KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes*

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability.

African trypanosomes such as Trypanosoma brucei and related subspecies are parasitic protists that cause sleeping sickness in humans and the wasting disease nagana in cattle. Between 2010 and 2013 there were ϳ7,000 new cases reported per year (1), although considerably larger numbers of people are at risk for infection (2), and disease in domestic livestock represents a major economic burden in impoverished regions of Africa. In addition to the medical and veterinary impact of these parasites, they also exhibit many fascinating and unusual biological features, and their study has led to novel discoveries such as glycosylphosphatidylinositol anchors for membrane proteins, trans-splicing of mRNAs, extensive editing of mitochondrial mRNAs, and global polycistronic transcription of gene arrays.
These parasites are flagellated in all life cycle stages, including the bloodstream forms (BFs) 4 that infect the mammalian host and in all of the developmental stages that infect the tsetse fly vector. Given the multiple important roles that flagella play for trypanosomes, these cellular appendages have been studied extensively (3,4). Although studies on axonemal and luminal proteins predominated originally, considerable work has been done more recently on proteins of the trypanosome flagellar membrane (5). One of the first such proteins identified was a receptor type adenylate cyclase (6) that is likely involved in sensing the extracellular environment and relaying signals via synthesis of cAMP, and recent work on this family of proteins has uncovered roles for evasion of host innate immunity (7) and in social motility in insect stage procyclic forms (PFs) (8 -10). Furthermore, a variety of putative transporters and channels are present in the flagellar membrane (5), where they may play roles in nutrient or ion uptake and potentially in nutrient or solute sensing (11). Other important roles for the flagellar membrane are attachment to the salivary gland epithelium in the tsetse fly (12), flagellar adherence of gametes during mating (13), and attachment of the tip of the new flagellum to the body of the old flagellum during parasite replication, where attachment of flagella is required for cleavage furrow formation and proper cell division (14 -16).
Although a host of proteins are known that are targeted selectively to the flagellar membrane of trypanosomes or related parasites such as Trypanosoma cruzi or Leishmania (17)(18)(19)(20), the mechanisms for this selective surface targeting are largely obscure. In studies of the flagellar specific glucose transporter 1 from Leishmania mexicana, LmxGT1, our laboratory has identified a novel protein KHARON (KH), 5 which interacts with the flagellar targeting signal and mediates trafficking of this permease to the flagellar membrane (21). Although this ϳ57-kDa protein did not exhibit detectable sequence similarity to proteins outside the kinetoplastid parasites nor did it possess any recognizable conserved sequence motifs (21), the existence of orthologs of KH in all kinetoplastid parasites whose genomes have been sequenced suggested that this protein might be important for flagellar targeting of multiple membrane proteins across this family of parasites. However, to date the role of KH in other parasites has not been investigated. Furthermore, various genetic advantages of T. brucei, such as the ability to readily knock down mRNAs by inducible RNAi, as well as the existence of a flagellar membrane proteome (5), suggested that this parasite might represent a particularly facile system with which to study targeting of flagellar membrane proteins. In the present study, we have investigated KH from T. brucei (TbKH) and established that it is involved in flagellar trafficking of a putative calcium channel, TbCaCh (also called FS179) (5). Notably, RNAi-mediated knockdown of KH mRNA not only prevented flagellar trafficking of TbCaCh but was also lethal to both BF and insect stage PF parasites, resulting in detachment of flagella from the cell body via the flagellar attachment zone and failure of the parasites to undergo cytokinesis. Subcellular localization of KH demonstrated that this protein is located at the base of the flagellar axoneme, where it is likely to mediate import of integral membrane proteins into the flagellar membrane, but also on the subpellicular microtubule cytoskeleton and unexpectedly on the mitotic spindle. Hence KH is a central component of flagellar membrane transport machinery and also associates with multiple components of the trypanosome cytoskeleton, where it is likely to serve diverse functions in parasite cell biology. The results reported here establish T. brucei as an ideal experimental system in which to study the multiple functions of KH and in particular to dissect the mechanism whereby it mediates trafficking of integral membrane proteins to the flagellar membrane.

Identification of TbKH and Subcellular Localization in BF
Trypanosomes-Recent studies by our laboratory identified the KHARON protein of L. mexicana, LmxKH, as a cytoskeletonassociated protein that mediates the translocation of the LmxGT1 glucose transporter to the flagellar surface membrane (21). Because a BLAST search employing GenBank TM and TriTrypDB revealed the presence of KH orthologs in kinetoplastid parasites but not in other organisms, we hypothesized that KH might play a fundamental biological role in this entire family of protists. Protein sequence alignment of LmxKH (gene ID in TriTrypDB: LmxM.36.5850) and TbKH (gene ID: Tb927.10.8940) revealed 27% (112/411) identity and an additional 11% similarity, and several segments of sequence that are present in LmxKH (520 amino acids) are absent from TbKH (411 amino acids). Hence, whereas KH exists in trypanosomes, it is relatively divergent in sequence between these two related parasites, thus underscoring the need to test the biological functions in both organisms. A conserved sequence signature encompassing amino acids 227-235 of TbKH (NESVDVLNL) is the largest block of contiguous sequence present in both KH orthologs.
To determine the subcellular localization of TbKH in BF parasites, transgenic parasites were generated in which the TbKH open reading frame was endogenously tagged with the Ty1 epitope on the N terminus or with the HA 3 epitope on the C terminus. To facilitate isolation of separated flagella for some experiments, these transgenic lines were also prepared in a parasite genetic background, the TbFLA1 RNAi line, allowing inducible RNAi (22,23) against the TbFLA1 gene (24). Inducing degradation of TbFLA1 mRNA results in separation of flagellar attachment to the cell body by disrupting the flagellar attachment zone, the interface that encompasses parts of both the flagellar and cell body membranes and associated structures where the two membranes adhere (25). This disruption allows the non-adherent flagella to be sheared off the cell body and purified by differential centrifugation (26). Immunofluorescence of the transgenic BFs demonstrated that epitope-tagged TbKH was located in three discrete compartments: 1) In purified flagella, Ty1::TbKH localizes at the base of the flagella (Fig.  1A). 2) In whole cell images, Ty1::TbKH localizes in the subpellicular microtubule cytoskeleton (Fig. 1B) as indicated by the overlap of the TbKH signal with ␣-tubulin signal; in addition, these two fluorescence signals also overlap in detergent-extracted cytoskeletons from BF parasites (Fig. 1C), consistent with TbKH being a cytoskeleton-associated protein. 3) Unexpectedly, Ty1::TbKH also localizes in the mitotic spindle in dividing BF parasites (Fig. 1D), where it exhibits intense fluorescence on a tubule that connects two replicated nuclei. To confirm that this localization represents the mitotic spindle, parasites were co-stained with the KMX-1 antibody (Fig. 1E) that preferentially recognizes ␤-tubulin associated with the spindle (27). These images show strong overlap of the Ty1::TbKH-and KMX-1-associated signals at the tubule that connects separating nuclei. In addition, spindle morphology has been shown to change during the cell cycle (28), with the rhomboid shaped spindle (Fig. 1E) observed early in mitosis developing into a dense central spindle (Fig. 1D) later in the nuclear replication process, similar to the morphologies observed for Ty1::TbKH fluorescence.
To confirm that the localizations observed above did not represent epitope tag-mediated mistargeting, similar images were also obtained from parasite lines expressing TbKH1 with a complementary epitope tag, the trimeric hemagglutinin epitope HA 3 , fused to the alternate C terminus of KH. These images confirmed localization at the base of the flagellum ( Fig.  2A), where the tagged protein is immediately adjacent to DAPI fluorescence signal that is associated with kinetoplast DNA, the highly catenated mitochondrial DNA that is physically attached to the basal body (29), the subpellicular microtubules (Fig. 2B), and the mitotic spindle (Fig. 2C) for both Ty1::TbKH and TbKH::HA 3 (compare Figs. 1 and 2). Whole parasites that are expressing TbKH::HA 3 do, however, also exhibit punctate staining in the interior of the cell in addition to staining on the subpellicular microtubules (Fig. 2B) or the spindle (Fig. 2C). The absence of cross-reacting bands on Western blots of lysates from wild type BF parasites (see Fig. 6G, lane 1) developed with anti-HA monoclonal antibody or immunofluorescence signal from wild type BF parasites stained with the same antibody (not shown) suggests that this punctate staining does not represent endogenous cross-reacting material. The additional puncta could represent a proportion of TbKH::HA 3 that is mistargeted, indicating that addition of HA 3 to the C terminus may represent a less optimal tag than addition of Ty1 to the N terminus.
Although spindle localization had not been detected for LmxKH in the original studies on L. mexicana (21), subsequent images of hydroxyurea synchronized promastigotes (Fig. 3) demonstrate that LmxKH is also associated with the mitotic spindle in that parasite and that this newly discovered localization is thus likely universal. Hydroxyurea synchronization was employed to increase the percentage of parasites captured in mitosis and thus facilitate the detection of mitotic spindles that are associated with LmxKH, but similar images were also observed with lower frequency using non-synchronized parasites (data not shown). L. mexicana promastigotes assumed an oval morphology following exposure to hydroxyurea rather than the elongated shape of untreated promastigotes, but LmxKH was nonetheless apparent on the mitotic spindle that separates dividing nuclei.
TbKHARON Is Essential for Viability of the BF Parasites-To determine whether TbKH is essential for BF parasites, we inhibited the expression of TbKH by RNAi. The transgenic BF/pHD1313 line (30), which expresses the tetracycline repressor, was transfected with the TbKH RNAi construct employing  and anti-␣-tubulin pAb (Tub, red) and demonstrates staining of Ty1::TbKH on the subpellicular microtubules, indicated by white arrowheads. C, detergent-extracted cytoskeletons from BF parasites expressing Ty1::TbKH were stained with BB2 mAb (TbKH, green) and with anti-␣-tubulin antibody (Tub, red). D, the Ty1::TbKH cell line was stained with BB2 mAb (TbKH, green). This image displays a parasite in which Ty1:TbKH staining is associated with both the subpellicular microtubules and the mitotic spindle that connects the two nuclei late during karyokinesis. E, another Ty1::TbKH-expressing parasite captured earlier during karyokinesis than the cell shown in D. In this image, the KMX-1 anti-␤-tubulin antibody (red) was employed, and it stained spindle microtubules as well as other microtubules (61) and displayed the rhomboid spindle that converges at two poles at opposite ends of dividing nuclei (62). All preparations were stained with DAPI that detects both nuclear and kinetoplast DNA (blue). White arrowheads in D and E point to the mitotic spindle.   3 were synchronized with hydroxyurea, released from the cell cycle block by washing out hydroxyurea, and cultured in hydroxyurea-free RPMI medium for 7 h. A-C show images of parasites that were stained with anti-␣-tubulin pAb (red) and anti-HA antibody pAb (green) and images with the red, green, and blue (DAPI-stained) images merged (merged). White arrowheads mark mitotic spindles that stain with both anti-␣-tubulin and anti-HA antibodies. the p3666 vector (22) that transcribes the hairpin loop RNA driven by the EP procyclin promoter fused to four tetracycline operators. Induction of expression of this hairpin loop RNA with doxycycline for 24 h reduced the level of TbKH mRNA by at least 6-fold (Fig. 4A, upper panel), as determined by densitometry, whereas T. brucei hexose transporter 1 (TbHT1) mRNA remained unchanged (Fig. 4A, bottom panel). A time course for expression of TbKH::HA 3 following induction of RNAi against TbKH mRNA (Fig. 4B) showed that the protein was almost completely eliminated by 48 h after induction. Upon induction of RNAi, parasite growth was severely compromised (Fig. 4C, open circles), resulting in death of BFs by 72 h. Whereas uninduced parasites exhibited normal morphology upon staining with anti-␣-tubulin antibody and DAPI (Fig. 4E), parasites where RNAi had been induced for 12 h (Fig. 4F), 24 h (Fig. 4G), or 48 h (Fig. 4H) continued to replicate nuclear and kinetoplast DNA, as determined by the multiple DAPI staining foci, but failed to undergo cell division, resulting in cells with multiple nuclei, kinetoplasts, and flagella. These parasites are similar to the "monster" cell phenotype originally described for RNAi directed against mRNAs encoding various flagellar axoneme proteins (31). Microscopic quantification of parasites with a normal complement of kinetoplasts and nuclei (1K1N) or with altered numbers of both genomes (e.g. 2K1N, 2K2N, etc.)  Fig. 6A) was blocked, resulting in BF parasites with TbCaCh in only a few (Fig. 6, B and C) of the multiple flagella, as indicated by white arrowheads that mark flagella that stain with anti-␣tubulin but not with antibody directed against the HA 3 epitope on TbCaCh::HA 3 . Isolation of sheared flagella followed by immunoblotting using antibody against the HA 3 tag also confirmed that TbCaCh was largely absent from the flagella under TbKH RNAi conditions (Fig. 6E, lane 1) but not when RNAi was directed against TbFLA1 mRNA (Fig. 6, D and E, lane 2), a different knockdown that also results in detachment of flagella from the cell body (24). Hence, mistargeting of TbCaCh is a specific consequence of down-regulating TbKH and not simply an outcome of flagellar detachment.
To examine whether TbKH may interact with flagellar membrane proteins that it targets to the flagellar membrane, we monitored possible co-immunoprecipitation of Ty1::TbKH and HA-tagged flagellar membrane proteins. In BFs expressing Ty1::TbKH, cells were cross-linked with formaldehyde and sonicated to disrupt cytoskeletons, followed by immunoprecipitation with BB2 monoclonal antibody directed against the Ty1 epitope. This procedure was carried out employing BF clones that also expressed TbCaCh::HA 3 . As negative controls, similar experiments were done in BF parasites expressing TbCaATPase::HA 3 , FS33::HA 3 , and FS133::HA 3 , proteins that do not depend on TbKH for trafficking to the flagellar membrane (data not shown). TbCaCh::HA 3 co-immunoprecipitated along with Ty1::TbKH (Fig. 6F, upper panel, lane 2), whereas none of the other tested flagellar membrane proteins did so (Fig. 6F, upper panel, lanes 1 and 3-5). Control immunoblots of input lysates before immunoprecipitation (Fig. 6G) demonstrated that these control lines did express the relevant tagged proteins, supporting the notion that the absence of signals in the blot in Fig. 6F was due to failure or any of these proteins to interact with TbKH and not simply to failure of the cell line to express the tagged flagellar membrane protein. Immunoblots of the immunoprecipitates were also probed with BB2 antibody (Fig. 6F, lower panel) to demonstrate that similar amounts of Ty1::KH had been captured in the immunoprecipitation step.
Loss of TbCaCh from flagella following induction of TbKH RNAi was due to failure to target this channel to flagellar membranes and not simply to degradation of the channel. Thus, immunoblots (Fig. 6H, TbKH RNAi ) of lysates from parasites that had been induced for RNAi against TbKH mRNA for 24 or 48 h showed that TbCaCh was still present at levels similar to that seen before induction of RNAi (0 h). Similar results were obtained for parasites where RNAi had been induced against TbFLA1 mRNA (Fig. 6H, TbFLA1 RNAi ).
Localization of TbCaATPase, a protein that traffics to puncta in both the flagellar membrane and the cell body (Fig. 7A), was also determined following induction of RNAi against TbKH mRNA. This flagellar membrane protein continued to traffic to the flagellar membrane when RNAi was induced against either TbKH (Fig. 7B) or TbFLA1 (Fig. 7C) mRNA. Immunoblots of isolated flagella probed with antibody against the HA 3 tag also demonstrated that TbCaATPase was still present in flagella following induction of RNAi against either TbKH or TbFLA1 mRNA (Fig. 7D, upper panel, lanes 1 and 2, respectively). This result demonstrates that induction of RNAi against TbKH mRNA does not cause global disintegration of the flagellar membrane and/or nonspecific removal of all flagellar membrane proteins but rather specifically impairs flagellar trafficking of TbCaCh.
TbKHARON Is a Functionally Important Component of the Mitotic Spindle in BF Parasites-To investigate the potential role of TbKH in the mitotic spindle of BF parasites, the mitotic spindle fate was followed by immunofluorescence microscopy, employing KMX-1 antibodies, following the induction of RNAi against TbKH mRNA for 12, 24, or 48 h (Fig. 8, A-D). Prior to induction of RNAi, 3 Ϯ 1% (n ϭ 2, 100 cells counted per sample) of BF parasites had observable spindles (Fig. 8E). Following induction of RNAi against TbKH, the percentage of BF parasites with spindles first increased to 17 Ϯ 2% at 12 h after induction, but at 24 and 48 h, no spindles were observed (Fig. 8, C and  D, and asterisks in E). This observation suggests that TbKH not only localizes to the mitotic spindles but is also necessary for their integrity and/or stability. Moreover, 24 h RNAi-induced parasites have multiple nuclei that appeared to be well separated (Fig. 8C), whereas parasites induced for 48 h had multiple clumped nuclei (Fig. 8D), suggesting that nuclear segregation may be impaired following sufficient reduction in the level of TbKH mRNA. In addition, synchronization of Ty1::TbKH parasites using hydroxyurea (33) resulted in an increase in the number of parasites showing specific Ty1::TbKH fluorescence signal on the mitotic spindle of dividing cells, from ϳ5% of total cells in asynchronous to ϳ20% in synchronous parasites, as determined by examining 100 cells by immunofluorescence microscopy. This observation further confirms the association of TbKH with the mitotic spindle.
TbKHARON Is a Cytoskeletal Protein Essential for Viability of the PF Parasites-To establish the subcellular localization of TbKH in PF parasites, a PF line was generated that carried a copy of TbKH ORF endogenously tagged on the N terminus with Ty1. TbKH localization in PFs was essentially the same as that observed in BFs. In isolated flagellar cytoskeletons (31), Ty1::TbKH was detected at the base of the flagella adjacent to the DAPI-stained kinetoplast DNA (Fig. 9A). In whole PF parasites, Ty1::TbKH was observed in the subpellicular microtubule cytoskeleton by overlapping fluorescence with the ␣-tubulin signal (Fig. 9B) and in the mitotic spindles of dividing PF (Fig. 9C, right panel) similar to the signal observed in parasites stained with KMX-1 antibody (Fig. 9C, left panel). In Fig. 9C, separate cells were stained with each primary antiserum, because the same secondary antiserum is employed to detect both primary monoclonal antibodies. Induction of RNAi resulted in loss of detectable TbKH mRNA within 48 h (Fig.  10A), and whereas parasite growth arrested by this time, PF cell death was not complete until 12.5 days (Fig. 10B), much longer than for BF parasites. Induction of RNAi also resulted in formation of polynucleated and polyflagellated PF parasites with disrupted attachment of the flagella to the cell body (Fig. 10, C, E, and F), unlike uninduced PFs (Fig. 10D). Hence, the phenotypes upon induction of RNAi against TbKH mRNA are similar in BF and PF parasites. This observation is notably distinct from the monster cell phenotype originally observed using RNAi directed against components of the flagellar axoneme (31), where the cytokinesis defect was only observed for BF but not PF trypanosomes. Of note, both BF and PF parasites undergoing RNAi against TbKH were still motile, as determined by phase contrast microscopy, even after these dramatic morphological changes.
Rescue of L. mexicana ⌬lmxkh[pGT1::GFP] Phenotype by TbKH-To determine whether TbKH could substitute in L. mexicana for LmxKH regarding trafficking of LmxGT1 to the flagellar membrane and support growth of intracellular amastigotes, the ⌬lmxkh[pGT1::GFP] knock-out line was complemented with the TbKH ORF expressed from an episome. Partial restoration of LmxGT1 flagellar membrane targeting (Fig. 11, A and B) and growth of intracellular amastigotes (Fig.  11C) was observed. These results indicate that TbKH can function, albeit suboptimally, in trafficking of flagellar membrane proteins in L. mexicana, despite the fact that sequence identity for the KH proteins is relatively low between the two species of parasite.

Discussion
Cilia and flagella are multifunctional organelles that are represented broadly in biology, from unicellular eukaryotes to differentiated mammalian cells. Over the past decade or so, the role of cilia and flagella as sensors of the extracellular environment has emerged, with a corresponding emphasis on the membrane proteins that mediate these sensory processes (34). In parallel, a burgeoning interest has developed in the mechanisms whereby membrane proteins are selectively targeted to cilia and flagella (35). Recent studies on African trypanosomes have uncovered novel unexpected functions for flagella in these parasites. Szempruch et al. (36) have reported that BF trypanosomes generate extracellular vesicles that originate from the flagellar membrane, contain flagellar proteins and virulence determinants such as serum resistance-associated protein, and deliver these vesicles to host cells including erythrocytes. This observation further underscores the importance of flagellar membrane proteins in the biology and virulence of African trypanosomes. Furthermore, flagellar membranes of PF trypanosomes can fuse with each other and transfer both membrane-bound and cytosolic proteins between the two flagellarattached parasites (37), suggesting that flagella may be organelles involved in cell-cell communication. In addition, flagellar adenylate cyclases are involved in the process of social motility (8 -10), a coordinated motility process that is important for migration of parasites within the tsetse fly and for successful colonization of this insect vector (38). Hence, understanding the function of parasite flagella and the processes for targeting integral membrane proteins to these organelles is central to the biology of these parasites and the diseases they cause. The current characterization of TbKH has resulted in a number of novel discoveries that extend beyond the initial discovery of KH in L. mexicana. First, KH is essential for viability of both BF and PF trypanosomes, establishing the critical role of this protein in a second kinetoplastid parasite, in this case in both mammalian and insect life cycle stages. Second, a novel putative Ca 2ϩ channel is dependent upon TbKH for flagellar targeting, demonstrating that this conserved trafficking machinery mediates flagellar targeting of at least two distinct membrane proteins in diverse parasites. Third, a new location for KH has emerged, namely its association with the mitotic spindle.
TbKH is involved in flagellar targeting of the putative Ca 2ϩ channel TbCaCh, and this function is likely mediated by TbKH located at the base of the flagellum. During biosynthesis, integral membrane proteins are first incorporated into the surface membrane at the flagellar pocket (39), from where they migrate to the plasma membrane surrounding the cell body or the flagellar membrane. The presence of KH in close apposition to the flagellar pocket membrane may thus mediate, by a still unknown mechanism, the entry of TbCaCh and LmxGT1 into the adjacent flagellar membrane. This function of TbKH in targeting flagellar membrane proteins is likely to be essential for viability of BF and PF parasites. Thus an early phenotype accompanying induction of RNAi against TbKH RNA is detachment of the flagellum from the cell body. Previous stud-  ies have also demonstrated that induction of RNAi against TbCaCh mRNA results in detachment of the flagella from the cell body and parasite death (5,40). This observation suggests that failure of TbKH RNAi knockdown parasites to target TbCaCh to its normal location in the component of the flagellar membrane that associates with the cell body would be sufficient to disrupt this junction between the flagellar and cell body membranes. Failure of BF parasites to divide, resulting in multinucleate, multiflagellated monster cells, was previously observed by Gull and colleagues (31) following induction of RNAi against mRNAs encoding proteins of the flagellar axoneme. These authors suggested that proper flagellar function is required for establishment of the cleavage furrow during mitosis, thus establishing an intimate link between flagella and cytokinesis. A number of other studies (41)(42)(43)(44) have also underscored a role for the flagellum in cytokinesis and parasite viability. Hence, it is not surprising that disruption of targeting for flagellar membrane proteins, such as TbCaCh and possibly others, following RNAi against TbKH mRNA would induce failure of cytokinesis and a lethal phenotype in BF parasites. It is, however, notable that the essentiality of flagellar function noted in the previous studies on other flagellar proteins was typically reserved to the BF stage, but induction of RNAi in the PF forms was not lethal. The role of TbKH is distinct in this regard, because RNAi induced against TbKH mRNA is lethal in both life cycle stages. If TbCaCh is a bona fide calcium channel, it may also play an important role in flagellar physiology that is dependent upon flagellar localization. One example of such an organelle-dependent function for Ca 2ϩ channels is provided by the cilia of mammalian kidney tubule cells, where several of these channels are thought to be involved in sensing fluid flow within this organ (45). We anticipate that some other flagellar membrane proteins will likely be dependent upon TbKH for targeting to this organelle. It should be possible to identify other potential cargo for TbKH by performing quantitative proteomics to detect proteins whose relative abundance in isolated flagella decreases after induction of RNAi directed against TbKH mRNA.
Although the specific functions of TbKH at the mitotic spindle and the subpellicular microtubules are not yet clear, this protein could also be required for critical processes at those loci. RNAi targeting of TbKH mRNA impairs mitotic spindle formation, a deficiency that would be expected to be lethal. However, the effect of TbKH RNAi on spindle integrity is only detected at 24 h, after the detachment of flagella and failure of cytokinesis, which are already detected at 12 h. Hence, loss of spindles is probably not the primary lethal defect of knocking down TbKH mRNA. Similarly, association of TbKH with the subpellicular microtubules might mediate critical functions for this extensive cytoskeletal network. In principle, lethality could be due to disrupted KHARON function at the base of the flagellum, on the mitotic spindle, or in the subpellicular cytoskeleton, or loss of KHARON at all three loci may contribute to the lethal phenotype, potentially with different kinetics. Hence one challenge will be to dissect the potentially distinct functions of TbKH at each of its subcellular loci. Experiments employing biotin proximity labeling (46) 6 suggest that both TbKH and LmxKH may associate with multiple additional subunits to form KHARON Complexes, and this possibility is being further investigated. These potential separate complexes may share KH and other subunits, but they may also incorporate other distinct subunits that are unique to the subcellular localization and specific function of each complex. If this model of KH structure and function is correct, then targeting complex-specific subunits using RNAi may allow dissection of the distinct functions of each complex and insight into the role of KH and its associated subunits at each of its three subcellular locations.

Experimental Procedures
Growth and Transfection of T. brucei Cell Lines-BF Lister 427 and PF TREU927 T. brucei cell lines were grown as described previously (30). For T7 RNA polymerase-indepen-dent driven expression (22), BF or PF parasites transfected with the pHD1313 plasmid expressing the tetracycline repressor, kindly provided by Dr. Christine Clayton (47), were generated and grown in 2.5 g ml Ϫ1 phleomycin. Subsequently, 5-10 g of linearized plasmid DNA was used to transfect mid-log phase parasites as described (48). Transfectants were isolated by limiting dilution according to published protocols (49,50).
Hydroxyurea Synchronization-Synchronization of BF Ty1:: TbKH cell line with hydroxyurea was performed as described by Forsythe et al. (33). Briefly, parasites were incubated in HMI-9 (51) culture medium containing 10 g ml Ϫ1 of hydroxyurea for 6 h at 37°C in 5% CO 2 . Later, parasites were pelleted by centrifugation at 1600 ϫ g for 10 min at room temperature to remove hydroxyurea and resuspended in fresh HMI-9 culture medium, and centrifugation was repeated. Finally, parasites were resuspended in 10 ml of fresh HMI-9 and incubated for 3 h at 37°C in 5% CO 2. Then parasites were processed for fluorescence microscopy as indicated.
RNA Isolation and Northern Blotting-Total RNA from T. brucei parasites was isolated using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Northern blotting analysis was performed as described elsewhere (52). Non-radioactive detection employing DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) was used according to the manufacturer's instructions, and the Image Quant LAS 400 (GE Healthcare) was employed for image acquisition. ImageJ software (National Institutes of Health) was used for densitometry analysis and Adobe Photoshop CS3 and Adobe Illustrator CS3 to create figures.
Primary Amino Acid Sequence Analysis-For DNA sequence analysis of TbKH and amino acid sequence alignment with the LmxKH ORF, MacVector software (Intelligenetics) was employed. Sequence similarity was analyzed using the BLAST search and GenBank TM or TritrypDB.
Inhibition of Gene Expression by RNAi-To inhibit the expression of TbKH in both BF and PF parasites, a construct was generated based on the p3666 vector harboring the blasticidin (BSD) resistance marker (22) that integrates into the rRNA gene repeat locus and generates a hairpin RNA derived from the TbKH ORF. A 521-bp gene fragment corresponding to nucleotides 340 -860 of the TbKH ORF (Tb927.10.8940) was employed, identified as an optimal fragment for RNAi using RNAit software. The TbKH RNAi construct was linearized and transfected into BF or PF/pHD1313 cell lines as described (30) to generate BF or PF/TbKH RNAi clonal lines. RNAi clones were selected by resistance to 2.5 g ml Ϫ1 phleomycin and 5 g ml Ϫ1 (10 g ml Ϫ1 for PF) blasticidin, and expression of hairpin loop RNAi was induced by addition of 1 g ml Ϫ1 doxycycline. Similarly, a TbFLA1 RNAi hairpin loop construct was generated using a 539-bp gene fragment corresponding to nucleotides 692-1230 bp of the TbFLA1 ORF (Tb927.8.4010), identified by RNAit software, and the p3666 plasmid as described above. A BF/TbFLA1 RNAi clonal line was produced as described for the TbKH RNAi clones. RNAi was induced by adding 1 g ml Ϫ1 doxycycline to the medium.
Endogenous Epitope Tagging-For endogenous tagging of TbKH at the N terminus with the Ty1 epitope tag (53), a DNA segment representing sequence 500 bp upstream of the start  codon of the TbKH ORF was ligated to the BSD/BB2 module (54) followed by another segment representing the first 170 bp corresponding to the N-terminal open reading frame. For endogenous tagging of TbKH at the C terminus with a trimeric hemagglutinin (HA 3 ) tag, 500 bp corresponding to C-terminal coding region of the TbKH ORF was ligated to the pMOTag2H plasmid (55), followed by a 500-bp segment downstream of the stop codon. Constructs for expression of flagellar membrane proteins FS33, FS60, FS133, and FS179 tagged with a HA 3 epitope at the C terminus were kindly provided by Dr. Kent Hill (5). Parasites were transfected with 5 g of linear construct as described (48) and selected using 5 g ml Ϫ1 blasticidin or 0.1 g ml Ϫ1 puromycin and cloned by limiting dilution.
Immunofluorescence Microscopy-For immunofluorescence microscopy, parasites (5 ϫ 10 6 ) were centrifuged at 700 ϫ g for 4 min and washed twice at room temperature with PBS, pH 7.2, containing 10 mM glucose. The cell pellet was resuspended in 4% paraformaldehyde in PBS, pH 7.2, and incubated for 15 min at room temperature; cells were centrifuged as described above, washed once with 10 mM glycine in PBS and once with PBS, resuspended in 100 l PBS, spotted onto poly-L-lysine-coated coverslips, blocked with 2% goat serum, 0.01% sodium azide, 0.01% saponin in PBS (blocking solution) for 1 h at room temperature, rinsed three times with PBS, and incubated with primary antibodies for 1 h at room temperature. The following primary antibodies were employed: 1:500 dilution anti-HA.11 clone 16B12 mAb (BioLegend, catalog no. MMS-101R, lot no. D13FF01558), 1:1000 dilution anti-HA polyclonal antibody (pAb) (Sigma-Aldrich, catalog no. H6908, batch no. 083M4837V), 1:500 dilution anti-Ty1 (BB2) mAb (53), 1:1000 dilution anti-␣-tubulin mAb (Sigma-Aldrich, catalog no. T5168, batch no. 104M4790V), 1:1000 dilution anti-␣-tubulin pAb (Sigma-Aldrich, catalog no. SAB3501071, lot no. 75971302), 1:1000 anti-␤-tubulin clone KMX-1 mAb (Millipore, catalog no. MAB3408, lot no. 2646950), and 1:1000 anti-GFP pAb (Molecular Probes, catalog no. A11122, lot no. 847818). Subsequently, the cells were rinsed as before and incubated with a 1:1000 dilution of secondary antibodies coupled to Alexa Fluor dyes (Molecular Probes) as follows: Alexa Fluor 488 goat anti-mouse IgG (HϩL) (catalog no. A11001, lot no. 1572559), Alexa Fluor 595 goat anti-mouse IgG (HϩL) (catalog no. A11005, lot no. 1524907), Alexa Fluor 594 goat antirabbit IgG (HϩL) (catalog no. A11012, lot no. 84B2-1), and Alexa Fluor 488 goat anti-rabbit IgG (HϩL) (catalog no. A11008, lot no. 84B1-1), as indicated, in blocking solution for 1 h at room temperature in the dark. Coverslips were rinsed three times with PBS and mounted onto slides using DAPI Fluoromount-G (SouthernBiotech). Fluorescence images were acquired using a wide field deconvolution system (Applied Precision Instruments, Inc.) consisting of an inverted Nikon TE 200 Eclipse microscope, a Kodak CH350 CCD camera and the Deltavision operating system. Images were acquired using a 60ϫ objective in a 1024 ϫ 1024 format and deconvolved with nine iterations using SoftWoRx software. Adobe Photoshop CS3 and Adobe Illustrator CS3 (Adobe Systems Inc.) were used to create image compositions. Images that included differential interference contrast (DIC) were acquired on a high resolution wide field Core DV system (Applied Precision TM ). This system is an Olympus IX71 inverted microscope with a proprietary XYZ stage enclosed in a controlled environment chamber; DIC transmitted light and a solid state module for fluorescence. The camera is a Nikon Coolsnap ES2 HQ. Each image was acquired as Z stacks in a 1024 ϫ 1024 format with a 60ϫ 1.42 NA PlanApo lens in three colors: Alexa Fluor 488, Alexa Fluor 594, and DAPI. The pixel size in XYZ was 0.107 ϫ 0.107 ϫ 0.2 microns. The images were deconvolved with the appropriate optical transfer function using an iterative algorithm of 10 iterations. The histogram was optimized for the most positive image and applied to all the other images for consistency before saving the images as 24-bit merged TIFF. A reference DIC image was acquired from the middle of the Z stack.
Flagellar Protein Purification, Formaldehyde Cross-linking, Co-immunoprecipitation, and Western Blotting Assay-Flagellar purification was performed as described (26) with slight changes. Briefly, BF/TbKH RNAi clones or BF/TbFLA1 RNAi clones were grown in 1 g ml Ϫ1 doxycycline for 48 or 72 h, respectively, and pelleted at 420 ϫ g for 20 min. Cell pellets were washed with buffer A (25 mM Na ϩ -Tricine, pH 7, 1% BSA, 0.1 mM CaCl 2 , 0.2 mM EDTA, 5 mM MgCl 2 , and 12 mM ␤-mercaptoethanol) containing 0.32 M sucrose and centrifuged at 420 ϫ g for 10 min. Cell pellets were gently resuspended at 3 ϫ 10 8 parasites ml Ϫ1 in buffer A plus 0.3 M sucrose, transferred into Eppendorf tubes and vortexed for 5 min or until microscopic verification of flagellum detachment, followed by centrifugation at 420 ϫ g for 10 min. Supernatants were recovered and centrifuged at 1600 ϫ g for 10 min at 4°C. Pellets containing the isolated flagella were resuspended in 25 l of PBS and used for Western blotting assays. Also, flagellar cytoskeleton purification from wild type BF and PF clones was performed by isolation of cytoskeletons using Nonidet P-40 extraction, followed by 1 M NaCl treatment according to Broadhead et al. (31) to dissociate the subpellicular cytoskeleton. The remaining flagellar cytoskeletons were processed for immunofluorescence microscopy as indicated.
For formaldehyde cross-linking and co-immunoprecipitation, cell lines were prepared that expressed TbKH endogenously tagged with the Ty1 epitope at the N terminus (Ty1::TbKH) and also co-expressed a flagellar membrane protein of interest endogenously tagged at its C terminus with an HA 3 epitope (FS33::HA 3 , FS60::HA 3 , FA133:HA 3 , and FS179::HA 3 ) (5). Parasites were washed once with PBS and pelleted at 1000 ϫ g for 10 min, and cell pellets were resuspended in 9.37 ml of PBS plus 0.63 ml of 16% formaldehyde-EM grade (Polysciences, Inc.) and incubated at 37°C for 10 min. Subsequently, 1 ml of 2.5 M glycine in PBS was added to the crosslinked samples and incubated at 37°C for 5 min to terminate cross-linking, and cross-linked parasites were pelleted at 1000 ϫ g for 10 min and washed twice with PBS. Finally, cell pellets were resuspended in 500 l of Tween 20/PBS buffer (0.2% Tween 20 in 1ϫ PBS, pH 7.2) containing 1ϫ of Halt TM protease inhibitor single-use mixture (Thermo Fisher). Crosslinked samples were sonicated on ice using two 15-s pulses at maximum output (Branson Sonifer 150). After disruption, samples were centrifuged at 16,000 ϫ g for 20 min at 4°C. Supernatants were transferred to new Eppendorf tubes and incubated overnight with 1:50 dilution of BB2 mAb at 4°C on a rocker. Later, 30 l of PureProteome TM protein A/G mix magnetic beads (Millipore) were incubated for 30 min at room temperature on a rocker to capture Ty1::TbKH proteins that had been cross-linked to other partner proteins. Two final washes of the magnetic beads for 30 s at room temperature with radioimmune precipitation assay buffer were performed to increase stringency and reduce background. Coimmunoprecipitated proteins were eluted, and cross-linking was reversed by adding 30 l of 1ϫ NUPAGE-LDS sample buffer (Life Technologies) containing 10 mM DTT followed by heating at 70°C for 30 min. Eluted protein samples were used for Western blotting analysis.
Macrophage infections were performed by infecting differentiated macrophages from the human acute leukemia monocyte cell line (THP-1) in triplicate samples, as described (57). Briefly, 3 ϫ 10 5 differentiated THP-1 cells were infected with stationary phase L. mexicana (10:1 promastigotes:macrophages ratio) for 4 h. Infected macrophages were then washed exhaustively to remove extracellular promastigotes. Cultures were incubated at 37°C in a 5% CO 2 environment, and infected macrophages were stained using the HEMA 3 STAT PACK staining kit as described by the manufacture (Fisher Scientific) at 1 and 7 days after infection. Stained infected macrophages were examined using a Nikon Eclipse 50i microscope equipped with a 100sϫ 1.25 NA oil objective (Nikon Instruments). The number of parasites per 100 macrophages was determined by counting 100 cells in each of the triplicate experiments.
Electron Microscopy-BF/TbKH RNAi clone was grown in the presence of 1 g ml Ϫ1 doxycycline to induce RNAi against TbKH mRNA, and parasite samples were harvested at 0, 18, 24, and 48 h postinduction. Parasites were fixed in 2.5% glutaraldehyde by addition of 1/10 volume of 25% glutaraldehyde directly to the culture and incubated at room temperature for 5 min. Fixed cells were subsequently collected and centrifuged for 10 min at 800 ϫ g. The cells were resuspended in 1 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 50 mM sucrose and pelleted for 10 min at 800 ϫ g. The cells were then resuspended in 1 ml of buffered fixative (2.5% glutaraldehyde, 2% formaldehyde, and 50 mM sucrose in 0.1 M phosphate buffer, pH 7.4), pelleted for 1 min at 21,000 ϫ g in a microcentrifuge, and stored at 4°C.
Fixed samples were prepared for transmission electron microscopy and serial block face scanning electron microscopy. Transmission electron microscopy samples were prepared and imaged as previously described (57). Serial block face scanning electron microscopy samples were washed in 0.1 M PIPES buffer and then embedded in a mix of 4% low melting point agarose and 4% gelatin from porcine skin. Excess agarose/gelatin was trimmed away, and samples were prepared according to Wilke et al. (58) with minor modifications: samples were incubated at 4°C for 1 h in a 1:1 mix of 3% potassium ferricyanide in 0.2 M PIPES buffer and 4% aqueous osmium tetroxide. Samples were washed with double distilled (dd) H 2 O between each of the subsequent heavy metal staining steps, which consisted of 1% thiocarbohydrazide in ddH 2 O for 20 min at room temperature, 2% aqueous osmium tetroxide for 30 min at 4°C, 1% uranyl acetate overnight at 4°C, and lead aspartate (0.02 M lead nitrate and 0.03 M aspartic acid) for 30 min at room temperature. Final washes in ddH 2 O were performed prior to dehydration through a graded series of ethanol and acetone incubations. The samples were then infiltrated with and embedded in Durcupan ACM epoxy resin. Serial block face images were captured with a Zeiss Merlin Compact field emission gun scanning electron microscope with variable pressure mode and a Gatan 3View system using digital micrograph with an accelerating voltage of 3 kV, variable pressure of 45 Pa, aperture size of 30 m, 4.7-nm lateral resolution, and 50-nm axial resolution and processed using ImageJ (59).
Synchronization of L. mexicana with Hydroxyurea-Promastigotes of L. mexicana were synchronized with hydroxyurea as described by da Silva et al. (60). Briefly, mid-log phase promastigotes were incubated in RPMI culture medium containing 5 mM hydroxyurea for 14 h at 26°C. Later, parasites were pelleted by centrifugation at 1600 ϫ g for 10 min at room temperature to remove hydroxyurea, resuspended in fresh RPMI, and incubated for 7 h at 26°C. Subsequently, parasite samples were processed for fluorescence microscopy as indicated.