Alexandra Chloé

Corinne Morel

Corinne Asencio

Bénédicte Blancard

Etienne Salin

Stéphane Gontier

Manuel Duvezin-Caubet

Frédéric Rojo

Emmanuel Bringaud

Morel

Corinne Blancard

Bénédicte Salin

Etienne Gontier

Stéphane Duvezin-Caubet

Manuel Rojo

Frédéric Bringaud

Emmanuel Tetaud
email: emmanuel.tetaud@u-bordeaux.fr

Identification of a novel and ancestral machinery involved in mitochondrial membrane branching in Trypanosoma brucei

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INTRODUCTION

Trypanosoma brucei is a parasite responsible for African sleeping sickness as well as the related cattle disease Nagana, affecting sub-Saharan Africa. T. brucei is transmitted to mammals, its final host, by an insect vector: the tsetse fly. During the trypanosome life cycle, the parasite exists in at least two replicative forms; the procyclic form (PCF), transmitted by the tsetse fly Glossina spp., and the bloodstream form (BSF) responsible for diseases in vertebrates. The pathogenic trypanosomes have a single mitochondrion, unlike most other eukaryotes that contain numerous ones (1).

Trypanosome's mitochondrion is highly elongated and reticulated and its genome (mitochondrial DNA) is restricted to a specific structure at the basis of the flagellum known as kinetoplast (kDNA). During the trypanosome life cycle and its adaptation to different hosts and environments, the shape of its mitochondrial compartment undergoes spectacular changes that reflect its functional plasticity. Indeed, the mitochondrion of this parasite exists in at least two major forms: (i) the fully active and developed one characteristic for the PCF that harbors the oxidative phosphorylation complexes (OXPHOS) for energy production [START_REF] Priest | Developmental regulation of Trypanosoma brucei cytochrome c reductase during bloodstream to procyclic differentiation[END_REF][START_REF] Van Hellemond | The extraordinary mitochondrion and unusual citric acid cycle in Trypanosoma brucei[END_REF] and (ii) the functionally less active and morphologically reduced form found in the bloodstream, with energy produced exclusively through glycolysis since OXPHOS is repressed [START_REF] Michels | Metabolic functions of glycosomes in trypanosomatids[END_REF]. These modifications correlate with the adaptation of the parasite to the glucose-rich blood of a mammalian host and the proline-rich hemolymph and tissue fluids of the blood-feeding tsetse fly [START_REF] Matthews | The developmental cell biology of Trypanosoma brucei[END_REF].

Studies in mammals and in yeast having unraveled tight links between mitochondrial bioenergetics, morphology and dynamics [START_REF] Duvezin-Caubet | Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology[END_REF][START_REF] Sauvanet | Energetic requirements and bioenergetic modulation of mitochondrial morphology and dynamics[END_REF][START_REF] Ramos | Bioenergetic roles of mitochondrial fusion[END_REF], it is tempting to assume that such close relationships not only regulate mitochondrial function and energy metabolism, but also the life cycle and pathogenic potential of trypanosomes. However, in contrast to fungi and metazoa, where mitochondrial morphology and fusion/fission dynamics have been thoroughly characterized [START_REF] Chan | Fusion and fission: interlinked processes critical for mitochondrial health[END_REF][START_REF] Westermann | Mitochondrial fusion and fission in cell life and death[END_REF], little is known about the morphology and dynamics of mitochondria in trypanosomes.

In most eukaryotes, mitochondria are very dynamic, alternating between two major events; fusion and fission. Mitochondrial dynamics allows to maintain mitochondrial morphology, distribution and size [START_REF] Sauvanet | Energetic requirements and bioenergetic modulation of mitochondrial morphology and dynamics[END_REF]. The main proteins involved in these events are large GTPase belonging to the dynamin family. The main GTPase responsible for mitochondrial fission in yeast and mammalian cells is Dnm1/Drp1, a soluble protein which is recruited to mitochondrial and peroxisomal membranes to exert its function [START_REF] Sesaki | In vivo functions of Drp1: lessons learned from yeast genetics and mouse knockouts[END_REF][START_REF] Otera | New insights into the function and regulation of mitochondrial fission[END_REF]. Mammalian mitochondrial fusion requires fusion of outer mitochondrial membrane (OMM) followed by fusion of inner mitochondrial membrane (IMM) which is carried out by mitofusins 1 and 2 (Mfn1 and Mfn2) and optic atrophy 1 (Opa1), respectively [START_REF] Malka | Separate fusion of outer and inner mitochondrial membranes[END_REF][START_REF] Song | Mitofusins and OPA1 mediate sequential steps in mitochondrial membrane fusion[END_REF].

Alteration in one of these processes and of the equilibrium between them provokes mitochondrial fragmentation or hyperfusion [START_REF] Sesaki | In vivo functions of Drp1: lessons learned from yeast genetics and mouse knockouts[END_REF][START_REF] Legros | Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins[END_REF] and is linked to diseases, notably neuropathies [START_REF] Bertholet | Mitochondrial fusion/fission dynamics in neurodegeneration and neuronal plasticity[END_REF].

Mitochondrial fusion and fission in trypanosomes have been poorly investigated to date. Unlike mammalian and yeast cells, fusion has not been directly observed [START_REF] Jakob | Mitochondrial growth during the cell cycle of Trypanosoma brucei bloodstream forms[END_REF][START_REF] Hughes | Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy[END_REF], but indirect evidence for mitochondrial fusion came 25 years ago from in vivo genetic exchange of mtDNA [START_REF] Gibson | Kinetoplast DNA minicircles are inherited from both parents in genetic crosses of Trypanosoma brucei[END_REF]. Another indication of fusion stems from the observation that the mitochondrial fragmentation provoked by the overexpression of mammalian Bax in trypanosomes is reversible [START_REF] Esseiva | Temporal dissection of Bax-induced events leading to fission of the single mitochondrion in Trypanosoma brucei[END_REF] and from the observation that, in BSF, the mitochondrial network mostly grows from two areas where loops and branches eventually merge to build a complex network [START_REF] Jakob | Mitochondrial growth during the cell cycle of Trypanosoma brucei bloodstream forms[END_REF]. Available evidence indicates that mitochondrial fission is not an endogenous/spontaneous process [START_REF] Jakob | Mitochondrial growth during the cell cycle of Trypanosoma brucei bloodstream forms[END_REF][START_REF] Hughes | Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy[END_REF] but it has to be artificially induced to be observed [START_REF] Esseiva | Temporal dissection of Bax-induced events leading to fission of the single mitochondrion in Trypanosoma brucei[END_REF][START_REF] Chanez | Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest[END_REF] and appears restricted to parasite division [START_REF] Hughes | Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy[END_REF][START_REF] Chanez | Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest[END_REF]. More recently, expression, in the mitochondrial matrix of trypanosome, of a putative dynamin from giant viruses has shown remodeling of the mitochondrion into a simple tubular structure, supporting that mitochondrial fission occurs in this parasite [START_REF] Sheikh | A Novel Group of Dynamin-Related Proteins Shared by Eukaryotes and Giant Viruses Is Able to Remodel Mitochondria From Within the Matrix[END_REF]. The absence of spontaneous, ongoing or continuous fission could explain the unique tubular and reticulated structure of the parasite mitochondrion, resembling that observed in yeast or mammalian mutants devoid of fission [START_REF] Sesaki | Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape[END_REF][START_REF] Bleazard | The dynaminrelated GTPase Dnm1 regulates mitochondrial fission in yeast[END_REF]. Finally, a recent study of the mitochondrial structure in another trypanosomatid (Crithidia fasciculata), revealed slow and discrete fission and fusion events during the cell cycle, suggesting that fusion/fission machineries must exist in trypanosomatids [START_REF] Dimaio | The single mitochondrion of the kinetoplastid parasite Crithidia fasciculata is a dynamic network[END_REF].

Two decades of research have revealed that numerous mitochondrial fusion and fission factors appear conserved in fungi and mammals with non-neglectable differences [START_REF] Sauvanet | Energetic requirements and bioenergetic modulation of mitochondrial morphology and dynamics[END_REF]. In plants, mitochondria fuse and divide, but factors directly involved in fusion have not been clearly identified until now, suggesting the existence of a different fusion machineries [START_REF] Logan | Plant mitochondrial dynamics[END_REF][START_REF] Arimura | Fission and Fusion of Plant Mitochondria, and Genome Maintenance[END_REF]. So far, only one dynamin-related protein (DRP), TbDlp, has been described and studied in T. brucei: its ablation impairs mitochondrial division and endocytosis and leads to cytokinesis arrest [START_REF] Chanez | Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest[END_REF][START_REF] Morgan | The single dynamin-like protein of Trypanosoma brucei regulates mitochondrial division and is not required for endocytosis[END_REF]. TbDlp thus appears to be the functional homolog of Drp1/Dnm1 in mammals and yeast, but its fission activity seems restricted to the cytokinesis [START_REF] Chanez | Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest[END_REF]. Another putative DRP displaying some similarity to mitofusins (TbMFNL, Tb927.7.2410) has been reported to influence the mitochondrial shape [START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF]. Its down-regulation led to a highly fenestrated mitochondrial network, suggesting that, despite its name, it plays a role in mitochondrial fission [START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF]. To date, no protein has been involved in mitochondrial fusion in trypanosomes.

In this study, we characterized the trypanosome DRP named TbMFNL [START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF]. We show that this DRP shows a strong similarity with a family of eukaryotic, prokaryotic and archaeal proteins that is absent from all organisms expressing Mfn or Opa1 homologues (fungi and mammals) and from plants, suggesting that it belongs to an ancestral membrane remodeling machinery. We show that it localizes to mitochondrial membranes and is able to modulate mitochondrial branching and shape. Its deletion does not alter mitochondrial morphology or distribution but its overexpression increases the size and branching of its mitochondrial compartment in a GTP-dependent manner. Taken together, our findings reveal that mitochondrial branching in trypanosomes is modulated by a member of a novel and ancestral membrane remodeling machinery that we have named TbDBF for Trypanosoma brucei Dynamin-related Branching Factor.

-Identification and sequence analysis of TbDBF

To identify potential fusion players, we started our search by careful re-examination of trypanosome genomic and proteomic databases for proteins showing sequence homology to known fusion factors (Opa1/Mfn1/2). We were only able to identify the previously reported dynamin-related protein TbMFNL (Tb927.7.2410) [START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF], that displays some similarities with both mammalian fusion factors of the inner (Opa1) or outer membrane (Mfn2) with 21 and 23 % of sequence identity, respectively (Figure 1, S1 and Table S1). Analysis of its sequence identified several motifs and domains found in other mitochondrial DRPs. Indeed, its N-terminal sequence displays a potential mitochondrial targeting signal (MTS) with potential cleavage sites (Figure S1), followed by a GTPase domain with conserved G1 to G4 motifs and, in its C-terminal region, two consecutive transmembrane domains (TM) (Figure 1 and S1). Interestingly, the MTS points to inner membrane targeting (such as the inner membrane fusion factor Opa1 localizing to the intermembrane space). In contrast to Opa1, that carries an N-proximal TM domain mediating inner membrane anchoring, no transmembrane domain was identified following the MTS, suggesting that it is not an Opa1 counterpart. The overall structure and domain organization of the protein displays more similarity to human Mfn (Figure 1) with two C-proximal transmembrane domains (TMs) that are reminiscent of the targeting to the outer mitochondrial membrane of Mfn. However, the presence of a N-terminal MTS makes it unlikely to be located in the outer membrane like Mfn in mammals. Altogether, these data suggest that the protein named TbMFNL (29) is not a homolog of fusion factors Opa1 or Mfn.

We therefore investigated whether this DRP could be conserved in other organisms.

As expected, it is well conserved throughout the kinetoplastida order (Figure S2 and Table S1 for some examples) but, surprisingly, we identify homologous proteins, with high similarity over the whole sequence, in many eukaryote lineages (mainly in the SAR clade Stramenopiles and Alveolata) (Figure 2A, S3 and Table S1) but never in Opisthokonta (clade including fungi and mammals) or in plants. Unexpectedly, we also identified many similar sequences in bacteria, mainly in the PVC group and in proteobacteria (Figure 2A). Finally, a sequence was also identified in Archaea, revealing that, unlike Opa1 and Mfn proteins, the proteins of this family are conserved in all the three domains of life (Figure 2A, S3 and Table S1). Remarkably, we could not identify sequences corresponding to Opa1 and Mfn in any of these organisms, the only sequences identified being restricted, as anticipated, to the well-conserved GTPase domain.

Phylogenetic analysis was carried out using the amino acid sequences of the proteins and a comprehensive tree based on sequence alignment is shown in Figure 2B. Structural analysis of several of these identified proteins, show again a very conserved structural organization on the whole sequence (Figure 2C andS3). Indeed, apart from the obvious absence of the MTS in prokaryotes, the general structure and domain organization are extremely well preserved (Figure 2C). Altogether, our analysis shows that these proteins form a phylogenetic group that is clearly separated from the wellknown fusion and fission dynamins of fungi and mammals and of NpBDLP, its closest bacterial homologue (Figure 2B). As these DRPs are conserved throughout the living world, but not in fungi, mammals, and plants, it is tempting to speculate that they represent a component of a novel and ancestral machinery involved in membrane dynamics. Their belonging to a protein family different from that of mitofusins and the functional characterization described below led us to rename this DRP TbDBF (for Trypanosoma brucei Dynamin-related Branching Factor).

-TbDBF is localized in the mitochondrion

Interrogation of the TrypTag database (cellular localization of trypanosome proteins [START_REF] Dean | TrypTag.org: A Trypanosome Genome-wide Protein Localisation Resource[END_REF]) with the TbDBF sequence (Tb927.07.2410) reveals mitochondrial localization for a C-terminal GFP-tagged version and a cytoplasmic, flagellar and nuclear lumen localization for an N-terminal GFP tagged construct. One potential explanation for this apparent discrepancy is that adding an N-terminal GFP sequence in front of a mitochondrial targeting sequence (MTS) is expected to perturb its addressing and therefore does not allow to conclude on its localization. To confirm the subcellular localization of TbDBF in T. brucei, we added a C-terminal 10xHA tag (to avoid disruption of mitochondrial targeting) to endogenous TbDBF (TbDBF::10HA) in both PCF and BSF using the pPOTV7 vector series (31) (Figure 3A) and verified that the tag has no impact on cell growth (Figure S4). It should be noted that the slight variation in growth observed between the wild-type and the tagged strain could be due to the difference between a non-clonal (wild-type) and clonal (TbDBF::10HA) population. Expression of TbDBF::10HA was confirmed by western-blot with anti-HA antibody in PCF and BSF (Figure 3B). The protein is about 2-fold more expressed in PCF than BSF which is in agreement with the data obtained by SILAC analysis for this protein [START_REF] Urbaniak | Comparative SILAC proteomic analysis of Trypanosoma brucei bloodstream and procyclic lifecycle stages[END_REF]. The localization of the TbDBF protein was studied in PCF by immunofluorescence using threonine dehydrogenase (Tdh, [START_REF] Millerioux | The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control[END_REF]) as a mitochondrial matrix marker (Figure 3C). TbDBF::10HA and Tdh clearly colocalized in the mitochondrial compartment, but TbDBF depicted a less homogeneous mitochondrial distribution (Figure 3C, Anti-HA/Merge). We also addressed, by immunofluorescence, TbDBF subcellular localization in BSF. For technical reasons (Tdh antibody do not react in bloodstream and the lack of primary antibody of non-murine origin), we could not perform a colocalization of the TbDBF::10HA protein with a mitochondrial protein. However, we performed a parallel labeling of the mitochondria, with an antibody directed against the mitochondrial Hsp60 or against the HA-tag. Under these conditions, we observe a tubular structure characteristic of the BSF mitochondrion both for TbDBF and Hsp60 (Figure 3D). The overall modular organization of TbDBF previously described and its subcellular localization confirm that TbDBF is a mitochondrial protein.

-Inactivation and silencing of TbDBF does not alter mitochondrial shape

Live microscopy of yeasts and cultured mammalian cells has revealed that mitochondrial morphology is continuously remodeled by antagonizing fission and fusion reactions [START_REF] Legros | Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins[END_REF][START_REF] Nunnari | Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA[END_REF]. Under normal conditions, the fusion/fission balance is established in favor of fusion and the mitochondria appear filamentous. Inhibition of fusion leads to mitochondrial fragmentation by ongoing fission [START_REF] Legros | Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins[END_REF] and reversely enhancing fusion leads to hyperfused mitochondria with increased branching and interconnection [START_REF] Sesaki | In vivo functions of Drp1: lessons learned from yeast genetics and mouse knockouts[END_REF]. In contrast, trypanosomes show a single permanently branched and interconnected mitochondrion, with very few mitochondrial fission events reported, which appear to be restricted to cell division and especially cytokinesis [START_REF] Hughes | Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy[END_REF][START_REF] Chanez | Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest[END_REF].

To investigate the role of TbDBF, we first inactivated the gene encoding TbDBF by CRISPR/Cas9 [START_REF] Soares Medeiros | Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins[END_REF] with an optimized protocol (see Materials and Methods). Both TbDBF alleles were inactivated by integration of a puromycin resistance marker cassette into both PCF and BSF cells (Figure 4A). Cloned parasites were analyzed by PCR to select cell lines carrying PAC insertions in both TbDBF alleles (TbDBF -/-) (Figure 4B). No significant impact on cell growth was observed in these PCF and BSF null-mutants over a period of 7 days compared to wild-type cells. We infer that the slight variation in growth observed between the wild-type and the inactivated strain reflects the difference between a non-clonal (wild-type) and clonal (TbDBF -/-) population (Figure S4). To investigate potential impact of TbDBF -/-on mitochondrial shape, we labeled PCF mitochondria with rhodamine-123 staining and analyzed mitochondrial shape in living cells. Since BSF were too mobile to obtain focused images with rhodamine 123, we stained mitochondria in fixed cells by immunofluorescence with mitochondrial Hsp60 antibodies. In both PCF and BSF, no significant difference in the structure of the TbDBF -/- mitochondrion was observed (Figure 4C and4E). To confirm that this strategy leads to TbBDF inactivation, we repeated this process in a strain endotagged with a C-terminal 3xTy1 tag in order to follow the expression of the protein by western-blot (Figure S5/A-D). Two separate inactivations were performed, one by inserting the puromycin resistance gene as before (Figure S5E-G) and the other one by deleting part of the gene (Figure S5H-J). In both cases, the protein was not detected in the inactivated clones and no modification of the mitochondrial structure was observed (Figure S5G/J).

In order to detect and quantify discrete modifications of the mitochondrial shape that may escape detection upon qualitative visual analysis, we developed an ImageJ macro (Supplemental MMS1) for the automatic analysis of the obtained images, which allows quantification of mitochondrial junctions (Figure 4D). This allowed us to establish that the number of mitochondrial junctions is equivalent in TbDBF -/-and wild-type PCF cells (8.5±4.9 vs 9.4±4.3) confirming the qualitative analysis of the microscopy images (Figure 4C). These findings regarding the consequences of TbDBF inactivation in PCF and BSF are not in line with a previous report describing a highly fenestrated mitochondrial network consecutive to a partial but significant inhibition (70%) of TbDBF (TbMFNL) expression by RNAi in BSF [START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF]. To exclude that the divergence arose from the different inactivation and silencing strategies, we conditionally down-regulated expression of TbDBF using a stem-loop RNAi construct. This approach led to a significant reduction (~70%) of the expression of endogenously tagged TbDBF::10HA in PCF (as observed in BSF ( 29)), but this was not paralleled by a modification of mitochondrial shape (Figure S6A-C). Since mitochondrial fenestration upon TbMFNL/TbDBF silencing was observed in BSF [START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF] we decided to repeat the experiment using the same strain (T. brucei 427 BSF), the same RNAi system (p2T7-177) and the same sequence used for down-regulation [START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF]. As previously reported [START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF], we observed a significant reduction of TbDBF expression (~80% after 4 days) of the endogenously tagged TbDBF::3HA in BSF (Figure S6D-F) that was not accompanied by mitochondrial shape modifications (Figure S6G). In our hands, neither inactivation nor silencing of TbMFNL/TbDBF alters mitochondrial morphology or induces a fenestration phenotype. At present, we ignore the reasons for the discrepancy with the results reported by Vanwalleghem et al. [START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF].

-TbDBF overexpression increases mitochondrial branching in a GTPdependant manner

We also investigated the possible role of TbDBF by conditionally expressing a Cterminally-tagged (3xTy1) TbDBF ( oe TbDBF::3Ty1) in PCF (Figure 5A). Expression was confirmed by western-blot analysis after 3 to 5 days of tetracycline induction (Figure 5B). Rhodamine-123 staining showed that wild-type and non-induced oe TbDBF::3Ty1 cells harbor the expected reticulated mitochondrial structure, while the mitochondrion appears significantly more reticulated and branched in induced oe TbDBF::3Ty1 cells (Figure 5C and Figure S7). For a more precise and quantitative description of the branching phenotype, we used our ImageJ macro allowing quantification of mitochondrial junctions. After 5 days of induction, the average number of mitochondrial junctions appeared more than 2times higher in induced oe TbDBF::3Ty1 cells compared to non-induced or wild type cells (Figure 5G). In order to estimate the level of TbDBF overexpression achieved in T.

brucei PCF, we repeated this experiment with cells expressing endogenously 3Ty1-tagged TbDBF (TbDBF::3Ty1, clone 2A11; Figure S5A-D). Western-blot analysis revealed that upon induction, exogenous TbDBF::3Ty1 protein is well overexpressed relative to its endogenous expression and induces alterations of mitochondrial shape (Figure S8A/B).

It is also noteworthy that the expression of TbDBF without the C-terminal 3Ty1 tag induces the same phenotype as the tagged TbDBF, confirming that the phenotype is not due to the use of a C-terminal tag (Figure S8A/B). These data clearly demonstrate that overexpression of TbDBF stimulates intermitochondrial connections.

All Dynamin-like proteins possess a GTP-binding domain that binds and hydrolyzes GTP and mutations in the highly conserved domains interacting with GTP provoke dynamin-dysfunction by lowering GTP-binding and hydrolysis efficiency. To determine whether TbDBF requires a functional GTPase domain to function, the key lysine 141 in the highly conserved G1 motif was converted to alanine, a mutation (K141A) significantly reducing GTP-binding of dynamins in other organisms [START_REF] Hales | Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase[END_REF][START_REF] Hermann | Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p[END_REF][START_REF] Santel | Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells[END_REF]. The K141A mutation was introduced into the sequence of TbDBF tagged with a C-terminal 3Ty1 ( oe TbDBFK141A::3Ty1, Figure 5D). Expression was confirmed by western-blot analysis after 3 to 5 days of tetracycline induction (Figure 5E) and overexpression of TbDBFK141A::3Ty1 was also confirmed as the wild-type TbDBF::3Ty1 (Figure S8A). The mutated protein localized to the mitochondria (Figure 6D), as expected, but did not alter mitochondrial shape (Figure 5F, Figure S7) nor the number of mitochondrial junctions (Figure 5G). This clearly demonstrates that TbDBF is a dynamin related protein that relies on a functional GTPase domain to modulate mitochondrial branching in trypanosomes.

-Submitochondrial localization of TbDBF

The localization of overexpressed TbDBF was also analyzed by immunofluorescence.

The overexpressed TbDBF::3Ty1 co-localized with Tdh in mitochondria (Figure 6A-d5.i), as previously described for the endogenous protein (Figure 3C). Analysis of mitochondrial shape by immunofluorescence confirmed that overexpression of TbDBF is linked to the appearance of hyper-reticulated mitochondrion (Figure 6A-d5 .i), as previously observed in living, rhodamine-123 labeled cells (Figure 5C). For more precise TbDBF localization, we performed Ultrastructure Expansion Microscopy (U-ExM), which increases the size of a sample while preserving its ultrastructure [START_REF] Gambarotto | Ultrastructure expansion microscopy (U-ExM)[END_REF][START_REF] Amodeo | Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei[END_REF]. By this approach, we show that both oe TbDBF::3Ty1 (Figure 6B) and the endogenous TbDBF::10HA (Figure 6C) have a different distribution from the matrix marker (Tdh) and localizes to the mitochondrial periphery. In addition, as already noticed with standard immunofluorescence microscopy (Figure 3C), TbDBF depicts a non-continuous, punctate distribution within the membrane as compared to the matrix protein Tdh. These findings suggest an association of the TbDBF protein with mitochondrial membranes. We then investigated the effect of TbDBF expression in the BSF form, where the mitochondrial compartment is smaller and composed of shorter and less interconnected mitochondria.

Overexpression of oe TbDBF::3Ty1 was monitored by western-blot (Figure 6E), but unlike PCF, the mitochondrial structure (labeled here with mitochondrial Hsp60) was not at all altered, remaining tubular and slightly reticulated like in wild-type cells (Figure 6F).

Induction over a longer period (up to 21 days) did not show mitochondrial alterations.

The localization of the wild-type TbDBF and mutant TbDBF (K141A) was also investigated by electron microscopy (EM) and immunogold-labeling using an anti-Ty1 antibody. After induction and immunogold-labeling, both proteins are clearly localized in the mitochondrion (Figure S9A3/B3) as compared to controls with non-induced cells (Figure S9A1/B1), which is also confirmed by gold-particles quantification (Figure S9D).

-Mitochondrial targeting and membrane anchoring of TbDBF 13

To investigate the mechanisms ensuring mitochondrial targeting of TbDBF, we first addressed the two domains that may determine TbDBF localization: (i) the N-terminal MTS that could mediate inner membrane targeting and intermembrane space localization (as the MTS of Opa1), and (ii) the C-proximal/terminal TMs that could mediate outer membrane anchoring (as the TMs of Mfn). To investigate their capacity to target proteins to mitochondria, we fused the MTS domain, or the two transmembrane domains of TbDBF, to GFP (Figure 7A). Fusion of the MTS at the N-terminus of GFP (MTS::GFP) fully addressed GFP to the mitochondria (Figure 7A). Interestingly, the size of MTS::GFP revealed by Western-blot is identical to that of cytosolic GFP without MTS (Figure 7B), indicating that the predicted MTS maturated by Matrix Processing Peptidase (MPP) (Figure S1) was cleaved upon import of MTS::GFP across the inner membrane. It is to note that oe TbDBF::3Ty1 and oe TbDBFK141A::3Ty1 showed the same apparent size as oe TbDBFDMTS::3Ty1 by western-blot analyses, indicating that the MTS is also cleaved in its natural context (Figure 7C) and thus, at least the N-terminus of TbDBF is imported into the matrix. We then investigated the addition of the 2 C-proximal transmembrane domains at the C-terminal extremity of GFP (GFP::TM). As expected, a size shift corresponding to the addition of transmembrane domains is observed by western-blot (Figure 7B). However, in contrast to MTS::GFP, the GFP::TM was not targeted to mitochondria, but to unknown distinct structures, possibly membrane or ER as well as the flagellum which is different from a cytosolic labeling (see GFP alone) or from a mitochondrion labeling (here revealed with the anti-Tdh antibody) (Figure 7A and Figure S10). Their inability to mediate mitochondrial targeting shows that the TMs of TbDBF do not represent mitochondrial targeting determinants like those targeting mitofusins to the mitochondrial outer membrane. Their capacity to target GFP from the cytosol to other organelles, suggests that they have the capacity to mediate membrane anchoring. It is probable that this membrane-anchoring function is exerted within mitochondria, upon MTS-mediated targeting of TbDBF.

In order to further characterize the role and relevance of these domains on TbDBF localization and function, we expressed tagged TbDBF-mutants without MTS ( oe TbDBFDMTS::3Ty1) or TMs ( oe TbDBFLDTM::3Ty1) in PCF using tetracycline-induction (Figure 7D and7G). Conditional overexpression of both recombinant TbDBF was confirmed by western blot, after 3 days to 5 days of induction (Figure 7E/7H/S8). In contrast to oe TbDBF::3Ty1, a significant proportion of oe TbDBFDMTS::3Ty1 doesn't colocalize with Tdh revealing that the N-terminal MTS is indeed required for proper mitochondrial targeting of TbDBF (Figure 7F, Figure S10). Interestingly, the labeling observed for oe TbDBFDMTS::3Ty1 is somewhat similar to the labeling of GFP::TM (Figure 7A/F/S10), suggesting unspecific membrane anchoring by C-proximal TMs. The fact that part of oe TbDBFDMTS::3Ty1 still remained close to Tdh signal may show that the DMTS mutant is also anchored to mitochondrial membranes. To analyze the localization of oe TbDBFDMTS::3Ty1 with more precision, we performed electron microscopy analysis with anti-Ty1 antibody. We observed a dual localization of oe TbDBFDMTS::3Ty1, and goldparticles quantification revealed that 60% of oe TbDBFDMTS::3Ty1 is located outside of the mitochondrion and 40% inside or close to the mitochondrion membranes (Figure S9C/D). MTS is therefore important for the correct localization of TbDBF, but without it, the protein is partially able to localize to membranes. We further observed that overexpression of oe TbDBFDMTS::3Ty1 does not alter mitochondrial structure, indicating that its proper, MTS-mediated, mitochondrial localization is essential for TbDBF-function 7F/Anti-Tdh and Figure S8). We then investigated the relevance of the Cproximal TMs of TbDBF by overexpressing a version truncated for the TM ( oe TbDBFDTM::3Ty1, Figure 7G) and analyzed the mitochondrial shape and their localization by immunofluorescence (Figure 7I). oe TbDBFDTM::3Ty1 is also well overexpressed (Figure 7H and Figure S8) and perfectly colocalizes with the mitochondrial marker, like MTS::GFP. This confirms that the N-terminal MTS sequence is sufficient for mitochondrial targeting of TbDBF. However, no modification of the mitochondrial shape was observed (Figure 7E3 and Figure S8) demonstrating that the transmembrane domains are required for proper function. Altogether, our data show that TbDBF is targeted to mitochondria by an N-terminal MTS and that C-proximal TMs are required for proper function. We infer that TMs probably mediate membrane anchoring and the establishment of proper membrane topology.

(Figure

-Impact of TbDBF overexpression on parasite morphology/biology

We then decided to characterize TbDBF activity and intermitochondrial branching with higher resolution. To this end, we used as serial block face scanning electron microscopy (SBF-SEM), an approach allowing to characterize the overall structure of membranes and organelles within the entire cell volume [START_REF] Hughes | Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy[END_REF]. With this technique, hundreds of images are collected to perform a 3D reconstruction of the whole cell, as well as spatial organization of the individual organelles within the cell. Four to five wildtype and oe TbDBF::3Ty1 PCF cells were reconstructed in 3D, with a particular emphasis on the mitochondrion, the nucleus, the flagellar pocket and the cell outline. We have not been able to correctly identify the kinetoplast DNA on the electron microscopy images, thus preventing its 3D reconstruction. However, since kDNA is in close contact with the flagellar pocket, it is easy to position it. Other organelles (glycosomes, acidocalcisomes, golgi, flagellum, etc.) are not represented either in order to not overload the 3D representation. As expected, wild-type PCF cells showed a reticulated mitochondrion along the whole cell (Figure 8A 8E) and surprisingly the whole cell volumes was also increased ~1.7-fold in oe TbDBF::3Ty1 cells (Figure 8E).

On the other hand, and as presented in the introduction, the shape of the mitochondrion undergoes spectacular changes during the life cycle of the parasite, reflecting adaptation to different environments. These adaptations are also accompanied by a profound change in their metabolism. We therefore investigated whether and how an alteration of the mitochondrial structure affects the metabolism of the parasite. To address this, we screened for the use of 119 different carbon and nitrogen sources in parasites overexpressing or not TbDBF. Two carbon sources, D-glucose and L-proline (Figure 9 and Figure S11, n°14/114), are significantly more consumed/used in oe TbDBF::3Ty1 cells compared to non-induced cells, suggesting that modifications of the mitochondrial structure require increased energy consumption or to the opposite, that mitochondrial shape restructuring impacts metabolism. Interestingly, ethanolamine is also significantly more consumed/used in oe TbDBF::3Ty1 cells (Figure 9 and Figure S11,n°61). This molecule is a precursor of phosphatidylethanolamine, an important component of mitochondrial membranes [START_REF] Richmond | Lipidomic analysis of bloodstream and procyclic form Trypanosoma brucei[END_REF], which in line with the observed increase in mitochondrial complexity. Altogether these data suggest that oe TbDBF::3Ty1 cells need more energy and metabolic precursors, from increased catabolism of carbon sources, to fuel the increased membrane biosynthesis.

DISCUSSION

We have identified a novel and ancestral DRP family whose first representative, TbDBF, has been characterized in the protozoan parasite T. brucei. We have demonstrated that TbDBF localizes to the mitochondrial periphery and is able to increase mitochondrial interconnectivity in a GTPase-dependent manner. Its overexpression also induces modifications in energy metabolism in the parasite, as well as membrane biogenesis and shaping. We propose that mitochondrial TbDBF may mediate and/or enhance fusion.

Membrane fusion and fission are often controlled by proteins of the dynamin superfamily (DRP) [START_REF] Daumke | Invited review: Mechanisms of GTP hydrolysis and conformational transitions in the dynamin superfamily[END_REF][START_REF] Ramachandran | The dynamin superfamily[END_REF]. These proteins consistently have a GTPase domain and contain domains required for membrane binding and oligomerization. In trypanosomes, only DRPs involved in fission have been characterized, and no fusion proteins similar to those of yeasts and mammals have been identified so far. This may suggest that trypanosome mitochondria fuse via a machinery that differs from that characterized in yeasts and mammals. Of note, the situation is similar in plants, where no fusion protein similar to known fusion factors has been identified until now. Indeed, the search for homologous sequences to TbDBF allowed us to identify close DRP homologs in several eukaryotic phyla lacking Mfn and Opa1, the fusion factors of metazoan and fungi. Unlike Mfn and Opa1, that have few homologues in some prokaryotes (cyanobacterial BDLP), homologues of TbDBF were also identified in numerous bacterial phyla and even in archaea. These data point to the existence in these organisms of a novel machinery involved in membrane fusion, shaping and/or remodeling that is not present in Opisthokonta and Plantae but still present in numerous prokaryotes. While studies of yeast and mammalian DRPs provide information about their functions and structures, little is known about bacterial DRPs, especially their cellular role. Indeed, some bacterial DRPs have been associated with a variety of processes involving membranes in vivo, such as a surveillance mechanism for membrane punctures caused by antibiotics and bacteriophages [START_REF] Sawant | A dynamin-like protein involved in bacterial cell membrane surveillance under environmental stress[END_REF][START_REF] De Sousa Borges | Bacterial dynamin as a membrane puncture repair kit[END_REF], membrane vesicle formation [START_REF] Michie | LeoA, B and C from enterotoxigenic Escherichia coli (ETEC) are bacterial dynamins[END_REF] or cytokinesis by promoting membrane curvature at the septum [START_REF] Schlimpert | Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation[END_REF][START_REF] Bramkamp | Structure and function of bacterial dynamin-like proteins[END_REF]. In addition, studies of the Bacillus subtilis DRP DynA have shown that this protein can promote membrane fusion in vitro [START_REF] Burmann | A bacterial dynamin-like protein mediating nucleotide-independent membrane fusion[END_REF]. The mechanism by which the fusion operates is not yet known, but bacterial DRPs are probably recruited to the sites where membrane fusion is needed [START_REF] Bramkamp | Structure and function of bacterial dynamin-like proteins[END_REF][START_REF] Jimah | Structural Insights into the Mechanism of Dynamin Superfamily Proteins[END_REF]. Interestingly, structural analysis of Campylobacter jejuni DLP1/DLP2 proteins allowed to propose a mechanism explaining how these proteins attach and bind distant and opposing membranes [START_REF] Liu | Structural basis for membrane tethering by a bacterial dynamin-like pair[END_REF]. Unfortunately, the cellular function of C. jejuni DLP1/DLP2 is currently unknown. DRPs identified in this study have not yet been characterized and no information on their localization and function is available. However, among the identified organisms, we found, with the second-best score (E-Value 2e-80, Table S1), the bacteria of the phylum Planctomycetes which are somewhat particular organisms. Indeed, these bacteria contain a compartmentalized cytosol, separated by an intracytoplasmic membrane [START_REF] Lindsay | Pirellulosomes: a new type of membranebounded cell compartment in planctomycete bacteria of the genus Pirellula[END_REF], in some cases surrounding the nucleus [START_REF] Fuerst | Membrane-bounded nucleoid in the eubacterium Gemmata obscuriglobus[END_REF] or forming an anammoxosome, a pseudo-mitochondrion that is responsible for the production of energy [START_REF] Jogler | The bacterial 'mitochondrium[END_REF][START_REF] Neumann | Isolation and characterization of a prokaryotic cell organelle from the anammox bacterium Kuenenia stuttgartiensis[END_REF]. It is then conceivable that the identified protein in Planctomycetes (PbDBF, Figure 2 andS3) is involved in membrane structuring.

The role of TbDBF in mitochondrial shaping was then confirmed by its overexpression in PCF. Indeed, overexpressed TbDBF modifies the structure of the mitochondrion, with a very strong increase in connections. Moreover, the inactivation of its GTPase domain (K141A mutation) also confirms its belonging to the large family of dynamins. Interestingly, a similar link between hyperconnection of a branching network and increased fusion processes has been reported in filamentous fungi, which grow as interconnected branching networks [START_REF] Herzog | Cell fusion in Neurospora crassa[END_REF]. It is therefore tempting to speculate that, in trypanosomes, excess TbDBF increases the interconnectivity of mitochondrial networks by enhanced fusion. The absence of fragmentation observed upon partial or total inactivation of TbDBF expression was rather expected and suggests that trypanosomes differ from mammalian and yeast cells by the absence or a very low level of regular/permanent mitochondrial fission. This is in agreement with previous studies suggesting that fission only occurs during cell division [START_REF] Chanez | Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest[END_REF][START_REF] Morgan | The single dynamin-like protein of Trypanosoma brucei regulates mitochondrial division and is not required for endocytosis[END_REF]. It should also be kept in mind that other proteins could be involved in the structuring of mitochondria as shown by studies on mitochondrial outer membrane proteome in T. brucei [START_REF] Niemann | Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology[END_REF][START_REF] Povelones | Mitochondrial shape and function in trypanosomes requires the outer membrane protein, TbLOK1[END_REF] and the presence of mitochondrial complexes such as recently characterized MICOS [START_REF] Kaurov | The Diverged Trypanosome MICOS Complex as a Hub for Mitochondrial Cristae Shaping and Protein Import[END_REF]. It should be noted that TbDBF seems to be part of a new fusion mechanism, so its inactivation may not have the same effects as for Mfn or Opa1. It is also possible that subtle changes in mitochondrial structure are present, but that we were not able to reveal with our approaches. Another intriguing point is the absence of phenotype in response to overexpression of TbDBF in the BSF, which seems to reflect the relatively low dynamics of the mitochondrial structuration in these parasitic forms. It is possible that TbDBF interacts with other partners, in a possible branching complex, that might only be expressed in the PCF. However, one cannot exclude that overexpression of TbDBF is not high enough to initiate an increase in interconnectivity.

Using several microscopy approaches, we have shown that TbDBF is localized in the mitochondrion and more precisely in the mitochondrial membranes. TbDBF contains two domains involved in its accurate mitochondrial addressing, an N-terminal sequence (MTS) and two C-terminal transmembrane domains. Proteins with an N-terminal MTS are mainly fully imported into the mitochondrial matrix, however, some of them, such as Opa1 and Apoptosis-inducing factor (AIF) face the inner membrane [START_REF] Otera | Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space[END_REF]. Indeed, Opa1 and AIF localize mainly to the IMS due to its TM domain located in close proximity to the MTS, interrupting transport across the inner membrane, which is not the case for TbDBF.

Moreover, TbDBF has a functional MTS, which is cleaved in the mitochondrial matrix and its absence impacts the activity of the protein, suggesting that at least part of the protein crosses OMM and IMM. However, with an MTS-less construct, the protein is still partially associated to the mitochondrion and other membranes through its two C-terminal transmembrane domains. It should be noted that the targeting specificity of C-terminal TM domains is often determined by the amino acids flanking the TMs (61, 62) which could explain the remaining TbDBF protein in mitochondrion when the MTS was deleted.

TbDBF TM domains should therefore be considered as membrane-anchoring domains.

Interestingly, the structure prediction of TbDBF by Alphafold [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF] suggests that the two TM domains form a loop that may allow anchoring in the inner membrane with a possible orientation of the remaining protein on the matrix side (Figure 10). This orientation is also probably similar to the prokaryotic DBFs which could also be anchored in the plasma membrane and facing the cytosol. Recently, Sheik et al. [START_REF] Sheikh | A Novel Group of Dynamin-Related Proteins Shared by Eukaryotes and Giant Viruses Is Able to Remodel Mitochondria From Within the Matrix[END_REF] clearly demonstrated that expression of a potential DRP from giant virus in T. brucei PCF strongly affects mitochondrial morphology within the matrix, with a close association with the inner membrane. This contrasts with the mode of action of human or yeast mitofusins, which are involved in the remodeling of mitochondrial outer and inner membranes, but never via the matrix side. All of these results point to a complex or even unique targeting and membrane topology of TbDBF, different from those observed for mammalian and fungi OM and IM fusion factors.

In many eukaryotes, mitochondria display a dynamic behaviour of constant fission and fusion, with the size, appearance and organization of mitochondrial membranes varying between species, tissues and physiological conditions [START_REF] Sauvanet | Energetic requirements and bioenergetic modulation of mitochondrial morphology and dynamics[END_REF]. Although there is considerable convincing evidence that mitochondrial dynamics and bioenergetics are linked, the precise relationship between the two is still not known [START_REF] Duvezin-Caubet | Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology[END_REF][START_REF] Sauvanet | Energetic requirements and bioenergetic modulation of mitochondrial morphology and dynamics[END_REF][START_REF] Ramos | Bioenergetic roles of mitochondrial fusion[END_REF]. It is therefore tempting to assume that such tight connections regulate not only mitochondrial function and energy metabolism, but also the life cycle and pathogenic potential of trypanosomes.

In order to identify possible metabolic variations induced by TbDBF overexpression, we screened 119 molecules that could be used by parasites as carbon and energy sources.

Only 3 molecules appeared to be more used/consumed in cells with altered mitochondria, i.e., glucose, proline and ethanolamine. Glucose and proline are the two main carbon sources that trypanosomes use in the mammalian host and the insect vector, respectively. This suggests a greater energy demand, required either to change the mitochondrial structure and/or to increase the mitochondrial content. The third molecule, ethanolamine, is probably not a carbon source per se, but reflects a change in phospholipid metabolism and more specifically in phosphatidylethanolamine (PE), which together with phosphatidylcholine (PC), are the main constituents of eukaryotic cell membranes. PE is preferentially distributed in the inner leaflet of mitochondrial membranes and plays an essential role in mitochondrial functions [START_REF] Gonzalvez | Cardiolipin: setting the beat of apoptosis[END_REF][START_REF] Ardail | Mitochondrial contact sites. Lipid composition and dynamics[END_REF] by maintaining the mitochondrial morphology [START_REF] Tamura | Ups1p and Ups2p antagonistically regulate cardiolipin metabolism in mitochondria[END_REF][START_REF] Osman | The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria[END_REF][START_REF] Kuroda | FMP30 is required for the maintenance of a normal cardiolipin level and mitochondrial morphology in the absence of mitochondrial phosphatidylethanolamine synthesis[END_REF]. Indeed, PE is a fusogenic phospholipid that confers negative curvature to the mitochondrial membrane [START_REF] Van Den Brink-Van Der Laan | Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile[END_REF] and has been proposed to play important roles in membrane fusion involving the mitofusin Mfn [START_REF] Joshi | Cardiolipin and mitochondrial phosphatidylethanolamine have overlapping functions in mitochondrial fusion in Saccharomyces cerevisiae[END_REF]. In PCF T. brucei, PE and PC are synthesized exclusively through the Kennedy pathway [START_REF] Signorell | Phosphatidylethanolamine in Trypanosoma brucei is organized in two separate pools and is synthesized exclusively by the Kennedy pathway[END_REF]. Mutations in lipid composition result in alterations in mitochondrial morphology and inhibition of respiration [START_REF] Guler | Mitochondrial fatty acid synthesis is required for normal mitochondrial morphology and function in Trypanosoma brucei[END_REF] [START_REF] Fridberg | Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei[END_REF]. Remarkably, RNAi-mediated downregulation of ethanolamine phosphate cytidylyltransferase expression, the second enzyme in the Kennedy pathway, has been shown to disrupt mitochondrial morphology and ultrastructure in T. brucei. The most immediate effect was a change in mitochondrial morphology and disruption of the inner mitochondrial membrane topology [START_REF] Signorell | Perturbation of phosphatidylethanolamine synthesis affects mitochondrial morphology and cell-cycle progression in procyclic-form Trypanosoma brucei[END_REF]. Since PE is essential for mitochondrial morphology, the increased ethanolamine utilization/consumption in TbDBF-overexpressing parasites likely reflects increased membrane lipid biogenesis to support the alterations in mitochondrial structure. SBF-SEM analysis and 3D reconstruction of parasites expressing TbDBFL showed a significant increase in mitochondrial membrane biogenesis with an almost doubling of the mitochondrial volume. This increased membrane biogenesis is particularly evident in the vicinity of the kDNA, which is completely enveloped by a hyper-reticulated globular mitochondrial structure. More work is needed to determine whether this part of the mitochondrion is a preferential place for membrane biogenesis. It is therefore likely that the increase in membrane biogenesis is not limited to the mitochondrial membranes, but also affects the other cell membranes which may explain the significant increase of the cell volume observed and thus the cellular surface. However, this increase in volume does not affect the parasite's growth rate, implying that the increase in volume is disconnected from mechanisms regulating cell division. More work is now required to understand how TbDBF induces these structural and metabolic changes, as well as its mode of action.

In conclusion, all these results clearly revealed the presence of a novel and ancestral machinery involved in mitochondrial branching in trypanosomes. In addition,

23

MATERIALS AND METHODS

Trypanosomes and cell cultures

The PCF of T. brucei EATRO1125.T7T (TetR-HYG-T7RNAPOL-NEO, where TetR stands for tetracycline resistance, HYG for hygromycin, T7RNAPOL for RNA polymerase T7, and NEO for neomycin) was cultured at 27°C with 5% CO2 in SDM79 medium containing 10%

(vol/vol) heat-inactivated fetal calf serum, 5 µg/ml hemin, 25 µg/ml hygromycin and 10 µg/mL neomycin. The bloodstream form of T. brucei 427 90-13 (TetR-HYG-T7RNAPOL-NEO) was cultured at 37°C with 5% CO2 in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS), 0.2 mM bmercaptoethanol, 36 mM NaHCO3, 1 mM hypoxanthine, 0.16 mM thymidine, 1 mM sodium pyruvate, 0.05 mM bathocuproine, 1.5 mM L-cysteine ( 75), 5 µg/ml hygromycin and 2,5 µg/mL neomycin. Overexpression cell lines were induced with tetracycline (10 µg/mL for BSF and 1 µg/mL for PCF). Growth was monitored by daily cell counting with the cytometer Guava® Muse® and Guava® easyCyte™.

Endogenous tagging and overexpression of TbDBF

For endogenous gene tagging, primers were designed as described in [START_REF] Dean | A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids[END_REF] and PCR was performed using pPOTv7 vector as template. The pPOT used here was pPOTv7 for Cterminus 10xHA tagging (blasticidin resistance). For overexpression, DBF gene (Tb427.07.2410) was inserted in both pLew100 (phleomycine resistance) [START_REF] Wirtz | A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei[END_REF] and cloned in the pLew100 with a 3xTy1 tag as previously described. Catalytic mutant K141 was replaced by an alanine, using PCR approach [START_REF] Ling | Approaches to DNA mutagenesis: an overview[END_REF] and PCR product cloned in the pLew100 with a 3xTy1 tag. EATRO1125.T7T PCF was transfected using Amaxa same conditions and selected in IMDM containing 5 µg/ml hygromycin, 2,5 µg/mL neomycin and 5 µg/mL blasticidin. Primers used for the constructions are presented in supplemental Table S2.

Endogenous tagging and inactivation of TbDBF by CRISPR-Cas9

Endogenous tagging and inactivation of TbDBF was achieved by CRISPR Cas9 technology according Soares Medeiros et al. [START_REF] Soares Medeiros | Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins[END_REF] but using SpCas9 instead of SaCas9 (more detailed description of optimized and adapted protocols will be published separately). Briefly, inactivation of the TbDBF was achieved by inserting the resistant marker puromycin (Pac) or a small sequence encoding for a BamHI restriction site plus 6 successive stop codons, flanked by 50 bp homologous to the 5' and 3' TbDBF sequences from the Cas9 cutting site. Similar approaches have been performed for endogenous tagging with the insertion of a sequence encoding 3xTy1 or 3xHA. The EATRO1125.T7T PCF and the 427 90.13 BSF (1x10 6 cells) were respectively transfected, using Amaxa nucleofectorII, with 1 µg of purified cassette (puromycin resistance marker, StopBamH1Stop, 3xTy1 or 3xHA), 30 µg Cas9 protein from IDT and pre-annealing TracrRNA (0.4 µmol) and gRNA (0.4 µmol). Cells were transfected using program X-001 and selected or not with puromycin (SDM79, 1 µg/mL, or IMDM 0.1 µg)/mL). Cells were cloned by using a cell sorter (TBM Core facility), and selection of double inactivated TbDBF gene (TbDBF -/-) or endogenously tagged clones was done by DNA extraction, with NucleoSpin Blood (Macherey-Nagel) and PCR amplification, see supplemental Table S2. Guide RNA were designed using EuPaGDT, from http://tritrypdb.org. Primers and guide RNA used are synthetized by Integrated DNA Technologies (IDT) and listed in supplemental Table S2. S2.

Down-regulation of TbDBF gene expression

Down

Mitochondria staining on living cells

Rhodamine-123 (30 µg/mL) was added on cell culture (5x10 6 -1x10 7 cells per mL) for 15 min at room temperature, then cells were washed twice with PBS and spread on slides.

Images were acquired with MetaMorph software on Axioplan 2 microscope and processed with ImageJ.

Immunofluorescence

Cells were washed twice with PBS, then fixed with 2% paraformaldehyde (PFA) for 10 min at room temperature and 0.1mM Glycine was added 10 min to stop the reaction. The cells were spread on slides and permeabilized with 0.05% triton X-100. After incubation in PBS containing 4% bovine serum albumin (BSA) 20 min, cells were incubated for 1 hour with primary antibodies diluted in PBS-BSA 4%, washed 4 times with PBS and incubated for 45 min with secondary antibodies diluted in PBS-BSA 4% followed by three washes. Then, kinetoplasts and nuclei were labelled with DAPI (10 µg/mL) for 5 min.

Slides were washed 3 times with PBS and mounted with SlowFade Gold (Molecular probes). Images were acquired with MetaMorph software on Zeiss Imager Z1 or Axioplan 2 microscope and processed with ImageJ.

Ultrastructure Expansion Microscopy (UExM)

The protocol of UExM realized exactly as described by Casas et al. 2022

(dx.doi.org/10.17504/protocols.io.bvwqn7dw). An expansion factor was determined using the ratio between the size of the coverslip (12mm) and the size of the gels after the first expansion. Images were acquired on a confocal Leica SP5-MP (Bordeaux Imaging Center) using a 63X oil objective and processed with ImageJ.

Immunoelectron microscopy

Harvested cells were placed on the surface of formvar-coated copper grids (400 mesh).

Each loop was quickly submersed in liquid propane (-180°C) and transferred into a precooled solution of 0.1% uranyl acetate in dry acetone for 3 days at -82°C. Samples were rinsed with acetone at -20°C, and embedded progressively at -20°C in LR Gold resin (EMS, USA). Resin polymerization was carried out at -20°C for 7 days under UV illumination. Ultrathin LR Gold sections were collected on nickel grids coated with formvar. Sections were first incubated for 15 min with NH4Cl 500mM in Tris-buffered saline (TBS) pH7.8, blocked 2x10 min with 2% BSA in TBS pH 7.8. The grids were incubated 1 hour at room temperature with anti Ty1 antibody diluted to 1:200 in TBS containing 2% BSA rinsed with TBS containing 2% BSA and with TBS containing 1% BSA.

The samples were then incubated for 1 hour at room temperature with anti-mouse IgG diluted to 1:20 conjugated to 10 nm gold particles (BioCell). The sections were rinsed with TBS containing 1% BSA, and fix with 1% glutaraldehyde in TBS. After rinsing with TBS, grids were contrasted through a 5 min incubation with 2% uranyl acetate in water, followed by 1 min incubation with 1% lead citrate. Observations were performed on a HITACHI 7650 electron microscope operated at 80 KV with a Gatan-11 MPx camera (PIE Bordeaux Imaging Center).

SBF-SEM

Cells were prepared as described by Blancard and Salin (80) except for freezesubstitution medium (0.1% potassium permanganate for 24 h followed by 0.1% tannic acid for 24 h, and 2% osmium tetroxide with 1% uranyl acetate for 3 days). Resinembedded samples were trimmed, mounted onto stubs and placed into a Zeiss GeminiSEM300 with a fitted Gatan 3View2XP system (Gatan, Abingdon, UK). Serial images of the block face were recorded at an accelerating voltage of 1.2 kV. All images were taken with the following scanning settings: Pixel size 10 nm, pixel time 5 µs, slice thickness 50 nm (Z-slice), image size 5000x5000. Between 700 and 900 images were collected for each run. Images were collected from Back scattered electron with a specific BSE Detector (On point -Gatan Inc., Pleasanton, CA, USA) using the software Digital Micrograph (Gatan). Images were combined to form a single stack. Data processing and statistical analysis: Segmentation was conducted using AMIRA (versions 5.2.0 Thermofisher Scientific). Manual segmentation of cells and organelles were conducted using the brush tool and was based on ultrastructural characteristics specific to each organelle. Each organelle was segmented using the organelle membrane as the outer edge. Surface models were produced for each segmented organelle. Volumes of organelles were automatically generated from the surface renderings of segmented data.

Western-blot

Total protein extracts (5x10 Immunodetection was performed using the primary antibodies, diluted in PBS-Tween-Milk (0.05% Tween20, 5% skimmed milk powder), summarized in supplemental Table S3. Revelation was performed using a second antibody coupled to the HRP and the Clarity Western enhanced-chemiluminescence (ECL) substrate as describes by the manufacturer (Bio-Rad). Images were acquired and analyzed with the ImageQuant Las 4000 luminescent image analyzer.

Biolog

Biolog Phenotype MicroArrays™ provide an easy-to-use technology for scanning and measuring the energy metabolism pathways present in a wide range of in vitro cultured cell types. The metabolic pathway activities are assayed with a simple colorimetric reagent that measures redox energy produced when a cell oxidizes a chemical (Tetrazolium reduction). Standard protocol described by the manufacturer (Biolog) has been used with the following modifications: cells were resuspended in Biolog IF-M1 buffer at 1x10 7 cells per mL and 50 µL of this cell suspension were pre-incubated in each well of the PM-M1 or PM-M2 plates for 1 hours at 27°C with 5% CO2. Then, 10 µL of Biolog Dye Mix (Mix MB) was added to all plate wells and load into the Omnilog® for kinetic reading for 18 hours at 27°C or endpoint measurement at 590 nm with a microplate reader after 18 hours.

Quantification of Mitochondrial junctions -ImageJ macro

To quantify number of mitochondrial junctions, an ImageJ macro was developed with the help of the Bordeaux Imaging Center (BIC). The script is presented in supplemental Materials and Methods data MMS1.

Statistical analysis

Experiments were performed at least in triplicates. Statistical analyses were performed using Prism (GraphPad) software. The results are presented as mean ± S.D. Where indicated the results were subjected to two-sided student's t-test to determine statistical differences against the indicated group (Confidence interval 95% -P-value style: 0.1234 (ns); 0.0332 (*); 0.0021 (**); 0.0002 (***); <0.0001 (****)).

Structural prediction software and sequence analysis 29

Structure predictions were performed using PSIPRED ("http://bioinf.cs.ucl.ac.uk/psipred/") (81) and prediction of mitochondrial targeting sequences by various algorythms (MitoprotII, MitoFates and iPSORT) [START_REF] Claros | Computational method to predict mitochondrially imported proteins and their targeting sequences[END_REF]. Phylogeny analysis was performed with Geneious software using default parameters and the neighbor-joining method was used for tree calculation.

  Figure 8E). As anticipated, PCF mitochondrial volume is nearly doubled (4.9±1 µm 3 ) compared to the mitochondrial volume of BSF (1.1-3 µm 3 )[START_REF] Jakob | Mitochondrial growth during the cell cycle of Trypanosoma brucei bloodstream forms[END_REF][START_REF] Hughes | Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy[END_REF]. As expected, mitochondrial volume in cells overexpressing TbDBF::3Ty1 is almost doubled by comparison to wild-type cells (Figure8E) and surprisingly the whole cell volumes was

  and pHD1336 (blasticidin resistance) (77) expression vectors. Fragments were amplified and cloned into HindIII and XbaI restrictions sites of the pLew100 and into HindIII and BamHI restrictions sites of the pHD1336 expression vectors. In addition, a 3xTy1 tag was added at the C-terminus of the protein in the pLew100-TbDBF, using the XbaI and BamHI restrictions sites. The truncated version of TbDBF without the first 41 amino acids (TbDBFDMTS) and without the two trans-membranes domain (TbDBFDTM) were amplified

13 (

 13 -regulation of DBF expression by RNAi in PCF was achieved by expression of stemloop "sense/antisense" RNA molecules targeting a 400-bp fragment of the DBF gene introduced into the pLew100 tetracycline-inducible expression vector. A PCR-amplified 450-bp fragment, containing the antisense DBF sequence was inserted between XhoI and BamHI restriction sites of the pLew100 plasmid. Then, the separate 400-bp PCRamplified fragment containing the sense DBF sequence was inserted upstream of the antisense sequence, using HindIII and XhoI restriction sites. The resulting plasmid, pLew100-DBF-SAS, contains a sense and antisense version of the DBF fragment separated by a 50-bp fragment. The EATRO1125 PCF was transfected with the pLew100-DBF-SAS and cells were selected in SDM79 medium containing 25 µg/ml hygromycin, 10 µg/mL neomycin and 5 µg/mL phleomycin. Expression of the RNAi was induced by tetracycline (1 µg/mL). Down-regulation of DBF expression by RNAi in BSF was achieved exactly as described[START_REF] Vanwalleghem | Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1[END_REF] with a 280-bp fragment derived from the TbDBF open reading frame inserted into the p2T7-177 plasmid[START_REF] Wickstead | Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei[END_REF]. Linearized plasmid was transfected in T. brucei 427 90-TetR-HYG-T7RNAPOL-NEO) BSF cells. Expression of the RNAi was induced by tetracycline (10 µg/mL). Primers used for the constructions are presented in supplemental Table

Figure 1 -

 1 Figure 1 -Structure comparison between mitochondrial dynamin-related proteins (DRPs): Human mitofusin 1 or 2 (HsMfn1/2), T. brucei TbDBF, and human Opa1 (HsOpa1). MTS, predicted mitochondrial targeting sequence; CC, coiled-coil region; green box, GTPase domain with the four GTP-binding motifs in yellow; TM, transmembrane span; Middle, dynamin middle domain; PH, pleckstrin homology domain; GED, GTPase effector domain. Structure predictions were performed using PSIPRED (Jones, 1999). More details and sequences alignment are shown in Figure S1.

M f n 2 H sD yn 1 H s D y n 2 H

 212

Figure 2 -

 2 Figure 2 -Homologous sequences to TbDBF, phylogenetic analysis and domain organization of the proteins. (A) Schematic view of the results of the database analysis. The black circles indicate the number of different organisms identified. Bold and underlined characters indicate taxon where homologous sequences were identified. (B) Phylogeny of the homologous TbDBF sequences. Trypanosome sequences are highlighted in brown, mammals in blue, yeast in red, other eukaryotes in orange, prokaryote in green and Archaea in black. Mammal, yeast and trypanosome mitochondrial fission and fusion factors were also included as well as the mammals Clathrin-mediated endocytosis dynamins (CME, HsDyn1 and HsDyn2). Phylogeny analysis was performed using Geneious software with default parameters (unrooted and scaled). (C) Domain organization of several identified proteins from various organisms. MTS, predicted mitochondrial targeting sequence; CC, coiled-coil region; green box, GTPase domain with the four GTP-binding motifs in yellow; TM, transmembrane span; Structure predictions were performed using PSIPRED. More details and sequences alignment are shown in Figure S3. TbDBF, Trypanosoma brucei DBF (ID: Tb927.7.2410); AhDBF, Achlya hypogyna (ID: OQR970001); PgDBF, Polarella glacialis (ID: CAE8737761.1); CtDBF, Chaetoceros tenuissimus (ID: GFH50037.1); HfDBF, Hondaea fermentalgiana (ID: GBG23909.1); BsDBF, Bactrachochytrium salamandrivorans (ID: KAH9255760.1); ChtDBF, Chrysochromulina tobinii (ID: KOO35358.1); PbDBF, Planctomycetaceae bacterium (ID: MBV8881356.1); PsDBF, Pedosphaera sp. (ID: MCH2381658.1); RcDBF, Rubrimonas cliftonensis (ID: WP_093253540.1); GpDBF, Gammaproteobacteria bacterium (ID: RTZ59088.1); LdDBF, Lentisphaerae bacterium (ID: NLB69313.1); KbDBF, Kiritimatiellae bacterium (MBO7223041.1); MbDBF, Myxococcota bacterium (MBU0550280.1). ArcDBF, Archaeon (ID: RYH26728.1); TbDlp1, Trypanosoma brucei Dlp1 (ID: Tb927.3.4720); HsDnm1, Homo sapiens Dnm1 (ID: AAH50279); ScDnm1, Saccharomyces cerevisiae Dnm1 (ID: AJV55509); HsDyn1 and HsDyn2, homo sapiens Clathrin-mediated endocytosis dynamins 1 and 2 (ID: KAI2554107.1 and ID: NP_001005360 respectively); HsOpa1, Homo sapiens Opa1 (ID: NP_056375); ScMgm1, Saccharomyces cerevisiae Mgm1 (ID: NP_014854); NpBDLP, Nostoc punctiforme (ID: WP_012412711); HsMfn1 and HsMfn2, Homo sapiens Mfn1 and 2 (ID: NP_284941 and ID: NP_001121132 respectively); ScFzo1, Saccharomyces cerevisiae Fzo1 (ID: NP_009738).

Figure 3 -

 3 Figure 3 -Subcellular localization of TbDBF. (A) Schematic representation of TbDBF endogenously tagged its at C-terminus extremity with 10xHA. Only one of the two alleles is tagged. (B) Western blotting of whole-cell extracts (5x10 6 cells) of T. brucei PCF or BSF wild-type cells and PCF or BSF expressing TbDBF ::10HA cells revealed with an anti-HA antibody. It should be noted that Enolase used as a loading control is about 3 to 4 times more expressed in BSF than PCF according to Hannaert et al. (82). (C) Subcellular localization of TbDBF ::10HA in PCF. Colocalization of TbDBF ::10HA (anti-HA antibody) with matrix mitochondrial threonine dehydrogenase (Anti-Tdh antibody) analyzed by standard immunofluorescence. (D) Subcellular localization of TbDBF ::10HA in BSF. Matrix mitochondrial Heat shock protein 60 (Anti-Hsp60 antibody) was used to labelled the mitochondrial compartment in wild-type and TbDBF ::10HA cells.

Figure 4 -

 4 Figure 4 -Inactivation of the TbDBF gene. (A) Schematic representation of TbDBF inactivation by insertion of the puromycine resistant marker (PAC). (B) PCR confirmation of TbDBF gene inactivation on both alleles in BSF and PCF. (C) The mitochondrial shape of living WT and TbDBF -/-PCF cells (right panel) was designed by an ImageJ macro (MJ, Mitochondrial Junctions) from rhodamine 123 staining (left panel). (D) Corresponding mitochondrial junctions quantification. (E) Antibody directed against Hsp60 was used to label and visualized mitochondrial shape in BSF cells. Top panel : BSF WT cell; bottom panel : BSF TbDBF -/-cell. Statistic : t test -Confidence interval 95% -P-value style : 0.1234 (ns); 0.0332 (*); 0.0021 (**); 0.0002 (***); <0.0001 (****).

Figure 5 -

 5 Figure 5 -Overexpression of TbDBF and GTPase mutant. (A) Schematic representation of overexpressing TbDBF protein tagged with 3Ty1 peptide at C-terminus of the protein ( oe TbDBF::3Ty1). (B) Western blotting of whole-cell extracts of T. brucei PCF overexpressing TbDBF::3Ty1 cells. Non-induced (ni) and induced (i) cells after 3 to 5 days (d3 and d5) were revealed with an anti-Ty1 antibody. Antibody directed against Enolase protein (Eno) was used as loading control. (C) Mitochondrial structure analysis using rhodamine 123 staining on living cells; mitochondrial structure of PCF WT and PCF oe TbDBF::3Ty1 cells before (ni) and 5 days after (i) tetracycline induction. (D) Schematic representation of overexpressing TbDBFK141A ::3Ty1 mutant protein tagged with 3Ty1 peptide at the C-terminus of the protein ( oe TbDBFK141A::3Ty1). (E) Western blotting of whole-cell extracts of T. brucei PCF overexpressing TbDBFK141A::3Ty1 cells; (ni) non-induced and (i) induced cells for 3 or 5 days (d3 and d5). Antibody directed against glycosomal Fumarate reductase protein (Frdg) was used as loading control. (F) Mitochondrial structure analysis using rhodamine 123 staining on living cells; mitochondrial structure of PCF WT and PCF oe TbDBFK141A::3Ty1 cells before (ni) and 5 days after (i) tetracycline induction. (G) quantification of mitochondrial junctions number using Rhodamine 123 microscopy images. Mitochondrial junctions number: WT, 9.4 ± 4.3; .ni oe TbDBF::3Ty1 8.7 ± 3.6; .i oe TbDBF::3Ty1 20.4 ± 8.9; .ni oe TbDBFK141A::3Ty1 8.6 ± 4.8 and .i oe TbDBFK141A::3Ty1 10 ± 5.2. Statistic : t test -Confidence interval 95% -P-value style : 0.1234 (ns); 0.0332 (*); 0.0021 (**); 0.0002 (***); <0.0001 (****).

Figure 6 -

 6 Figure 6 -Overexpression of TbDBF and GTPase mutant. Subcellular localization of oe TbDBF::3Ty1 in PCF by immunofluorescence and Ultra Expansion Microscopy (UExM); Colocalization of oe TbDBF::3Ty1 (anti-Ty1 antibody) with matrix mitochondrial Threonine dehydrogenase (Anti-Tdh antibody) was analyzed by standard immunofluorescence (A) and by UExM with an expansion factor of ~4.4 (B), after 5 days induction (d5.i). (C) Subcellular localization of TbDBF::10HA in PCF analyzed by UExM. (D) Subcellular localization of oe TbDBFK141A::3Ty1 in PCF analyzed by standard immunofluorescence. (E) Western blotting of whole-cell extracts of T. brucei BSF overexpressing TbDBF::3Ty1 cells. Non-induced (ni) and induced (i) cells after 3 to 7 days (d3, d5 and d7) were revealed with an anti-Ty1 antibody. Antibody directed against Enolase protein (Eno) was used as loading control. (F) Subcellular localization of oe TbDBF::3Ty1 in BSF by immunofluorescence. Antibody directed against Hsp60 was used to label and visualized mitochondrial shape in wild type and TbDBF::3Ty1 overexpressing cells.

Figure 7 -Figure 8 -

 78 Figure 7 -Role of TbDBF domains. (A) Localization of the GFP according to the different tags fused. Colocalization by standard immunofluorescence of GFP with matrix mitochondrial Threonine dehydrogenase (Anti-Tdh antibody) after 5 days induction. MTS, mitochondrial targeting signal; TM, transmembrane domains. (B) Western-blot analysis of GFP expression. cGFP, constitutive and cytosolic GFP. Non-induced (ni) and induced (i) cells after 5 days. (C) TbDBF size analysis by western-blot using anti-Ty1 antibody. (D) Schematic representation of N-terminus truncated TbDBF protein (first 41 amino acids) tagged with 3Ty1 peptide at the C-terminus of the protein ( oe TbDBFΔMTS::3Ty1). (E) Western blotting of whole-cell extracts of T. brucei PCF overexpressing TbDBFΔMTS::3Ty1 cells; (ni) non-induced and (i) induced cells for 3 or 5 days (d3 and d5). (F) Subcellular localization of oe TbDBFΔMTS::3Ty1 in PCF after 5 days induction. Colocalization of oe TbDBFΔMTS::3Ty1 (anti-Ty1 antibody) with matrix mitochondrial Threonine dehydrogenase (Anti-Tdh antibody) analyzed by standard immunofluorescence. (G) Schematic representation of truncated transmembrane domains of TbDBF protein (45 amino acids) tagged with 3Ty1 peptide at the C-terminus of the protein ( oe TbDBFΔTM::3Ty1). (H) Western blotting of whole-cell extracts of T. brucei PCF overexpressing TbDBFΔTM::3Ty1 cells; (ni) non-induced and (i) induced cells for 3 days. (I) Subcellular localization of oe TbDBFΔTM::3Ty1 in PCF after 5 days induction, analyzed by standard immunofluorescence.

Figure 9 -Figure 10 -

 910 Figure 9 -Carbon sources used in cells overexpressingoe TbDBF::3Ty1. The metabolic pathway activities are assayed using a colorimetric reagent that measures redox production when a cell oxidizes a substrate (Biolog Phenotype MicroAr-rays™). Non-induced (black) and induced (grey) cells were incubated for 18 hours at 27°C with various substrates and redox production was measured by an endpoint absorbance at 590 nm. Only the 3 carbon sources showing significant variation are presented among the 119 molecules used. All the molecules (carbon and nitrogen sources) are show in FigureS11. The negative control corresponds to an absence of carbon sources. Statistic : Two way ANOVA -Confidence interval 95% -P-value style : 0.1234 (ns); 0.0332 (*); 0.0021 (**); 0.0002 (***); <0.0001 (****), n=3.

  ). MTS, predicted mitochondrial targeting sequence; CC, coiled-coil region; green box, GTPase domain with the four GTP-binding motifs in yellow; TM, transmembrane span; Middle, dynamin middle domain; PH, pleckstrin homology domain; GED, GTPase effector domain. Structure predictions were performed using PSIPRED[START_REF] Jones | Protein secondary structure prediction based on position-specific scoring matrices[END_REF]. More details and sequences alignment are shown in FigureS1.

	STRAMENOPILES	SAR	EXCAVATA
	Oomycota		G1 G2 Discoba (Trypanosomes)	G3	G4
	HsMfn1/2 Ochrophyta Bigyra		Metamonada Malawimonadida		GTPase		TM	TM	741 aa
	Dinoflagellates Ciliates TbDBF ALVEOLATA Apicomplexa		G1 G2 INCERTAE SEDIS MTS OPISTHOKONTA	G3	GTPase	G4	TM	TM	600 aa
	HsOpa1 RHIZARIA		Animals Fungi Microsporidia						960 aa
			AMOEBOZOA
			Ancathamoeba
	PLANTAE		Slime moulds Entamoeba
	Land Plants
	Green algae
	Red algae
	Glaucophytes		EUKARYOTA
	BACTERIA
	PVC group
	Lentisphaere
	Proteobacteria
	Myxococcota
	Kiritimatiellaeota
	Thermodesulfobacteria

suggests an important role in the biology of these organisms.

ACKNOWLEDGMENTS

We thank Keith Gull (University of Manchester) for providing us the anti-Ty1 and anti-PFR antibodies and Derrick Robinson, Mélanie Bonhivers, Elina Casas and Nicolas Landrein (University of Bordeaux) for the pPOTv7 expression vector and extremely valuable help in the expansion microscopy experiment. Cell sorter analysis were performed at the TBMCore facility (FACSility) on BD FACSAria™ III Sorter and we thank Atika Zouine for data acquisition and interpretation. We also thanks Bordeaux Imaging Center (BIC), which is a member of the FranceBioImaging national infrastructure (ANR-10-INBS-04), for helping us to design the ImageJ macro and microscopy acquisitions.

Supplemental material

Figure S1 shows alignment between human and trypanosome mitochondrial dynaminrelated proteins.

Figure S2 shows alignment of various kineoplastids DBF proteins.

Figure S3 shows alignment between TbDBF and various eukaryotes and prokaryotes.

Figure S4 shows growth curves of parental, over-expressing and mutant cells.

Figure S5 shows the inactivation of TbDBF expression by CRISPR/Cas9 system.

Figure S6 shows the down-regulation of TbDBF expression by RNAi.

Figure S7 shows results of the ImageJ plugin on rhodamine-stained cells.

Figure S8 shows the level of overexpression of different TbDBF variants. Table S1 shows results from BLASTp analysis of several identified organisms.

Table S2 shows all the primers and guide RNA used in this study.

Table S3 shows all the antibodies used in this study.

Materials and Methods: MMS1 described the ImageJ macro.