 repeat orientation awaits direct molecular validation. These findings will interest researchers studying mitochondrial DNA inheritance, organelle organization, and the cell biology of kinetoplastid parasites.
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Abstract
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 230 to 625 nm in zoid cells and up to 874 nm in NP40-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 p197 alone defines the BB-OMM distance, we propose that p197 is a length-variable connector: the length of a tandem array of α-helical repeats depends on 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.
Significance statement
Using cryo-electron tomography, we describe structural features and dimensions of the tripartite attachment complex (TAC) that forms a stable link between the basal body and the mitochondrial DNA in Trypanosoma brucei . We show that p197, which spans this link and which we find to be the largest known TAC protein in T. brucei , forms filaments of different lengths at a single basal body, and we propose that it accommodates the forces of a moving basal body by varying its own length. The TAC is specific to trypanosomatids, but the problem of mechanical flexibility in dynamic cellular contexts is widespread and poorly understood. Previous work on mechanically compliant proteins has largely isolated molecules; cryo-electron tomography characterizes p197 in situ .
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eLife Assessment
This study provides valuable insights into the architecture of the tripartite attachment complex in Trypanosoma brucei and how it may preserve mitochondrial genome anchoring during basal body movement. Cryo-electron tomography and structural modelling provide solid support for the observed variability in filament length and basal body positioning, although the proposed mechanism involving p197 repeat orientation awaits direct molecular validation. These findings will interest researchers studying mitochondrial DNA inheritance, organelle organization, and the cell biology of kinetoplastid parasites.
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Reviewer #1 (Public review):
Summary:
In this manuscript, the authors employed Cryo-ET to visualize the structural organization of the tripartite attachment complex or the TAC, which is a unique structure connecting the flagellar basal body to the mitochondria in Trypanosoma brucei, a protozoan parasite causing human sleeping sickness in sub-Saharan Africa. The TAC is essential for segregation of the mitochondrial DNA, or kDNA, via the connection to the segregating basal bodies during the cell cycle. Although molecular characterization of the TAC and its components has yielded insights into the function of the TAC, the structural organization of the TAC, especially the largest protein p197 within the TAC, remains poorly defined. Using intact zoid cells, the authors provided, for the first time, a high-resolution image of the cytoplasmic …
Reviewer #1 (Public review):
Summary:
In this manuscript, the authors employed Cryo-ET to visualize the structural organization of the tripartite attachment complex or the TAC, which is a unique structure connecting the flagellar basal body to the mitochondria in Trypanosoma brucei, a protozoan parasite causing human sleeping sickness in sub-Saharan Africa. The TAC is essential for segregation of the mitochondrial DNA, or kDNA, via the connection to the segregating basal bodies during the cell cycle. Although molecular characterization of the TAC and its components has yielded insights into the function of the TAC, the structural organization of the TAC, especially the largest protein p197 within the TAC, remains poorly defined. Using intact zoid cells, the authors provided, for the first time, a high-resolution image of the cytoplasmic part of the TAC in its native context, resolved the basal body, the mitochondrial membranes, the exclusion zone filament connecting the BB to the outer mitochondrial membrane, and quantified the geometry of the region surrounding the TAC. These observations led to a proposed role of p197 as a length-variable connector to reconcile stable kDNA anchoring with the mechanical demands of BB movement. Overall, the work advances our understanding of the structure and function of p197 within the TAC in trypanosomes.
Strengths:
A major strength of the manuscript is the application of Cryo-ET in imaging the structure of the cytoplasmic part of the TAC, with a focus on the largest protein p197, which provided novel insights into its molecular function. Trypanosome cells are too thick for Cryo-ET, and the authors adopted intact zoid cells for Cryo-ET, allowing the resolution of the TAC structure in native conditions.
Weaknesses:
While this is the first attempt to image the TAC by Cryo-ET, which provided new insights into the potential role of p197 in mediating the connection between the BB and the mitochondria, there is a lack of mechanistic understanding of how p197's length is regulated and how the varying length of p197 coordinates with basal body movement. Also, it is unclear why the cell needs to regulate the length of p197, given that the main function of p197 is to connect the BB and the mitochondria for the segregation of kDNA via the BB.
The proposed length variation for p197 is an interesting idea, but it is unclear what exactly the authors meant by saying that p197 is length variable. Do they mean that the central repeats in p197 can fold/extend to variable lengths, or do they mean that p197 with variable lengths contains different numbers of the central repeats? This should be clarified.
The current work can be strengthened by performing some molecular and/or biochemical experiments to assess the function of p197 to complement the Cryo-ET-based structural characterization reported in the manuscript. Expression of some smaller-sized p197 proteins with varying numbers of central repeats in p197 RNAi cells will provide evidence for the proposed function of p197 length variation.
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Reviewer #2 (Public review):
While the aim of this study is interesting and the methods are advanced, the novel insight and conclusions rather feel limited at present, particularly considering the literature already available on the functional role of p197. There are some notable methodological issues in quantifying the EZF that need to be addressed.
The claims accompanying p197 and its role in EZF tethering to the BB and pBB to the OMM, as well as its effect on mitochondrial membranes, as displayed in Figure 3, should be augmented via additional comparative measurements with tdzoid cell tomograms, given that tomograms were generated for tdzoid cell lines, but are only utilised in a limited manner for Figures S4 and S5. This represents an underutilised opportunity to use this technique to contrast BB, pBB and mitochondrial membrane …
Reviewer #2 (Public review):
While the aim of this study is interesting and the methods are advanced, the novel insight and conclusions rather feel limited at present, particularly considering the literature already available on the functional role of p197. There are some notable methodological issues in quantifying the EZF that need to be addressed.
The claims accompanying p197 and its role in EZF tethering to the BB and pBB to the OMM, as well as its effect on mitochondrial membranes, as displayed in Figure 3, should be augmented via additional comparative measurements with tdzoid cell tomograms, given that tomograms were generated for tdzoid cell lines, but are only utilised in a limited manner for Figures S4 and S5. This represents an underutilised opportunity to use this technique to contrast BB, pBB and mitochondrial membrane separation in a comprehensive manner beyond what previous studies could achieve.
The authors need to justify specifically why different methodologies were employed for minimum filament length measurements between NP-40 wild-type cells and non-detergent-treated Zoid cells, preventing these datasets from being broadly comparable.
The reliance on mutant cell lines (primarily zoids) for conclusions needs to be acknowledged to a greater extent, such as when comparing results with previous works (Line 334) and naturally within the limitations mentioned in the discussion.
The integration of the discussion with relevant literature concerning Cryo-EM and trypanosomatid cytoskeletal studies feels somewhat limited; for example, no mention or comparison with the formative Höög et al. 2012 study for Cryo-EM within trypanosome flagella and basal bodies.
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Reviewer #3 (Public review):
Summary:
The authors use the advanced technique of cryo-electron tomography to understand the connections between the basal body and the mitochondrion in trypanosomes. They show that this connection is mediated by flexible and extendable filaments.
Strengths:
The authors use an array of advanced cryo-electron microscopy techniques that provide solid evidence that p197 forms the filaments between the basal body and the mitochondrion and demonstrate that these filaments are flexible and able to span a wide range of distances. This provides an important insight into how the trypanosome basal body, which has a dynamic localisation through the cell cycle, can manage its critical connection to the mitochondrion. The filaments provide the necessary flexibility to maintain the basal body - mitochondrion connections …
Reviewer #3 (Public review):
Summary:
The authors use the advanced technique of cryo-electron tomography to understand the connections between the basal body and the mitochondrion in trypanosomes. They show that this connection is mediated by flexible and extendable filaments.
Strengths:
The authors use an array of advanced cryo-electron microscopy techniques that provide solid evidence that p197 forms the filaments between the basal body and the mitochondrion and demonstrate that these filaments are flexible and able to span a wide range of distances. This provides an important insight into how the trypanosome basal body, which has a dynamic localisation through the cell cycle, can manage its critical connection to the mitochondrion. The filaments provide the necessary flexibility to maintain the basal body - mitochondrion connections as the basal body is repositioned.
Weaknesses:
The authors were unable to directly connect the filaments to the structural models for their protein component, p197 and how the filament flexibility relates to the changes in the repeat region of the protein. In part, this is due to the flexibility of these filaments and demonstrates the limitation of cryo-EM to resolve such structures, but this in itself is an important outcome, as it will inform the community's use of this technology.
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