https://orcid.org/0000-0002-8846-8526
Figures
Abstract
The Tripartite Attachment Complex (TAC) is essential for mitochondrial DNA (kDNA) segregation in Trypanosoma brucei, providing a physical link between the flagellar basal body and the mitochondrial genome. Although the TAC’s hierarchical assembly and linear organization have been extensively studied, much remains to be discovered regarding its complete architecture and composition – for instance, our identification of a new TAC component underscores these knowledge gaps. Here, we use a combination of proteomics, RNA interference (RNAi), and Ultrastructure Expansion Microscopy (U-ExM) to characterize the TAC at high resolution and identify a novel component, TAC53 (Tb927.2.6100). Depletion of TAC53 in both procyclic and bloodstream forms results in kDNA missegregation and loss, a characteristic feature of TAC dysfunction. TAC53 localizes to the kDNA in a cell cycle-dependent manner and represents the most kDNA-proximal TAC component identified to date. U-ExM reveals a previously unrecognized tubular architecture of the TAC, with two distinct TAC structures per kDNA disc, suggesting a mechanism for precise kDNA alignment and segregation. Moreover, immunoprecipitation and imaging analyses indicate that TAC53 interacts with known TAC-associated proteins HMG44, KAP68, and KAP3, forming a network at the TAC–kDNA interface. These findings redefine our understanding of TAC architecture and function and identify TAC53 as a key structural component anchoring the mitochondrial genome in T. brucei.
Author summary
Trypanosoma brucei is the causative agent of human African sleeping sickness and nagana in cattle. The eukaryotic parasite relies on the faithful inheritance of its singular mitochondrial genome, known as kinetoplast DNA (kDNA), to survive and proliferate. This genome is physically tethered to the flagellar basal body by the Tripartite Attachment Complex (TAC), a unique cytoskeleton-organelle connection. While previous studies described the TAC’s linear architecture, its full composition and spatial organization remained incomplete. Here, we use proteomics, RNAi, and ultrastructure expansion microscopy to identify TAC53 as a novel and essential TAC component. TAC53 localizes closest to the kDNA of all known TAC proteins and is required for correct mitochondrial genome segregation. High-resolution imaging reveals that the TAC is not a simple linear structure but instead forms a tubular architecture with two distinct TAC assemblies per individual genome, aligning with the cell’s basal body pairs. Our findings revise the current model of mitochondrial genome segregation in T. brucei and highlight TAC53 as the likely final component of the TAC, offering new insights into organelle genome inheritance mechanisms in early-branching eukaryotes.
Citation: Jetishi C, Aeschlimann S, Schimanski B, Käser S, Mullner R, Oeljeklaus S, et al. (2025) Connecting basal body and mitochondrial DNA: TAC53 and the tubular organization of the tripartite attachment complex. PLoS Pathog 21(9): e1013521. https://doi.org/10.1371/journal.ppat.1013521
Editor: Cynthia Y. He, National University of Singapore, SINGAPORE
Received: June 5, 2025; Accepted: September 7, 2025; Published: September 15, 2025
Copyright: © 2025 Jetishi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository and are accessible using the dataset identifiers PXD063227, PXD063233, PXD061646 and PXD062506 [71,72].
Funding: o This work was supported by the Swiss National Science Foundation (179454 and 207525 to T.O.) and the Uniscientia foundation. The work in the lab of A.S. was supported in part by NCCR RNA & Disease, a National Centre of Competence in Research (205601 to A.S.) and by project grant SNF (205200 to A.S.) both funded by the Swiss National Science Foundation. Work in the lab of B.A. was supported by a Wellcome Discovery Award (227243/Z/23/Z to B.A.). For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Trypanosoma brucei is a single-celled eukaryotic parasite. Major discoveries made in trypanosomes like GPI anchors, trans splicing, RNA editing and acidocalcisomes were later also found to be important features in other eukaryotes [1–4]. Trypanosomes have served as a model system to study mitochondrial biogenesis in an organism evolutionary distinct from the mainstream systems like yeast and human cells. Unlike yeast and mammals, T. brucei cells harbor only one single mitochondrion, with a single nucleoid that is named kinetoplast and contains the mitochondrial DNA (kinetoplast DNA, kDNA) [5–8].
The kDNA consists of two different genetic components, the maxi- and the minicircles, which are topologically interlocked and form a dense disc-shaped structure. While the maxicircles encode for ribosomal RNAs and proteins required in the respiratory chain and the mitochondrial ribosome, the minicircles encode for guide RNAs (gRNAs) which are required for editing the maxicircle encoded transcripts [9–11]. The replication of trypanosomal kDNA, unlike other eukaryotic mitochondrial genomes, is coordinated with the nuclear cell cycle and the segregation of replicated kDNA discs is completed before mitosis [12,13]. The single-unit nature of the kDNA requires a precise mechanism to ensure that both daughter cells inherit one kDNA disc. This is facilitated by a stable connection of the kDNA disc with the flagellum’s basal body. This linkage ensures the segregation of both the old and new flagellum along with the replicated kDNA [14].
The Tripartite Attachment Complex (TAC) is the structure that physically connects the basal body to the kDNA disc and positions the kDNA at the posterior end of the cell [15]. The TAC consists of three distinct subdomains: i) the exclusion zone filaments (EZF) between the basal body of the flagellum and the outer mitochondrial membrane (OMM), ii) the differentiated membranes (DM) where the inner mitochondrial membrane (IMM) is closely opposed to the OMM and iii) the unilateral filaments (ULF) that connect the IMM to the kDNA disc [6,16,17]. Although mitochondrial inheritance in other organisms involves cytoskeletal elements like actin and microtubules, a stable, permanent connection between mitochondrial nucleoids and the cytoskeleton has only been described in T. brucei [17–20]. Therefore, the TAC provides a direct physical link between the mitochondrial DNA and the cytoskeleton, making it the only known example of such a permanent DNA-cytoskeleton connection.
So far eight TAC proteins have been described in T. brucei [21]. The component closest to the BB and potentially directly connected to it, is the approximately 880 kDa protein p197 [22–25]. It has been shown that p197 filaments determine the distance between the BB and the OMM, where it directly binds TAC65 [24]. TAC65 is a peripherally OMM associated protein that interacts with the OMM integrated pATOM36. Interestingly, pATOM36 not only operates as a TAC protein, but also functions in biogenesis of a subset of OMM proteins. Therefore, pATOM36 is located throughout the entire OMM [26,27]. TAC40, TAC42 and TAC60 form a complex in the OMM. TAC40 and TAC42 are β-barrel proteins, whereas TAC60 is an alpha-helical protein with two transmembrane domains (TMDs) [28,29]. Both the N- and C-termini of TAC60 are located in the cytoplasm, resulting in the formation of a loop in the intermembrane space (IMS) [28]. For the IMM region of the TAC, only one protein is described, p166. Being the first TAC protein to be identified in 2008 [30], it was recently shown to have a mitochondrial targeting sequence (MTS) and being integrated into the IMM with a single C-terminal TMD. The very C-terminus of p166 reaches into the IMS and interacts with the loop formed by TAC60 [31,32]. The N-terminus of p166 directly interacts with TAC102, a protein of the ULFs [33]. TAC102 was identified as the most kDNA-proximal TAC component, however, direct kDNA binding could not be assigned [23,34]. In addition to the TAC proteins, two TAC-associated proteins, HMG44 and KAP68, were suggested to interact with TAC102. These proteins were shown to interact with each other in vitro, and both their depletion results in kDNA loss. Since HMG44 and KAP68 are located closer to the kDNA than TAC102, it was proposed that they anchor the TAC to the kDNA [35].
A shared feature of all TAC proteins is that their depletion leads to a missegregation phenotype, where the replicated kDNA will stay attached to the “old” TAC, whereas the “new” TAC emanating from the newly assembled pro-basal body only has fractions of kDNA or no kDNA. Ultimately this leads to kDNA loss in the daughter cells, while the mother cell retains the over-replicated kinetoplast [28]. Additionally, TAC proteins localize to the TAC regions in intact cells as well as isolated flagella [17] and they are dispensable in an engineered cell line of the bloodstream form of T. brucei (γL262P), which can survive without the kDNA [36].
Since the TAC assembles in a hierarchical manner, the depletion of upstream components (basal body proximal) will lead to mislocalization and depletion of downstream components (kDNA proximal proteins) [23]. We made use of the TAC hierarchy, and performed TAC-depletome experiments with isolated flagella on p197 and TAC42, identifying a new TAC protein, TAC53.
Ultrastructure Expansion Microscopy (U-ExM) with improved structural integrity and better preservation of ultrastructural details [37] were successfully established in the protozoan model systems like Trypanosoma brucei, Leishmania major, Toxoplasma gondii, Giardia lamblia and several Plasmodium species, providing insights into their unique cellular architecture [38–44]. In T. brucei, U-ExM not only allowed intermolecular information, but also allowed the localization of two distinct domains within the same protein [24].
In this study, we employed U-ExM to investigate the TAC, which has been traditionally observed from a lateral perspective. However, for the first time, we also analyzed it from the axial perspective, revealing that the TAC forms a tubular structure, indicated by the ring-like formation seen from the axial perspective. This finding provides new insights into the structural organization of the TAC and suggests a more complex architecture than previously thought.
Results
TAC depletomes reflect TAC biology
To identify new TAC components, we performed TAC depletomic assays as previously described. In brief, the kDNA remains attached to the basal body via the TAC during isolation of the flagellum [15,27,35]. We have also shown that the TAC assembles in a hierarchical fashion such that if you deplete a component of the TAC only the kDNA proximal part of the TAC will be lost while the basal body proximal part remains [23]. We thus can compare the composition of the TAC and its associated proteins from cells depleted of individual TAC components by quantitative mass spectrometry (MS). For this, stable isotope labelling by amino acids in cell culture (SILAC) in combination with quantitative MS has been performed using isolated flagella [23,35]. To avoid the detection of proteins like replication factors that are only connected to the TAC via the kDNA, a DNase digestion was performed before samples were analyzed by MS.
p197 depletomics
In the depletomic experiment targeting p197 (Tb927.10.15750, Tb11.v5.0394) we detected 1665 proteins, 43 of which were depleted >1.5 fold including all known TAC components (Fig 1A and S1 Table). Other proteins that were affected by the depletion of p197 include a basal body protein, several components of the oxidative phosphorylation chain as well as proteins of unknown function. From the latter we selected seven candidates for further analysis based on their predicted localization to the mitochondrion or the basal body according to TrypTag [45–47]. To test their potential function in the TAC we targeted the seven candidates by RNAi in the γL262P cell line (S1 Fig) [36]. We monitored growth as well as kDNA replication and segregation using DAPI staining. Depletion of only one candidate (Tb927.5.2970) showed loss and over-replication of the kDNA as well as a growth arrest. While the over-replication might hint towards a function in the TAC the rapid growth defect in the γL262P cell line indicates that the protein is involved in basal body biogenesis rather than mitochondrial genome inheritance. In summary none of the tested candidates fulfill the criteria of a TAC component.
(AB) Volcano plots showing the mean log10 ratios (induced/uninduced) of normalized protein abundance from four (A) or three (B) biological replicates plotted against the - log10 p-values (two-sided Student’s t-test). The dashed vertical lines refer to the 1.5-fold change threshold and the dashed horizontal lines mark the significance line (p < 0.05). Complete lists of all proteins that were depleted more than 1.5 times are shown in the S1 and S2 Tables. Known TAC components are depicted in pink; proteins without a TAC assignment that were depleted >1.5 with a p-value < 0.05 present in both datasets (A and B) are depicted in green, and the newly identified TAC53 is shown in orange. (C) Schematic representation of the Tripartite Attachment Complex TAC. TAC - and TAC- associated proteins are shown. Connecting lines indicate known interactions, dashed line possible interactions. BB, basal body; pBB, pro-basal body; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane; EZF, exclusion zone filaments; DM, differentiated membranes; ULF, unilateral filaments. Color coding as in (AB). (D) Table of proteins depleted by more than 1.5-fold in both datasets. The table includes fold change values from both experiments, gene annotation, molecular weight (kDa), isoelectric point (pI). Color coding as in (AB).
TAC42 depletomics
In the TAC42 (Tb927.7.3060) depletomic experiment we detected 2575 proteins, 52 of which were depleted >1.5 fold including five known TAC components (Fig 1B and S2 Table). Other proteins that were affected by the depletion of TAC42 include a basal body protein, several proteins involved in flagellar biogenesis as well as proteins of unknown function. From the latter we again selected seven candidates for further analysis based on their predicted localization to the mitochondrion according to TrypTag [45–47]. We tagged the proteins and defined their localization in situ and selected three candidates that localize to the kDNA for functional studies by RNAi (Tb927.10.8980, Tb927.1.2730, Tb927.8.3160) (S2 Fig). Targeting these candidates by RNAi leads to a growth defect, but the kDNA is not affected. Again, none of the tested candidates fulfill the criteria of a TAC component.
We also noticed that aside from the known TAC components four proteins were significantly depleted >1.5 fold in both data sets (Tb927.2.6100; Tb927.9.5020, HMG44; Tb927.11.16120, KAP68; Tb927.11.16400, KAP3). These four proteins have previously been studied in detail and have been proposed to be involved in functions other than the TAC. HMG44 and KAP68 are part of a distinct complex connecting the kDNA to the TAC [35]. KAP3 is a histone H1 like protein involved in kDNA organization and Tb927.2.6100 was shown to be involved in kDNA maintenance [48–50]. Interestingly, all four proteins were significantly depleted >2-fold in a RNAi cell line targeting TAC102, the protein previously thought to be the closest TAC component to the kDNA [35]. The above experiments suggest that all four proteins are positioned closer to the kDNA within the TAC hierarchy.
Since Tb927.2.6100 was repeatedly identified in our screens for new TAC components we decided to further characterize this protein [23,33,35].
Tb927.2.6100 (TAC53) is an essential protein of the TAC
The 53 kDa Tb927.2.6100 is a protein essential for normal growth with a pI of 10.77 and a predicted N-terminal mitochondrial targeting signal. It has putative homologs in most trypanosomatids including Leishmania and Paratrypanosoma (S3 Fig). Interestingly, minor groove DNA-binding motifs such as AT-hook and SPKK motifs are present in some homologs, suggesting a putative role in interaction with the kDNA [51,52]. We generated an inducible RNAi cell line targeting Tb927.2.6100. Consistent with previous findings, depletion of Tb927.2.6100 resulted in a slow growth phenotype three to four days post-induction [50] (Fig 2A). Additionally, cells depleted of Tb927.2.6100 show a kDNA loss phenotype with less than 20% of cells harboring a kinetoplast three days after RNAi induction in PCF (Fig 2A).
A) Growth curve of the procyclic TAC53 RNAi cell line. The orange line depicts the percentage of cells with kDNA, while the black line shows the growth curve of uninduced cells, and the grey line shows RNAi-induced cells (induced with tetracycline). B) Fluorescence DAPI stain of two representative cells after TAC53 RNAi [2 days induced]; The cell on the top shows a tiny kDNA, the bottom cell shows an over-replicated kDNA. C) Integrated kDNA intensities were measured in uninduced and RNAi-induced cells [2 days]. The red line marks the mean. D, E) Growth curves and loss of kDNA of TAC53 RNAi in the depicted bloodstream form cell lines. Color code of graph as in (A).
Using fluorescence microscopy, we also observed kDNA missegregation, which was not reported in the previous study but is typical for the loss of function phenotype of TAC proteins [50] (Fig 2B). To quantify the observations, we measured integrated kDNA intensities in cells where Tb927.2.6100 was depleted by RNAi for two days and compared it to uninduced cells (Fig 2C).
To further test if Tb927.2.6100 functions as a TAC protein, we repeated the RNAi experiments in wildtype NYsm bloodstream forms and in the γL262P bloodstream form cell lines [17,36]. In both cell lines depletion of Tb927.2.6100 leads to loss of kDNA in more than 90% of the cells. While this leads to a severe growth defect in the NYsm, the γL262P cell line growth is not affected (Fig 2D and 2E).
Based on these findings, we conclude that Tb927.2.6100 is an essential TAC protein and will be named TAC53.
TAC53 localizes at the kDNA in a cell-cycle dependent manner
To investigate the subcellular localization of TAC53, a polyclonal antibody was generated, and its specificity was validated by RNAi in conjunction with immunofluorescence microscopy and western blot analysis (S4 Fig). Subsequent immunofluorescence assays utilizing this antibody, in combination with DAPI staining, confirmed the subcellular localization of TAC53 at the kDNA (Fig 3A). Quantification of the integrated fluorescence signals, normalized to the kDNA, showed that TAC53 signals vary during the cell cycle. TAC53 is significantly enriched in cells undergoing kDNA division (1kdiv1N) and in cells with two kinetoplasts and one nucleus (2k1N), compared to cells with a single kinetoplast and nucleus (1k1N) or two kinetoplasts and two nuclei (2k2N) (Fig 3B). In contrast, TAC102 levels remained unchanged throughout the cell cycle (Fig 3C).
A) Immunofluorescence images of wildtype cells in different cell cycle stages stained with the TAC53 antibody. The upper lane shows DAPI signal, the lower lane shows signal for TAC53. B) Quantification of integrated signal intensities for TAC53 in different cell cycle stages, normalized to the kDNA. C) Quantification of integrated signal intensities for TAC102 in different cell cycle stages, normalized to the kDNA. D) U-ExM of TAC53 signal in 1k1N cells (left) and 2k1N cell (right) in magenta. kDNA is depicted in cyan. E) U-ExM co-staining of TAC102 (yellow) and TAC53 (magenta), with kDNA in cyan. Scale bars in D) and E) represent 1 µm (not adjusted for Expansion). F) Quantification of the punctate signals seen in U-ExM images for TAC53 per kDNA, n = 37 cells.
To further resolve the precise spatial distribution of TAC53, U-ExM was employed, a high-resolution imaging technique that enhances spatial resolution through physical expansion of the specimen [37]. In 1k1N cells TAC53 was detected as a punctate signal distributed throughout the entire kDNA disc, however, in 1kdiv1N cells, TAC53 signal appears on both kDNA lobes with a similar pattern as in 1k1N with little to no presence in center of the kinetoplast (Fig 3D). Additionally, co-staining with a monoclonal antibody against TAC102 revealed that TAC53 is positioned more proximally to the kDNA compared to TAC102 (Fig 3E). This observation, along with findings from depletomic assays, indicates that TAC53 represents the TAC component most closely associated with the kDNA. In the expanded cells, we additionally quantified the punctate signals from TAC53 staining as shown in Fig 3F. Our analysis, based on 37 measured kDNA lobes, indicated that the majority of kDNA lobes exhibited predominantly 11 foci, with a range of nine to 16 foci observed.
TAC53 incorporation into the nascent TAC depends on the kDNA
Since the TAC53 signal fluctuates throughout the cell cycle and is most abundant in segregating kDNAs, we sought to determine whether the kDNA is required for the proper localization of TAC53. To explore this, we utilized the γL262P cell line, which is particularly advantageous for studying the TAC because it permits a reversible disruption of TAC biogenesis. Upon tetracycline-inducible RNAi against a TAC component, the TAC structure is disrupted, leading to loss of kDNA. Once the kDNA has been eliminated, the TAC component targeted by RNAi is re-expressed by removal of tetracycline from the media, allowing TAC reassembly to occur in the absence of kDNA [35].
Since the TAC assembles hierarchically from the basal body to the kinetoplast, depletion of basal body-proximal TAC components leads to the loss of more distal components. Consequently, p197 RNAi depletes all TAC proteins, including TAC102 and TAC53. Immunofluorescence microscopy revealed that five days after p197 RNAi induction, only approximately 10% of the cells retained a detectable TAC102 signal, while no cells exhibited TAC53 signal (Fig 4A, middle lane). In contrast, in uninduced cells, both proteins were detected in over 95% of the population (Fig 4A, upper lane). Upon p197 re-expression, nearly 90% of cells successfully reassembled TAC102 into the TAC, consistent with previous findings [35], whereas only 20% of cells restored TAC53 incorporation at this time point (Fig 4A, lower lane).
A) Immunofluorescence analysis of TAC53 and TAC102 relative to the kDNA during TAC depletion and reassembly in the γL262P-p197 RNAi cell line. The top row shows uninduced cells, the middle row shows cells after five days of RNAi induction, and the bottom row shows cells following re-expression of p197. TAC102 is shown in yellow, TAC53 in magenta, and DNA in cyan. The scale bar is 1µm. B) Quantification of signal intensities shown in (A). C) Immunofluorescence analysis of TAC53 and TAC102 in extracted flagella, either untreated (top row) or treated with DNase-I (bottom row). Color coding as in (A). The scale bar is 1µm.
To assess whether TAC53, once assembled into the TAC, still requires the kDNA for its stable association with the structure, we conducted immunofluorescence microscopy on DNase I-treated isolated flagella. The results demonstrated, in agreement with the SILAC pulldown experiments (Fig 1), that TAC53 remains attached to the isolated flagella (Fig 4C), similar to previous findings for TAC102 [35]. This suggests that once TAC53 is incorporated into the TAC, its localization is no longer dependent on the presence of kDNA.
Immunoprecipitation and U-ExM reveal protein interactions and localization
TAC53 appears to be the kDNA most proximal component of the TAC structure and thus is in close proximity to the recently described HMG44 and KAP68 [35]. To test if HMG44 and KAP68 form a complex with TAC53 in situ, we used immunoprecipitation assays with cell lines expressing HA- and myc-tagged KAP68 and HMG44, respectively. Mitochondrial enriched fractions were sonicated, and the resulting soluble fractions were subjected to immunoprecipitations using anti-myc or anti-HA beads. In the KAP68 immunoprecipitation we identified 27 significantly enriched proteins (enrichment >2 fold). Among those, 16 have a mitochondrial annotation and seven are predicted to be localized to the kDNA including HMG44 (Fig 5A and S3 Table). In the HMG44 immunoprecipitation experiment we identified 11 significantly enriched proteins (enrichment >2 fold). Among these proteins, five have a predicted mitochondrial localization, including KAP68 and TAC53 (Fig 5B and S4 Table).
A) Volcano plots showing the log2 fold changes (KAP68/ wt) of protein abundance plotted against the -log10 p-values from four technical replicates. The dashed vertical lines refer to the 2-fold change threshold and the dashed horizontal lines mark the significance line (p < 0.05). B) Volcano plots showing the log2 fold changes (HMG44/ wt) of protein abundance plotted against the -log10 p-values from three technical replicates. The dashed vertical lines refer to the 2-fold change threshold and the dashed horizontal lines mark the significance line (p < 0.05). C) Volcano plots showing the log2 fold changes (TAC53-induced/ TAC53-uninduced) of protein abundance plotted against the -log10 p-values from three technical replicates. The dashed vertical lines refer to the 2-fold change threshold and the dashed horizontal lines mark the significance line (p < 0.05). D) Schematic representation of the interactions based on the immunoprecipitation experiments. E) U-ExM images of TAC53, HMG44, KAP68 and KAP3 shown from a lateral and an axial view. TAC53 and KAP68 are shown in yellow, HMG44 and KAP3 are shown in magenta, kDNA is shown in cyan. The scale bar is 1µm (not adjusted for Expansion).
To identify TAC53 interaction partners, we used a cell line overexpressing TAC53-HA upon tetracycline induction, in the background of a 3’ UTR RNAi targeting endogenous TAC53. This construct partially rescues the 3’ UTR RNAi phenotype, showing reduced and non-significant kDNA missegregation, and a delayed growth phenotype compared to the 3’ UTR RNAi condition (S5A-S5D Fig). While the TAC53-HA construct localizes similarly to the endogenous protein, it also induces a growth phenotype when overexpressed in the 29–13 host cell line (S5E and S5F Fig). The observed growth phenotype may result from the HA-tag at the C-terminus of the TAC53-HA construct, which potentially disrupts the protein’s normal function. Additionally, the overexpression of TAC53 (which normally is regulated dependent on the cell cycle), could lead to a disruption of its developmental control due to elevated protein levels.
In the immunoprecipitation a total of 35 proteins were significantly enriched including the bait TAC53 (Fig 5C and S5 Table). Although many of the enriched proteins are annotated with cytoplasmic or nuclear localization, two proteins, in addition to TAC53, are predicted to localize to the kinetoplast. These are KAP3 (Fig 1D) and the kDNA-associated protein Tb927.10.8980. However, the latter was identified by MS with a p-value > 0.05 (S2 Fig and S5 Table).
Additionally, we performed U-ExM for a better understanding of the relative orientation of the four proteins TAC53, HMG44, KAP68 and KAP3 at the interface of the TAC and the kDNA. From the lateral perspective, all observed proteins are closely associated with the kDNA. However, from the axial view, HMG44 and KAP68 exhibit a distribution pattern similar to TAC53, forming distinct punctate structures throughout the kDNA. In contrast, KAP3 seems to laterally encircle the kDNA. These findings indicate that the four proteins establish an interaction network, as evidenced by immunoprecipitation assays and U-ExM.
U-ExM of the TAC reveals a tube-like structure
To characterize the overall structure of the TAC we pursued U-ExM. Using double tagged cell lines in combination with available antibodies we imaged the overall architecture of the TAC from two main orientations relative to the kDNA disc, lateral and axial.
The TAC is organized in three regions: the EZF represented by p197, the DM represented by TAC65, pATOM36, TAC40, TAC42, TAC60 and p166 which transitions into the ULF that also contain TAC102 and TAC53 (Fig 1C).
The C-termini of p197 form a circular structure with a mean diameter of 0.89 ± 0.08 µm and are close to the proximal end of the pro- and mature basal bodies, which have a mean diameter of 0.85 ± 0.07 µm (expanded cells, Figs 6A, 6I, and S7). The N-termini of p197 also form a circular structure with a mean diameter of 1.07 ± 0.14 µm and are close to the OMM (Figs 6A and S7). Thus, both the mature basal body and the pro-basal body each have one distinct TAC structure per kDNA prior to replication of the disc (Fig 6A).
Rows A–I show different TAC protein combinations. Each row corresponds to one protein pair, with a lateral view (left) and an axial view (right). Within each column, color coding remains consistent across all panels. Proteins imaged are indicated in the respective panels. In the lateral view, the first column displays the first protein in yellow and DNA in cyan, the second column shows the second protein in magenta with the same DNA signal, and the third column presents the merged image. In the axial view, the first and second columns show only the respective proteins (yellow or magenta), while the third column shows the merge of both proteins. The scale bar is 1µm (not adjusted for Expansion).
In the DM, TAC65 follows the circular architecture of p197, with two distinct rings visible for each kDNA (Fig 6B and 6C). Similarly, other OMM components of the TAC including TAC40 and TAC60 show distinct circular structures (Fig 6C-6F). Also, the IMM and ULF protein p166 exhibits a circular architecture (Fig 6F-6G). The diameters of the TAC structure seem to enlarge and diffuse from the basal body towards p166 (Figs 6J and S7). While the TAC diameter marked by the C-terminus of p197 (0.89 ± 0.08 µm, expanded cells) is still very close to the basal body diameter (0.85 ± 0.07 µm, expanded cells), it is about 30% larger for the TAC components of the OMM (e.g., TAC40, 1.21 ± 0.17 µm, expanded cells, Fig 6J).
While p166 in the ULF still forms a circular structure, the signal for TAC102 which directly associates with p166 is more diffuse (Fig 6G-6H). The newly discovered TAC component TAC53 is visible as individual puncta with no specific arrangement (Fig 6H-6I).
We additionally imaged cells just prior to kDNA segregation when the kDNA is essentially fully replicated and used a combination of tagged p197 (both termini) and TAC53 (S6 Fig) to visualize the positions of these proteins. For the C-terminus of p197 we detected four distinct TAC structures with a clear separation. While the N-terminus of p197 only shows this degree of separation between the basal body pairs and less between pro- and mature basal bodies. TAC53 on the other hand keeps the punctate signal but is not present between the two segregating kDNA discs.
These findings support the presence of two distinct TAC structures prior to kDNA replication, forming a tube-like configuration.
Discussion
This study advances our understanding of the architecture and molecular composition of the tripartite attachment complex in T. brucei, offering significant insights into the mechanisms underlying mitochondrial genome segregation in this eukaryotic model organism. By employing a multidisciplinary approach integrating quantitative proteomics, RNA interference, and ultrastructure expansion microscopy, we delineated the structural organization of the TAC and identified TAC53 as a previously uncharacterized component of the complex.
Application of U-ExM revealed that, from an axial viewpoint, the TAC exhibits a tubular morphology reminiscent of a truncated cone, indicative of a more intricate three-dimensional organization than previously appreciated. Starting from the C-terminal region of p197, a protein localized within the exclusion zone filaments, the tubular structure conforms closely to the barrel-shaped architecture of the basal body. Progressing toward the N-terminal region of p197 and approaching the outer mitochondrial membrane, the TAC displays a gradual increase in diameter (Figs 6 and S7). This morphological gradient suggests an expansion along the basal body–kDNA axis, forming a structure resembling a truncated cone. At the outer mitochondrial membrane, proteins including TAC65, TAC40, and TAC60 are arranged in a circular manner that align with the N-terminus of p197 (Fig 6). This structural expansion is especially prominent in TAC40, which presents a diameter approximately 30% greater than that of the basal body. Such an enlargement may be necessary for the TAC to establish a robust physical interface with the more voluminous kDNA structure. While the inner mitochondrial membrane protein p166 maintains a circular geometry this structural definition diminishes in the unilateral filaments as evidenced by the diffuse U-ExM signal of TAC102 and the punctate pattern of TAC53, the newly identified TAC component.
Building on a previously proposed model of kDNA replication – in which each kDNA network comprises a diploid set of minicircles which is distributed across each lobe of the kDNA network – our findings provide compelling morphological evidence in support of this model [53]. Specifically, U-ExM analysis demonstrated that in non-dividing cells each kDNA network is associated with two TAC structures: one connected to the mature basal body and another to the pro-basal body (S6E Fig). This architectural configuration may constitute the structural basis for spatial segregation of the largely diploid mitochondrial genome within the kDNA disc. Furthermore, such spatial organization may also underlie the formation of the two replication foci known as the antipodal sites.
Given the TAC’s hierarchical organization – wherein proximal components near the basal body are prerequisite for the assembly of more distal elements – we employed a SILAC-based depletomic strategy to systematically identify novel TAC components. This approach, which enables unbiased detection of protein dependencies, successfully recapitulated the identification of several previously characterized TAC components (Fig 1A and 1B). Through extensive validation of novel candidate proteins derived from two independent screening datasets, we identified TAC53 as a bona fide and likely final structural component of the TAC. RNAi-mediated knockdown of TAC53 resulted in pronounced kDNA missegregation phenotypes, including loss of kDNA and kDNA over-replication in both the procyclic and bloodstream life cycle stages of T. brucei (Fig 2). Importantly, these defects did not compromise cell viability in the γL262P bloodstream form, a mutant capable of surviving without kDNA, thereby affirming TAC53’s specific role in kDNA maintenance (Fig 2E). TAC53 ticks all the boxes for the suggested operational definition of a TAC component [17]. This includes the localization between the basal body and the kDNA, the RNAi-mediated kDNA missegregation phenotypes, including loss of kDNA and kDNA over-replication in both the procyclic and bloodstream life cycle stages of T. brucei as well as the specificity for this process that is seen in all but one component of the TAC.
Except for TAC53 several additional putative TAC candidates were identified in the SILAC RNAi experiments. However, none of them show the expected localization or RNAi phenotypes that would be expected for typical TAC subunits. This suggests that, within the limit of the proteomic analysis, all TAC subunits that function exclusively in the TAC have been discovered.
TAC53 localizes in close proximity to the kDNA, as observed via U-ExM, and occupies a position distal to TAC102 within the TAC hierarchy. Furthermore, the localization of TAC53 is TAC102-dependent, while TAC102 itself localizes independently of TAC53. Moreover, the abundance of HMG44 and KAP68 decreases upon depletion of TAC53 (S8A and S8B Fig) [35]. Based on its position at the kDNA, TAC53, along with HMG44, KAP68, and KAP3, might mediate the interaction between the kDNA and the TAC. This model is further substantiated by the presence of canonical DNA-binding motifs within TAC53, including AT-hook and SPKK domains. Immunoprecipitation assays indicate that these proteins form an interacting network. However, it is important to note that immunoprecipitation experiments involving TAC subunits are challenging, as the fully assembled TAC complex is insoluble [32,54]. As a result, the immunoprecipitation assays likely capture only intermediate or subcomplex interactions, rather than the fully assembled TAC.
In replicating kDNA networks (1kdiv1N stage), TAC53 is conspicuously absent from the central region of the duplicated kDNA disc, an area predominantly composed of maxicircles that replicate immediately prior to segregation [55]. This observation suggests that the interaction between TAC53 – and by extension the TAC – and the kDNA may be preferentially mediated through minicircles rather than maxicircles (S6C and S6D Fig).
Although TAC53 shares functional parallels with other TAC proteins, it also displays distinct features. Notably, the relative abundance of TAC53 increases during mitochondrial genome replication, possibly reflecting a role in the reattachment of newly synthesized minicircles to the segregation apparatus. We propose a two-step model in which TAC53 and its interacting partners - HMG44, KAP68, and KAP3 - initially associate with replicated minicircles independently of upstream TAC components. In a second step, a subset of these TAC53-containing complexes that are bound to minicircles become integrated into the maturing TAC, while the remainder are targeted for degradation. This model is further supported by experimental evidence showing that early-stage TAC53 assembly is dependent on the presence of kDNA (Fig 4A and 4B), whereas this dependence diminishes significantly once TAC53 is integrated into the TAC (Fig 4C).
Collectively, we present a refined three-dimensional model of the tripartite attachment complex including its most distal component TAC53 and its interactors that mediate minicircle-specific tethering prior to integration into the mature TAC structure. The data strongly supports a model in which the mitochondrial genome is present in a diploid state and where each haploid genome is delineated by one TAC structure.
Materials and methods
Cell lines
All procyclic cell (PCF) lines are derivates of the T. brucei single marker inducible 427 cell line described in [24]. PCF cells were grown at 27°C in SDM-79 [56] and supplemented with 10% (v/v) fetal calf serum (FCS). Bloodstream form (BSF) cell lines are based on the New York single marker (NYsm) strain or on a derivative thereof named γL262P [36]. BSF were grown in HMI-9 [57] containing 10% (v/v) FCS at 37°C with 5% CO2. The tetracycline-inducible RNAi of p197 and TAC42 have already been described in [24] and [28], respectively. For tetracycline-inducible RNAi of the candidate genes, cells were stably transfected with a NotI-linearized pLew100-derivate plasmid containing stem-loop sequences covering parts of the ORF or the 3’UTR of the respective genes (see S6 Table for specifications). C-terminally in situ HA- or myc tagging of the candidate genes was done by stable transfection of PCR products using plasmids of the pMOtag series [58] as templates with primers defining the sites of homologous recombination leading to expression of HA- or myc tagged full-length proteins.
TAC depletomic assay using SILAC based MS analysis of isolated flagella
Cells capable of inducible RNAi against p197 or TAC42 were grown in SDM80 [59] supplemented with either light (12C6/14Nχ) or heavy (13C6/15Nχ) isotopes of arginine (1.1 mM) and lysine (0.4 mM) (Eurisotop) and 10% dialyzed FCS (BioConcept, Switzerland). To ensure complete labelling, cells were growing in this medium for 5 days in total. 3 days before harvesting, the cells growing in either light or heavy isotopes were induced with tetracycline for RNAi. Induced and uninduced cells were mixed in a 1:1 ratio, and 1x108 cells in total were subtracted to flagellar preparation as described in [60]. In brief, cells were harvested by centrifugation after EDTA (100nM) was added to the culture, directly resuspended in extraction buffer (10mM NaH2PO4, 150mM NaCl, 1mM MgCl2, pH 7.2) containing 0.5% Triton X-100 and incubated for 10 min on ice. After centrifugation pellets were incubated on ice for 45min with extraction buffer containing 1mM CaCl2. Extracted flagella could then be pelleted for 10min at 10’000g and were flash frozen in liquid nitrogen until used for liquid chromatography (LC)-MS analysis Experiments were performed in three (TAC42 RNAi) or four (p197 RNAi) biological replicates including a light/heavy label-switch. For LC-MS analysis, samples were thawed on ice and the extracted flagella were resuspended in urea buffer (8 M urea dissolved in 50 mM NH4HCO3). Cysteine residues were reduced by incubation with 5 mM Tris(2-carboxy-ethyl)phosphine/10 mM NH4HCO3 (30 min at room temperature) and free thiol groups were subsequently alkylated using 50 mM iodoacetamide/10 mM NH4HCO3 (30 min at room temperature in the dark). The reaction was quenched by addition of dithiothreitol at a final concentration of 33 mM. For tryptic in solution digestion, samples were diluted to approximately 1.6 M urea using 50 mM NH4HCO3 and proteins were digested overnight at 37°C. Peptide mixtures were desalted on StageTips [61], dried in vacuo, resuspended in 0.1% trifluoroacetic acid and analyzed on a Q Exactive (p197 SILAC RNAi samples) or an Orbitrap Elite (TAC42 SILAC RNAi samples) mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), each coupled to an UltiMate 3000 RSCLnano HPLC system (Thermo Fisher Scientific, Dreieich, Germany). Proteins were identified and quantified using MaxQuant/Andromda (version 1.5.5.1; [62]. MS/MS data were searched against all protein sequences for T. brucei TREU927 provided by the TriTrypDB database (https://tritrypdb.org; version 8.1) [47] using MaxQuant default settings except that only one unique peptide was required for protein identification. Lys8 and Arg10 were set as heavy labels and the options “requantify” and “match between runs” were enabled. Relative protein quantification was based on unique peptides and a minimum of one SILAC peptide pair. The mean log10 of normalized proteins abundance ratios (+/- Tet) was calculated, and for all proteins quantified in at least two replicates, p-values were determined using a two-sided Student’s t-test.
Immunofluorescence microscopy
For whole cell Immunofluorescence analysis cells were settled on a glass slide (Superfrost Microscope Slides, Epredia) for 10 minutes, fixed with 4% paraformaldehyde (PFA) for 10 minutes and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 5 minutes. Cells were washed twice with PBS in between and blocked with 4% bovine serum albumin (BSA) in PBS. Primary and secondary antibodies (see below for specifications) were diluted in PBS containing 2% BSA and were incubated for 45 minutes in between PBS washes. After the last wash with PBS, DAPI prolong (Thermofisher, P36931) was added and sealed with a cover glass.
For flagellar extraction, the cells were harvested by centrifugation after adding EDTA to a final concentration of 5 mM to the culture. The cells were pelleted and resuspended in extraction buffer (10 mM NaH₂PO₄, 150 mM NaCl, 1 mM MgCl₂, pH 7.2) containing 0.5% Triton X-100 and incubated on ice for 10 minutes. After centrifugation, the pellets were incubated on ice for 45 minutes in extraction buffer supplemented with 1 mM CaCl₂. The extracted flagella were pelleted by centrifugation at 3000 × g for 5 minutes at 4°C, resuspended in PBS, settled onto a coverslip for 10 minutes, and fixed with 4% PFA. After fixing, the described Immunofluorescence protocol was applied.
Immunofluorescence assay images for whole cells and extracted flagella were acquired on a LEICA DM5500B Widefield Microscope with a 100x (1.4 NA) objective and analyzed in ImageJ.
TAC53 sequence analysis
Protein sequences and accession numbers for TAC used in this study were retrieved from the TriTryp database [47]. Searches for TAC53 homologs were done using BLAST in the TriTryp database or using hmmsearch using custom hmm profiles (HMMER version 3.0) [47,63]. Multiple sequence alignment was performed with MAFFT (L-INS-i method, version 7) and visualized with the Clustalx coloring scheme in Jalview (version 2.11) [64,65]. AT-hook and SPKK motifs were identified by manual inspection [51,52]. Phylogenetic reconstruction was done on a curated alignment with PhyML with Smart Model Selection using the new generation phylogenetic service platform [66,67].
U-ExM
Coverslips (12 mm, CB00120RA120MNZ0, Epredia) were functionalized with poly-D-lysine (A3890401, Gibco) at room temperature for 30 minutes, followed by three washes with deionized water. Stained cells (prepared as per the immunofluorescence assay protocol) were spread onto the functionalized coverslips and allowed to settle at room temperature in the dark for 10 minutes.
Cells were anchored using 0.7% formaldehyde (FA, 36.5–38%, F8775, SIGMA) and 1% acrylamide (AA, 40%, A4058, SIGMA) in PBS at 37°C for 2 hours. Then, gelation was performed with a freshly prepared gelation solution consisted of 19% sodium acrylate (SA, 97–99%, 408220, SIGMA), 10% acrylamide (AA, 40%, A4058, SIGMA), and 0.1% N,N’-methylenebisacrylamide (BIS, 2%, M1533, SIGMA), supplemented with 0.5% ammonium persulfate (APS, 17874, Thermo Fisher) and 0.5% tetramethylethylenediamine (TEMED, 17919, Thermofisher). Gelation was carried out on ice. A 35 µl drop of gelation solution was placed on parafilm in a precooled humidity chamber, and cells were incubated on ice for 5 minutes, followed by final gelation at 37°C for 30 minutes.
Gels were detached from the coverslips by gentle shaking in 1 ml of denaturation buffer (200 mM SDS, 200 mM NaCl, and 50 mM Tris in deionized water, pH 9) at room temperature for 15 minutes. Gels were then incubated in the denaturation buffer at 95°C for 30 minutes. Afterward, gels were washed three times for 5 minutes in PBS, and DNA was stained with 5 µg/ml DAPI (D9542-5MG, SIGMA) in PBS (diluted 1:1000) with gentle agitation for one hour. Samples were then expanded in deionized water overnight.
Mounting and acquisition for ExM images
The gel was cut and mounted on poly-D-lysine functionalized 25 mm glass-bottom dishes (D35- 20-1.5-N, Cellvis, 35 mm glass bottom dish with 20 mm microwell #1.5 cover glass). Z-stacks were acquired using a Nikon Ti2 Kinetix Widefield Microscope (equipped with a Photometrics Kinetix 500 fps camera) on a 100x (NA = 1.45) objective. Following parameters were used: z step size was set to 0.2 µm and pixel size was 65 nm. Images were deconvolved using Huygens software HRM and analyzed with ImageJ. To quantify the images, z – projections were done using ImageJ with Maximum Intensity as Projection type. Measurements of TAC diameters were analyzed using GraphPad Prism version 9.5.0 (www.graphpad.com). Statistical analysis was performed using an Ordinary One-way ANOVA using the Bonferroni multiple comparisons test (ns = non-significant, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).
Immunoprecipitation
Our results indicate that sonication is the most effective method for solubilizing kDNA-proximal proteins, outperforming chemical fractionation methods like digitonin or RIPA buffer, as shown for KAP68 and HMG44 (S9 Fig). Mitochondria-enriched pellets (with 0.015% digitonin in SoTe (20 mM Tris HCl pH 7.5, 0.6 M sorbitol, 2 mM EDTA)) (5 x 107 cells each) were sonicated using a Bioruptor Plus sonication device by diagenode. Sonication was performed on level H for 10 times 30 seconds on/off at 4°C. Samples were then centrifuged at 4 ° C for 20 minutes and supernatants were applied to either anti-c myc (ETview Red Anti-myc Affinity Gel, Sigma-Aldrich, Merck, E6654) or anti-HA beads (EZview Red Anti-HA Affinity Gel, Sigma-Aldrich, Merck, E6779) in an overnight incubation at 4° C. Beads were then washed 3 times in bead wash buffer (2x lysis buffer (40mM Tris-HCL, ph 7.4, 0.2mM EDTA, 200mM NaCl, 50mM KCl, H2O), 25x cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, Merck, 04693159001) and H2O, centrifuged and sent for MS analysis.
Antibodies
For this study, the following primary and secondary antibodies were used (1:1000 for Immunofluorescence assays; 1:500 for Expansion Microscopy): Anti-HA.11 Epitope Tag Antibody, mouse (BioLegend, MMS-101R); Anti-c-Myc Monoclonal Antibody, mouse ((Thermofisher, 13–2500); Anti-c-Myc antibody, rabbit ((SigmaAldrich, C3956); HA tag antibody, rabbit (Thermofisher, 71–5500); Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermofisher, A-11001); Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermofisher, A-11005); Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermofisher, A-11008); Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermofisher, A-11012).
The TAC53 antibody was produced at Eurogentec targeting the following peptide sequence: 473–487 (H - CAT TPQ RIT NTG LLR L – OH).
Northern blots
Total RNA was extracted using acid guanidinium thiocyanate-phenol-chloroform according to [68] and separated on a 1% agarose gel. Northern blot probes for the corresponding RNAi targeting fragment were radioactively labelled using the Prime-a-Gene labelling system (Promega). Immunoblots- and Northern blots were done as previously described [69].
MS sample preparation for immunoprecipitation assays
The samples were processed via in-gel digestion as described previously [70], whereby denatured proteins were run on a 4–12% NuPage Bis-/Tris Gel (Thermo Scientific) at 180 volts for 8 min in 1x MES SDS Running Buffer (Thermo Scientific). Proteins in the gel were fixed with MS fixation buffer (7% acetic acid, 40% methanol) and stained with Coomassie Blue (10% acetic acid, 45% ethanol, 0.25% w/v brilliant blue (Roth)) for 5 minutes. The gels were destained overnight in water. The next day, each sample of the gel was cut into 1x1 mm squares, and the gel pieces were further destained with 50% ethanol and 25 mM f.c. ammonium bicarbonate digestion buffer (ABC pH 8.0) for several rounds and dehydrated with acetonitrile. Disulfide bridges were reduced with 10 mM f.c. DTT in 50 mM ABC at 56°C for 60 min and alkylated with 50 mM f.c. iodoacetamide in 50 mM ABC at 25°C for 45 min in the dark. The gel pieces were washed with 50 mM ABC and dehydrated three times with acetonitrile. The proteins in the gel pieces were subsequently digested overnight with 1 µg of MS-grade trypsin (Serva) in 50 mM ABC (pH 8.0) at 37°C. The next day, the digested proteins were extracted from the gel pieces with two rounds of extraction buffer (30% acetonitrile in 50 mM ABC buffer) and dehydrated with three rounds of acetonitrile for 15 min at 25°C. The supernatants were combined, and the samples were placed in a concentrator to evaporate the acetonitrile until the volume was < 200 µl. They were then desalted by StageTipping as described previously [61], where two layers of Empore C18 material (3M) were pushed into a 200 µl pipette tip, activated with 50 µl of methanol, equilibrated with 50 µl of Buffer B (80% acetonitrile, 0.1% formic acid), and washed with 50 µl of Buffer A (0.1% formic acid). The samples were added and washed with Buffer A. To run on the mass spectrometer, samples were eluted from the stage tip with 30 µl of Buffer B, placed in a concentrator to remove the acetonitrile until the remaining volume was 7 µl, and diluted with Buffer A for a final volume of 14 µl.
MS measurement and data analysis for immunoprecipitation assays
Five microliters of sample were injected on an Easy nLC-1000 (Thermo Scientific) coupled to a Q Exactive Plus (Thermo Scientific). On the Easy nLC-1000, tryptic peptides were separated through reversed-phase liquid chromatography on a 20 cm column with 75 µm inner diameter, (New Objective) self-packed with ReproSil-Pur 120 C18-AQ 1.9 (Dr. Maisch GmbH), mounted on an electrospray ion source. The samples were eluted from the column at a 225 nL*min-1 flow rate with a 105 min gradient ranging from 2–95% Buffer B (0.1% formic acid, 80% acetonitrile). Peptides were sprayed into the Q Exactive Plus set to positive ion mode operated with a TOP10 data-dependent acquisition scheme. The full scan was conducted with a resolution of 70,000, and the MS/MS scans had a resolution of 17,500. The raw MS data were processed with MaxQuant version 2.4.2.0 with the default settings. Proteins were quantified via the LFQ quantification algorithm (without FastLFQ) with 2 LFQ ratio counts unique + razor peptides. The false discovery rate was set to 0.01. Contaminants, protein groups identified only by site, reverse database hits, and proteins with fewer than two peptides removed prior to further analysis. Missing values were imputed.
Supporting information
S1 Fig. Analysis of Candidates from the p197-Depletome Assay.
(A) Table of candidate proteins depleted by more than 1.5-fold upon p197-RNAi induction. The table includes accession number, fold change, annotation, molecular weight (kDa), isoelectric point (pI), predicted domains, their localization according to TrypTag.org, and the tested method. (B) Growth curves (GC, upper panel) over 6 days for uninduced (-Tet, black) and RNAi-induced (+Tet, red) cells, along with DAPI stainings (lower panel) of the corresponding tested candidates. The tested gene and cell type are indicated above the GC. Time points of DAPI stainings are marked on the left of the images. GC inset: RNAi efficiency was tested using a northern blot (NB) probed against the respective RNAi target. Ethidium bromide-stained rRNAs serve as the loading control. Scale bar is 5 μm.
https://doi.org/10.1371/journal.ppat.1013521.s001
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S2 Fig. Analysis of Candidates from the TAC42-Depletome Assay.
(A) Table of candidate proteins depleted by more than 1.5-fold upon TAC42-RNAi induction. The table includes accession number, fold change, annotation, molecular weight (kDa), isoelectric point (pI), predicted domains, their localization according to TrypTag.org, and the tested method. (B) Immunofluorescence images showing the localization of the respective candidate genes. Cells were stained with DAPI (blue), anti-ATOM40 (green) as a mitochondrial marker, and anti-HA (red) for the in-situ HA-tagged candidate proteins. Localization patterns are indicated in the images showing the HA staining. Scale bar: 5 μm. (C) Growth curves (upper panel) over 6 days of uninduced (-Tet, black) and RNAi-induced (+Tet, red) cells, along with DAPI stainings (lower panel) of the corresponding tested candidates. The tested gene and cell type are indicated above the growth curve. Time points of DAPI stainings are marked on the left of the images. Growth curve left inset: RNAi efficiency was tested using a northern blot (NB) probed against the respective RNAi target. Ethidium bromide-stained rRNAs serve as the loading control. Growth curve middle and right inset: RNAi efficiency has been tested using a WB probed against the RNAi target and ATOM40 as loading control. Scale bar is 5 μm.
https://doi.org/10.1371/journal.ppat.1013521.s002
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S3 Fig. Structural and Evolutionary Analysis of TAC53.
(A) Multiple Sequence Alignment of TAC53 Homologs in Trypanosomatids. The alignment highlights the presence of AT-hooks and SPKK motifs in some TAC53 homologs. AT-hooks are shown in blue, while SPKK motifs are shown in red. (B) AlphaFold-predicted structure of TAC53. The N- and C-termini are indicated, and the location of the SPKK motif is highlighted. The model is colored according to the predicted local distance difference test (pLDDT) confidence scores, with the corresponding color scale. (C) S3 Phylogenetic reconstruction with branchpoint support of representative sequences of TAC53.
https://doi.org/10.1371/journal.ppat.1013521.s003
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S4 Fig. The TAC53 Antibody Specifically Binds TAC53 in Western Blot and Immunofluorescence Assays.
(A) Western blot showing TAC53 signal in RNAi-uninduced and induced cells [2d]. EF1α is used as a loading control. (B) Immunofluorescence assay showing TAC53 signal in RNAi-uninduced and induced cells [2d]. TAC53 antibody signal is shown in yellow, and DAPI staining is shown in cyan. Scale bar is 1 μm.
https://doi.org/10.1371/journal.ppat.1013521.s004
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S5 Fig. Analysis of TAC53 RNAi and Overexpression.
(A) Growth curve of procyclic form cells targeting the 3’ UTR of TAC53. Black line: uninduced cells; orange line: induced cells. (B) Integrated kDNA intensities measured in uninduced and induced [2d] cells from panel A. The red line marks the mean. (C) Growth curve of TAC53-HA expression in the background of the 3’ UTR RNAi (exclusive expression). Black line: uninduced cells; orange line: induced cells. Inset: Western blot showing HA signal after two days of induction, with EF1α as the loading control. (D) Integrated kDNA intensities measured in uninduced and induced [2d] cells from panel C. (E) Growth curve of TAC53-HA overexpression in 29–13 cells. Black line: uninduced cells; orange line: induced cells. (F) Immunofluorescence analysis of TAC53-HA overexpression. The HA tag is shown in yellow, the TAC53 antibody signal is shown in magenta, and DAPI is shown in cyan. BF = Brightfield. Scale bar is 1 μm.
https://doi.org/10.1371/journal.ppat.1013521.s005
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S6 Fig. Expansion Microscopy Images of Replicated kDNAs Displaying Four TAC Structures.
(A) Lateral perspective of replicated kDNAs showing the p197 C- and N-terminus. The C-terminus is shown in yellow, the N-terminus in magenta, and DAPI is shown in cyan. (B) Replicated kDNA shown from an axial perspective with p197 C-terminus in yellow and p197 N-terminus in magenta. DAPI is shown in cyan. (C) (D) Replicated kDNA shown from two different lateral perspectives with p197 C-terminus in yellow, TAC53 in magenta, and DAPI in cyan. Scale bar for A-D is 1 μm. (E) Illustration of the observed structure in expansion microscopy showing pro- and basal bodies (pBB and BB), outer- and inner mitochondrial membranes (OMM-IMM), the TAC, and the kDNA in 1k1N and 1kdiv1N cells. Color coding is found in the figure.
https://doi.org/10.1371/journal.ppat.1013521.s006
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S7 Fig. Ring diameter measurements of the basal body and TAC components in Expansion Microscopy.
Diameters of ring-like structures were measured from Expansion Microscopy images for the basal body and several TAC proteins, including the C- and N-terminus of p197, TAC65, TAC60, TAC40, and p166. The y-axis shows the measured ring diameters in micrometers (µm), and the x-axis indicates the specific proteins. Each data point represents an individual measurement. Red line marks the mean. Statistical analysis was performed using GraphPad Prism with ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test.
https://doi.org/10.1371/journal.ppat.1013521.s007
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S8 Fig. Protein dependencies.
(A) HMG44 and KAP68 are depleted after TAC53 RNAi [2d], while TAC102 remain unchanged. Western Blot analysis of TAC102, HMG44 and KAP68 signals following TAC53 depletion. VDAC serves as a loading control. (B) Immunofluorescence analysis of TAC53-depleted cells [2d], showing TAC102 signals in green, DAPI in cyan, and DIC. Scale bar is 5 μm.
https://doi.org/10.1371/journal.ppat.1013521.s008
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S9 Fig. Solubilization Assay Comparing Sonication and Chemical Fractionation for HMG44 and KAP68.
(A) Western blot analysis showing pellet and supernatant fractions for HMG44 and KAP68 after treatment with 1% digitonin or RIPA buffer. Enriched mitochondria were either treated with DNase-I or left untreated. (B) Western blot analysis showing total cells, pellet, and supernatant after sonication. ATOM40 serves as the loading control.
https://doi.org/10.1371/journal.ppat.1013521.s009
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S1 Table. List of proteins downregulated upon p197 depletion.
Proteins showing >1.5-fold downregulation are included, along with gene name, depletion factor, significance (p ≤ 0.05), TriTrypDB annotation, localization from TrypTag.org, and indication of detection in the TAC42 RNAi dataset. Proteins downregulated >1.5-fold in both datasets are color-coded as in Fig 1.
https://doi.org/10.1371/journal.ppat.1013521.s010
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S2 Table. List of proteins downregulated upon TAC42 depletion.
Proteins showing >1.5-fold downregulation are included, along with gene name, depletion factor, significance (p ≤ 0.05), TriTrypDB annotation, localization from TrypTag.org, and indication of detection in the p197 RNAi dataset. Proteins downregulated >1.5-fold in both datasets are color-coded as in Fig 1.
https://doi.org/10.1371/journal.ppat.1013521.s011
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S3 Table. KAP68 Immunoprecipitation List.
All proteins upregulated by more than 2-fold and with a p-value ≤ 0.05 in KAP68HA-tagged versus wild-type (wt) immunoprecipitation are listed. The table includes accession number, log2 fold change, gene description, molecular weight (kDa), isoelectric point (pI), and localization according to TrypTag.org.
https://doi.org/10.1371/journal.ppat.1013521.s012
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S4 Table. HMG44 Immunoprecipitation List.
All proteins upregulated by more than 2-fold and with a p-value ≤ 0.05 in HMG44myc-tagged versus wild-type (wt) immunoprecipitation are listed. The table includes accession number, log2 fold change, gene description, molecular weight (kDa), isoelectric point (pI), and localization according to TrypTag.org.
https://doi.org/10.1371/journal.ppat.1013521.s013
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S5 Table. TAC53 Immunoprecipitation List.
All proteins upregulated by more than 2-fold with a p-value ≤ 0.05 in TAC53-HA induced versus uninduced conditions are listed. The table includes accession number, log2 fold change, gene description, molecular weight (kDa), isoelectric point (pI), and localization according to TrypTag.org.
https://doi.org/10.1371/journal.ppat.1013521.s014
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S6 Table. List of RNAi Targets.
The table includes accession number, gene description, RNAi target, position of target, and length of RNAi target.
https://doi.org/10.1371/journal.ppat.1013521.s015
(TIF)
Acknowledgments
We want to thank the Microscopy Imaging Center (MIC) of the University of Bern and the team of the Proteomics and Mass Spectrometry Core Facility (PMSCF) at the Department for BioMedical Research (DBMR) for their valuable service. We additionally want to thank Bianca Berger and Markus Gerber for fruitful discussions.
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