A comparative analysis of trypanosomatid SNARE proteins☆

The Kinetoplastida are flagellated protozoa evolutionary distant and divergent from yeast and humans. Kinetoplastida include trypanosomatids, and a number of important pathogens. Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. inflict significant morbidity and mortality on humans and livestock as the etiological agents of human African trypanosomiasis, Chagas' disease and leishmaniasis respectively. For all of these organisms, intracellular trafficking is vital for maintenance of the host–pathogen interface, modulation/evasion of host immune system responses and nutrient uptake. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are critical components of the intracellular trafficking machinery in eukaryotes, mediating membrane fusion and contributing to organelle specificity. We asked how the SNARE complement evolved across the trypanosomatids. An in silico search of the predicted proteomes of T. b. brucei and T. cruzi was used to identify candidate SNARE sequences. Phylogenetic analysis, including comparisons with yeast and human SNAREs, allowed assignment of trypanosomatid SNAREs to the Q or R subclass, as well as identification of several SNAREs orthologous with those of opisthokonts. Only limited variation in number and identity of SNAREs was found, with Leishmania major having 27 and T. brucei 26, suggesting a stable SNARE complement post-speciation. Expression analysis of T. brucei SNAREs revealed significant differential expression between mammalian and insect infective forms, especially within R and Qb-SNARE subclasses, suggesting possible roles in adaptation to different environments. For trypanosome SNAREs with clear orthologs in opisthokonts, the subcellular localization of TbVAMP7C is endosomal while both TbSyn5 and TbSyn16B are at the Golgi complex, which suggests conservation of localization and possibly also function. Despite highly distinct life styles, the complement of trypanosomatid SNAREs is quite stable between the three pathogenic lineages, suggesting establishment in the last common ancestor of trypanosomes and Leishmania. Developmental changes to SNARE mRNA levels between blood steam and procyclic life stages suggest that trypanosomes modulate SNARE functions via expression. Finally, the locations of some conserved SNAREs have been retained across the eukaryotic lineage.


Introduction
Kinetoplastids are flagellated protozoa of the Excavata supergoup and evolutionarily distant from model eukaryotes such as fungi, animals and plants [1]; the order contains many pathogenic species. Major kinetoplastid pathogens include the African trypanosomes, represented by Trypanosoma brucei, causing African trypanosomiasis in humans and nagana in livestock and largely restricted to sub-Saharan Africa, the American trypanosome, Trypanosoma cruzi, the etiological agent of Chagas' disease, and also the Leishmania species, that cause various forms of leishmaniasis in Southern Europe, Africa, Asia and America [2]. Globally, approximately 25 million people are affected by trypanosomatid infections, while the number at risk exceeds 250 million [3]. Available kinetoplastid genome sequences indicate significant conservation of gene complement and synteny [4], but different lineages cause highly distinct diseases and survive in discrete biological environments; for example T. brucei is exclusively extracellular while T. cruzi and Leishmania major invade host cells [5].
Intracellular trafficking is responsible for the transport and sorting of lipid and protein cargo between membrane-bound intracellular compartments. Trafficking requires spatially and temporally co-ordinated protein-protein interactions and is fundamental to cell growth and differentiation, nutrient uptake, immune evasion, signaling and many other processes [6]. In trypanosomes, intracellular trafficking is especially important in evading the mammalian host immune system and maintaining the surface proteome. Specifically the copy numbers of proteins and other molecules that participate directly in immune defense or other pathogenesis associated events are significantly varied during life cycle progression. A potent example of this phenomenon is T. brucei, where antigenic variation [7] requires high-level surface expression of the variant surface glycoprotein, but in addition, immune evasion is augmented by recycling of surface antigens and immunoglobulin degradation via the endocytic pathway [8,9].
Among the key proteins mediating intracellular trafficking are the Rab and ARF small GTPases, vesicle coat proteins and soluble N-ethylmaleimide-sensitive factor attachment protein receptors or SNAREs [10]. SNAREs are 10-30 kDa, subcellular compartmentspecific, type II membrane proteins, characterized by a highly conserved SNARE motif, a~70 amino acid block comprising hydrophobic heptad repeats [11,12]. The SNARE motif, usually located towards the C-terminus and connected to a trans-membrane domain by a short linker, is critical for forming the SNARE complex during membrane fusion [13]. Many SNARE proteins also contain additional domains at the N-terminus, that serve to regulate SNARE complex assembly, and some SNAREs deviate from this prototypical organization. For example, Homo sapiens SNAP-23, SNAP-25, SNAP-29, Syn11 and Saccharomyces cerevisiae Ykt6 all lack a trans-membrane domain but are membrane anchored via prenylation or palmitoylation [14,15]. Human SNAP-25, which contains two SNARE motifs, attaches to membranes by noncovalent association with trans-membrane domain SNAREs [16,17].
Classification of SNAREs is based on the conservation of an amino acid residue in the central polar layer of the coiled-coil SNARE complex [18]. This residue is either a glutamine (Q) or an arginine (R), and defines Q-and R-SNARE subclasses [19]. Based on the relative positions of these critical residues within the SNARE complex, Q-SNAREs are further sub-classified into Qa-(syntaxins), Qb-and Qc-SNAREs [11]. Q-SNAREs are also differentiated by their N-terminal organization. Syntaxins and a few Qb-and Qc-SNAREs contain an Habc domain three-helix bundle [20] that is thought to act as a binding site for regulatory SM proteins [19]. The Habc domain may also fold back onto the SNARE domain to give a 'closed' conformation, preventing interaction of cognate SNARE partners [21]. R-SNAREs are sub-classified into short vesicle-associated membrane proteins (VAMPs; brevins) and long VAMPs (longins) based on the presence of a short and variable domain or a conserved longin domain at the N-terminus respectively [22].
Comparative genomic and phylogenetic analyses have, to some degree, defined a SNARE complement for the last eukaryotic common ancestor (LECA) and thus set expectations for the complement likely present in a given eukaryotic genome. Five Qa-SNARE subfamilies appear to be ancient [54]: Syntaxin 5, 16, 18, as well as the SynPM and SynE clades, which have undergone lineage-specific expansions in animals and yeast [55,56]. The LECA Qb-SNARE complement consists of at least Vti1, Gos1, Bos1 and Sec20, while the Qc complement holds Syntaxin 6, 8, and Bet 1 as a minimum [57]. Finally, the R-SNARE complement consists of three longin subfamilies Sec22, Ykt6 and Vamp7. Vamp7 is expanded in several eukaryote lineages [55,58], and also gave rise to the brevins, Vamp1-6, 8 and Snc1/2, which are believed to be opisthokont-specific [59].
Given that intracellular membrane transport is so critical for immune evasion and other cellular processes in trypanosomes, a detailed understanding of the process is clearly of importance. The roles of many proteins in trafficking in T. brucei and additional trypanosomatids have been described [23,24], but the contributions made by members of the SNARE repertoire remain to be elucidated. Building on an earlier investigation of L. major SNAREs [25], we identified and classified the putative SNARE complement in predicted proteomes of T. brucei and T. cruzi. These, together with L. major and opisthokont reference sequences, allow a classification for trypanosome SNAREs to be derived. Additionally, we predicted the domain structures and investigate the expression profile of the T. brucei SNAREs. Finally, by determining the subcellular location of a select cohort of the SNAREs that are conserved between trypanosomes, animals and fungi, we provide evidence for retention of a similar location of orthologous SNAREs across the eukaryota.

Genome searches for candidate SNARE open reading frames
The predicted proteomes of T. brucei and T. cruzi were obtained from EuPathDB (http://eupathdb.org/eupathdb/) and formatted into BLAST searchable databases. Validated Leishmania major SNAREs [25] were used to query the formatted databases using BLASTP [26] with cut-off E-value of 0.0001, given the short length of the proteins. Domain content predictions for the retrieved sequences were generated at the PFAM [27] and PROSITE [28] domain databases. Only sequences predicted to contain the SNARE domain were retained as potential homologues. These sequences were aligned using MUSCLE (62) and manually edited using JALVIEW (63) and subsequently used to create a Hidden Markov Model (HMM) profile that was used to exhaustively reinterrogate the T. brucei and T. cruzi genomes for distant homologues using the HMMER package [29]. Additionally, in cases where one kinetoplastid ortholog of a clade was not initially identified, BLASTp searches using the relevant sequences of the other trypanosomatids were performed. Trans-membrane (TM) domain topology prediction was performed using SMART [60]. Fold recognition was performed using the fold threading software PHYRE (www.sbg.bio.ic.ac.uk/~phyre).

Sequence alignment and phylogenetic reconstruction
Multiple sequence alignments were generated using MUSCLE [30] and manually edited in MacClade v4.08 to only retain unambiguously aligned regions [31]. Phylogenetic reconstruction was performed using two separate methods. To obtain the best Bayesian trees, topology and posterior probability values, the program MrBayes v3.2.1 [32] was used with the following run parameters; prset aamodelpr = fixed(WAG); mcmc ngen = 10,000,000; samplefreq = 1000; nchains = 4; startingtree = random; sump burnin = 2500; sumt burnin = 2500. Posterior probabilities were used as a measure of node robustness. All calculations were checked for convergence by running the analysis to split frequencies of b 0.1. Maximum-likelihood analysis was performed using the program PhyML v3.0 [33] with the following parameters; nb bootstrapped datasets = 100; substitution model = LG; proportion invariable sites = 0.0; and nb categories = 4. The model of sequence evolution prior to each PhyML analysis was determined using Prot-Test v3.2.1 [34] and included corrections for rate variation used to determine the best substitution model and invariable sites where applicable. Trees were rendered using FigTree v1. 2 [35]. To identify SNAREs that are conserved between trypanosomes, humans and yeast, opisthokont landmark sequences were included in the analyses. In some cases selected opisthokont-specific duplications of subfamilies were excluded to alleviate phylogenetic artifact. For R-SNAREs, only longin landmark sequences were used.

Transfection of T. brucei
Transfections were performed using the Amaxa human T-Cell Nucleofector® kit (Amaxa, Koeln, Germany) following the manufacturer's guidelines with a few modifications. Briefly, 3 × 10 7 log phase cells were harvested at 800 ×g for 10 min at 4°C and resuspended in 100 μL of ice-cold Amaxa Human T-Cell solution. Linearized DNA plasmid (10 μg) was placed in a cuvette to which the cells were immediately added. The sample was transfected using the Amaxa Human Nucleofector®II running program X-001. Electroporation mixtures were immediately transferred to flasks containing prewarmed HMI-9 complete medium. After 12 h, selection antibiotic was added to each and the culture was distributed into a 24-well plate and subsequently incubated at 37°C. Positive transformants were selected on the 5th or 6th day after transfection.
2.6. Quantitative real-time polymerase chain reaction 1 × 10 8 cells were harvested at 800 ×g for 10 min at 4°C and washed with ice-cold PBS and quick frozen in dry ice for 1 min. RNA was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions and quantified using a ND-1000 spectrophotometer and Nanodrop software (Nanodrop Technologies). qRT-PCR was performed using iQ-SYBRGreen Supermix on a MiniOpticon Real-Time PCR Detection System (Bio-Rad). Quantification was done using Opticon3 software (Bio-Rad).

Western blot analysis
Cells were harvested at 800 ×g for 10 min at 4°C and washed once with ice-cold phosphate-buffered saline (PBS). Samples were then re-suspended in 20 μl 2 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, heated to 95°C for 10 min, and then subjected to SDS-PAGE. Separated proteins were then electroblotted onto Immobilon-P membrane (Millipore Corp.). Membranes were then blocked with 5% skimmed milk in TBS (137 mM NaCI, 2.7 mM KCI, 25 mM Tris base, pH 7.4, 0.2% Tween 20) for 1 h at room temperature. Probing with the primary antibody (mouse anti-HA epitope immunoglobulin G (IgG) at 1:10,000 dilution; Santa Cruz Biotechnology) was then carried out overnight at 4°C. Membranes were washed twice with TBS and probed with secondary antibody (rabbit anti-mouse peroxidase-conjugate at 1:10,000 dilution) for 1 h at room temperature. Bound antibodies were detected by enhanced chemiluminescence using Biomax MR-1 films (Kodak). Films were scanned and, where relevant, quantitated using ImageJ software (NIH).

Immunofluorescence analysis (IFA)
For immunofluorescence analysis, bloodstream parasites in exponential growth were harvested by centrifugation at 800 ×g for 10 min at 4°C and washed with ice-cold Voorheis's-modified phosphatebuffered saline (vPBS; PBS supplemented with 10 mM glucose and 46 mM sucrose, pH 7.6). Cells were then fixed in 3% parafomaldehyde in vPBS for 10 min at 4°C. Fixed cells were then applied to poly-lysine microscope slides (VWR International) sectioned with an ImmEdge Pen (Vector Laboratories) for 40 min. For permeabilization, cells were incubated with 0.1% Triton-X100 in PBS for 5 min at room temperature and washed three times for 5 min with PBS. Samples were then blocked with 20% FCS in PBS for 1 h at room temperature. Fixed cells were incubated with primary antibodies for 1 h followed by three washes for 5 min in PBS. Secondary antibodies were then applied for 1 h at room temperature and washed again three times with PBS. Samples were then dried and coverslips were mounted using Vectashield mounting medium supplemented with DAPI (Vector Laboratories, Inc.). Coverslips were sealed with nail varnish (Max Factor Inc.). Both the primary (mouse anti-HA epitope immunoglobin G (IgG); Santa Cruz Biotechnology Inc.) and the secondary (anti-mouse Oregon Green; Molecular Probes or anti-mouse Alexafluor-red as appropriate) were used at a dilution of 1:1000. Cells were examined on a Nikon Eclipse 400 epifluorescence microscope fitted with a Hamamatsu CCD digital camera. Image acquisition was performed with Metamorph software (Molecular Devices, Version 6). Images were processed for presentation with Adobe Photoshop (Adobe Systems Inc.). Quantitation was performed on the raw image data with no prior processing.

Evolutionary relationships of trypanosomatid SNAREs
Homology searching of the predicted proteomes of T. brucei [39] and T. cruzi yielded a putative SNARE complement of 26 in both cases. Similar searches into L. major yielded a complement of 27 SNAREs, consistent with the previous analysis by Besteiro and co-workers [25]. By contrast, Yoshizawa and co-workers [57], using a different methodology, identified 58 SNAREs in T. cruzi. The discrepancy is likely due to their use of an earlier and lower quality release of the genome database, which is also known to be partially polyploid and with frequent duplications.
Phylogenetic analysis was undertaken to ascertain evolutionary relationships between the predicted SNAREs of T. brucei, L. major and T. cruzi, as well as to classify the proteins into established eukaryotic SNARE subfamilies. A landmark set of SNAREs from H. sapiens and S. cerevisiae was included, as the functions of the majority of the SNAREs in these two organisms have been described. An initial analysis including SNAREs from these five organisms robustly segregated into four subclasses [11,18,40], but with poor intraclade resolution (Qa, Qb, Qc and R, data not shown). To improve resolution, SNAREs from each subclass were subsequently analyzed independently.
without identifiable opisthokont affiliation. In the Qc-SNARE tree (Fig. 1C), a clade uniting the opisthokont Bet1 sequences with two robustly supported kinetoplastid subclades was reconstructed, although without internal resolution. Additionally, we observed three Qc clades (Qc-1-3) for which opisthokont orthologs could not be robustly assigned. Qc1 and Qc2 were also reconstructed as encompassing two separate subclades each containing the three trypanosomatids examined. In the R-SNARE tree (Fig. 1D), three opisthokont SNAREs formed clades with trypanosomatid sequences; ER-Golgi Sec22, involved in anterograde transport from the ER, the Golgi-vacuole localized Ykt6, and endosomal Vamp7. Additionally, the clade of R1 contained proteins from all three trypanosomatids, but was not robustly assignable to an opisthokont ortholog (data not shown). From these reconstructions we observed a few cases of genomespecific expansion and also of failure to identify a particular ortholog. However, overall we largely found a 1:1:1 ortholog among the trypanosomatid SNAREs, indicating general stability of the SNARE complement. This contrasts with the Rab GTPases which are represented by a larger cohort in T. cruzi and L. major than in T. brucei. In just under 50% of the cases, we were unable to identify an opisthokont ortholog for a particular clade of kinetoplastid SNAREs. Whether this is due to true biological novelty or failure of the phylogenetic methodology to resolve relationships between distantly related proteins awaits more in depth analysis, possibly with improved phylogenetic methods when they become available. Nonetheless, we were able to identify ortholog relationships of trypanosomatid SNAREs with opisthokont sequences in 10 of 19 cases; these trypanosome SNAREs are candidates for assuming equivalent cellular functions.

T. brucei SNARE architecture
The majority of T. brucei, T. cruzi and L. major SNAREs exhibit prototypic SNARE features, i.e. a C-terminal trans-membrane domain linked to a SNARE motif by a short linker, plus, in several, a helical N-terminal domain (Fig. 2). However, several SNAREs in both T. brucei and T. cruzi do not conform to this standard architecture. One of the non-prototypic T. brucei candidates, Tb927.8.3470 (TbQb2), is predicted to contain two putative SNARE domains at the N-and C-termini respectively. This is a unique finding given that such an architecture of N-and C-terminal SNARE domains has been reported for SNAP-23, SNAP-25, SNAP-29, Sec9p and Spo20p, but these are mainly restricted to animals, higher plants, fungi, and ciliates [61]. Further investigation of this Fig. 2. Schematic illustration of the structural organization of T. brucei SNAREs and representation among the TriRyps. Red ellipses represent the C-terminal SNARE motif, the transmembrane domain is represented by dark purple rectangles. The Habc domain is represented by green ellipses while the N-terminus longin domain of R-SNAREs is represented by cyan ellipses. Designations are taken from GeneDB accessions. The N-terminus of the protein is drawn towards the left. Dots represent presence (black) or absence (white) from a detectable ortholog in T. brucei, L. major and T. cruzi. A numeral within a circle represents the presence of more than one ortholog. TbR1 is shown spaced from the main body as this SNARE could not be assigned using phylogenetics, but only on BLAST and domain searches.
All T. brucei Qa-SNAREs were predicted to contain the N-terminal Habc domain (Fig. 2). This domain regulates SNARE activity by preventing coiled-coil formation. Although generally restricted to the Qa-SNAREs, the Habc domain was putatively identified in several Qc-SNAREs (TbQc1A, TbQc2A and TbQc3). Finally, the R-SNAREs appeared to possess the canonical domain structure for this subclass. Only in TbR1 did we fail to predict a longin domain.

Differential expression of T. brucei SNAREs
To investigate if the identified T. brucei SNARE genes are transcribed, real-time PCR was performed, using gene-specific primers, against total RNA from both the bloodstream (BSF) and procyclic forms (PCF) of the parasite. Significant levels of transcription were found for the entire cohort. While our transcriptome data suggests that TbSyn5, TbR1 TbSyn16A and TbQb2A are constitutively expressed, a subset of T. brucei SNAREs are differentially expressed at the mRNA level between lifecycle stages. Further, consistent with earlier data [47], we also find that the SNAREs analyzed in this study are differentially expressed, with the majority being up-regulated in the BSF relative to the PCF (Fig. 3). This dynamic expression is also consistent with the earlier study by Bestiero et al. [25], which demonstrated that L. major SNAREs are differentially regulated, suggesting that this may be a general phenomenon of the trypanosomatid SNARE cohort. As membrane trafficking requirements are variable between life stages, these transcriptional changes may reflect significant changes to individual transport steps. In T. brucei, SNAREs must play a critical role in recycling of VSG, a process that requires both high rates of endocytosis as well as recycling/exocytosis. While we did observe strong up-regulation of TbVAMP7B, we saw little evidence for changes in the expression of the remaining cohort of putative endosome-associated SNAREs. By contrast to the endosomal SNAREs, there is prominent up-regulation in the BSF of TbSec22 and TbYKT6 which suggests potential modulation of specific ER exit pathways, and which may be coupled to the presence of two Rab1 orthologs and a Rab 2 ortholog in T. brucei and hence complexity in ER exit [48].

Subcellular localization of trypanosome SNAREs
The sequences of several differentially expressed T. brucei SNAREs that were also found to have an ortholog in either H. sapiens or S. cerevisiae. TbSyn16B, TbVAMP7C, TbQc2A, TbVAMP7A, TbSyn5 and TbQc1B were chosen for genomic tagging in order to identify the subcellular location of the protein [47]. Multiple attempts to fuse a C-terminal hemagglutinin (HA) epitope tag to TbVAMP7A and TbQc2A were unsuccessful, but the remaining four SNAREs were successfully tagged and expressed. Intracellular localization of the HA-tagged SNARE proteins was assessed by staining with an anti-HA antibody and by co-staining cells using a selection of markers, including early endosomal epsinR, the lysosome marker p67, the plasma membrane and endosomal markers ISG65 and ISG75 and the endosomal/post-Golgi proteins clathrin, Rab5 and Golgi-located GRASP [49][50][51][52][53].
Immunofluorescence revealed juxtaposition between TbVAMP7C and ISG65, clathrin, epsinR and Rab5A, with the majority of the immunoreactivity localized to the region between the nucleus and kinetoplast (Fig. 4). These co-localizations indicate a possible endosomal localization for TbVAMP7C, consistent with the phylogenetic analysis. TbQc1B demonstrated a location very close to the posterior face of the nucleus, but expression levels were rather low and as a consequence localization was equivocal (Fig. 5). TbSyn5 is juxtaposed to GRASP (Fig. 6), suggesting localization to Golgi-associated structures. This was expected given the orthologous relationship with the Golgi located human Syn5 (Fig. 1A). Additionally, LmSyn5 has been experimentally localized at the Golgi [25], while TbSyn16B is also juxtaposed to the Golgi (Fig. 6). This was expected given the orthologous relationship with the Golgi localized human STX16 (Syn16) (Fig. 1A). These data suggest that for three SNAREs where orthologous relationship could be established, the locations of the trypanosome proteins suggest retention of targeting specificity with their mammalian and yeast orthologs.

Conclusions
The SNARE repertoire appears well conserved between L. major, T. brucei and T. cruzi, with a restricted number of losses or expansions between these organisms. It is therefore unlikely that the SNARE complement plays a major role in defining the highly divergent life styles and specific pathogenesis and immune evasion mechanisms of these parasites. This contrasts with a more restricted Rab protein repertoire in African trypanosomes compared with T. cruzi and Leishmania, and further underscores the importance of Rab proteins in mediating evolution of new trafficking pathways. Any contribution from SNAREs to adaptation of the trypanosomatid trafficking system is likely in expression levels, specific amino acid changes and/or precise mechanistic aspects. Endocytosis is significantly developmentally regulated in African trypanosomes, but significantly we observed little upregulation of SNAREs assigned as endocytosis orthologs. Experimental investigation of the three SNAREs conserved between trypanosomatids and opisthokonts suggests that the subcellular locations of the orthologs are conserved. This mirrors the conservation observed among the vast majority of Rab GTPases, and while location and function need not been fully concordant, this evidence does suggest a likely functional equivalence has been retained, in at least some aspects; direct experimental evidence is needed to verify this hypothesis. Further our phylogenetic evidence indicates that a substantial proportion of trypanosome SNAREs may be orthologous with those in other eukaryotes and consequently possibly perform similar functions. SNAREs could therefore serve as excellent cellular markers in many organisms for the definition of intracellular compartments. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.parint.2013.11.002.  10.790 (TbVAMP7C) relative to organelle markers ISG65, clathrin, epsinR and Rab5A. The tagged protein was visualized with a mouse monoclonal anti-HA antibody (green). Organelles were stained with rabbit polyclonal antibodies against specific trypanosome marker proteins (red). The nucleus and kinetoplast were stained blue with DAPI. Scale bar: 2 μm. Fig. 5. Localization of HA-tagged Tb927.9.3820 (TbQc1B) protein in the bloodstream form T. brucei. Shown is the localization of Tb927.9.3820 (TbQc1B) relative to known organelle markers ISG75, clathrin, epsinR and p67. The tagged protein was visualized with a mouse monoclonal anti-HA antibody (green). Organelles were stained with rabbit polyclonal antibodies against specific trypanosome marker proteins (red). The nucleus and kinetoplast (blue) were stained with DAPI. Scale bar: 2 μm.  10.1420 (TbSyn5) and Tb927.9.13030 (TbSyn16B) relative to DAPI or DAPI and GRASP. The tagged protein was visualized with a mouse monoclonal anti-HA antibody (red). Organelles were stained with rabbit polyclonal antibodies against specific trypanosome marker proteins (green). The nucleus and kinetoplast (blue) were stained with DAPI. Scale bar: 2 μm.