A CLK1-KKT2 Signaling Pathway Regulating Kinetochore Assembly in Trypanosoma brucei

ABSTRACT During mitosis, eukaryotic cells must duplicate and separate their chromosomes in a precise and timely manner. The apparatus responsible for this is the kinetochore, which is a large protein structure that links chromosomal DNA and spindle microtubules to facilitate chromosome alignment and segregation. The proteins that comprise the kinetochore in the protozoan parasite Trypanosoma brucei are divergent from yeast and mammals and comprise an inner kinetochore complex composed of 24 distinct proteins (KKT1 to KKT23, KKT25) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2, and KKT3. We recently reported the identification of a specific trypanocidal inhibitor of T. brucei CLK1, an amidobenzimidazole, AB1. We now show that chemical inhibition of CLK1 with AB1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. Here, we show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 by CLK1 to be essential for KKT2 function and for kinetochore assembly. Additionally, KKT2 protein kinase activity is required for parasite proliferation but not for assembly of the inner kinetochore complex. We also show that chemical inhibition of the aurora kinase AUK1 does not affect CLK1 phosphorylation of KKT2, indicating that AUK1 and CLK1 are in separate regulatory pathways. We propose that CLK1 is part of a divergent signaling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2.

IMPORTANCE In eukaryotic cells, kinetochores are large protein complexes that link chromosomes to dynamic microtubule tips, ensuring proper segregation and genomic stability during cell division. Several proteins tightly coordinate kinetochore functions, including the protein kinase aurora kinase B. The kinetochore has diverse evolutionary roots. For example, trypanosomatids, single-cell parasitic protozoa that cause several neglected tropical diseases, possess a unique repertoire of kinetochore components whose regulation during the cell cycle remains unclear. Here, we shed light on trypanosomatid kinetochore biology by showing that the protein kinase CLK1 coordinates the assembly of the inner kinetochore by phosphorylating one of its components, KKT2, allowing the timely spatial recruitment of the rest of the kinetochore proteins and posterior attachment to microtubules in a process that is aurora kinase B independent.
KEYWORDS Trypanosoma, cell signaling, kinetochore, mitosis, protein kinases A t the onset of cell division, the accurate distribution of genomic material is crucial for cell survival and development (1). Central to this process are the kinetochores, a centromere macromolecular protein complex that drives chromosome segregation in eukaryotes by connecting chromosomes to microtubules (2). The kinetochore is a large, highly dynamic machine assembled from multiple pathways that are temporally controlled (3). Kinetochores gather on opposite sides of a centromere region of each

RESULTS
CLK1 inhibitor AB1 disrupts kinetochore dynamics in bloodstream-form T. brucei. Given the importance of kinetochore movement during metaphase in eukaryotes (23), we assessed the impact of T. brucei CLK1 (TbCLK1) activity on kinetochore dynamics using AB1 as a chemical tool. The expression and localization of kinetochore proteins, labeled with mNeonGreen (mNG), were assessed by confocal microscopy in the bloodstream form of the parasite (Fig. 1A). Similar to previous observations in procyclic form cells (15,17), we observed different kinetochore timings and patterns of expression throughout the cell cycle. By using the kinetoplast (K) and nucleus (N) configuration to define each cell cycle stage (24), we observed that KKT2 and KKT3 are constitutively expressed until anaphase. KKT1 and KKIP1 gradually load from S phase onwards until the end of mitosis, while KKT4 and KKT5 expression is restricted to metaphase. Furthermore, KKT9 and KKIP7 expression diminish during anaphase, suggesting both proteins are acting as scaffolds for the recruitment of multiple other components. Treatment with 5Â the 50% effective concentration (EC 50 ) of AB1 for 6 h caused dispersal, to various degrees, for KKT1, KKT2, KKT5, KKT9, KKT13, KKT14, and KKT20 from the defined foci of the kinetochore within the nucleus, while KKT3, KKT7, KKT11, KKIP1, and KKIP7 remained in distinct foci ( Fig. 1B and C; see also Fig. S1A in the supplemental material). Automated focus detection using subpixel precise single-particle localization combined with image segmentation (25) and intensity quantification (26) determined that there was a significant reduction in focus intensity for KKT1, KKT2, KKT4, KKT5, and KKT9 but not KKT3 ( Fig. 1D and Fig. S1B and C). No degradation of these proteins was observed after treatment (Fig. S1D). These results suggest that although KKT2 and KKT3 are centromere-anchored proteins (15), they respond differently to CLK1 inhibition, and that TbCLK1 is a critical regulator of inner kinetochore component dynamics.
CLK1 phosphorylates KKT2 at position S508. KKT2 and KKT3 protein kinases are likely components of the trypanosome inner kinetochore with functional equivalence to the constitutive centromere-associated network (CCAN), a canonical component of the eukaryotic inner kinetochore (27). Defective KKT2 clustering was also observed after CLK1 RNA interference (RNAi) (22). It has been reported that phosphorylation of kinetochore proteins has critical roles in kinetochore organization and interaction during mitosis in mammals and yeast (28). Indeed, cell cycle-regulated changes in the phosphorylation of T. brucei kinetochore components have been reported recently, where the regulation is coordinated with phosphorylation of essential protein kinases, including CLK1 (19).
We speculated that KKT2 provides a platform on which the kinetochore multiprotein complex assembles and that phosphorylation orchestrates this process. To address whether KKT2 is a CLK1 substrate, we first analyzed mobility shifts of phosphorylated forms of KKT2 and KKT3 using Phos-tag gel electrophoresis (29). A low-mobility, nonphosphorylated form of KKT2 was detected after treatment with AB1 or after CLK1 depletion by RNAi, while KKT3 remained unaffected (Fig. S2). Six phosphorylation sites have been identified in KKT2 (S 5 , S 8 , S 25 , S 507 , S 508 , and S 828 ) (30), and we tested if these are important for KKT2 function by generating a KKT2 RNAi line (Fig. S3) with a recoded hemagglutinin (HA)-tagged version of KKT2 integrated into the tubulin locus. This constitutively expressed KKT2 (KKT2 R ) is not susceptible to RNAi-mediated degradation, and KKT2 R complements the loss of function of KKT2 48 h after RNAi induction ( Fig. 2A). Replacement of Ser for Ala in KKT2 at positions S 5 , S 8 , S 25 , and S 828 resulted in FIG 1 Legend (Continued) Fixed parasites in metaphase or anaphase were considered for analysis of kinetochore pattern and intensity. (B) Localization of inner (top) and outer (lower) kinetochore core components after CLK1 inhibition by AB1. Parasites were incubated or not for 6 h with 5Â EC 50 AB1. Representative fluorescence microscopy micrographs show bloodstream-form parasites endogenously expressing N-terminal mNeonGreen (mNG)-tagged kinetochore proteins. Cells with 2K1N and 2K2N kinetoplast/nucleus configuration are shown. Cells were counterstained with DAPI to visualize DNA (cyan). The right panel shows the corresponding Nomarsky (differential interference contrast [DIC]) images. Scale bar, 2 mm. (C) Percentage of cells in metaphase (1N2K) and anaphase (2N2K) showing a defined kinetochore localization before and after AB1 treatment as described for panel A (n . 100 cells in each stage). (D) Intensity of KKT foci detected before (DMSO) and after AB1 treatment. The data represent 75% of total focus intensity (n = 80 kinetochores under each condition). Error bars, standard errors of the means (SEM); **, P , 0.01, ***, P , 0.001; ns, not significant. (Mann-Whitney U test). In vitro growth profile of KKT2 RNAi, KKT2 R , and KKT2 R phosphomutants and active-site mutant. Bars show cumulative fold over uninduced control counts over time following tetracycline induction of cell lines in culture. Error bars represent means 6 SEM from three replicates; P values were calculated using a two-tailed Student's t test: **, P , 0.01; ***, P , 0.001. (Lower) Western blot of HA-KKT2 mutants. The expression of KKT2 phosphomutants mutants was detected using an anti-HA antibody. EF-1 alpha protein expression was used as the loading control. (C) Cell cycle profile of KKT2 RNAi, KKT2 R , and KKT2 R phosphomutants. Bars showing G 2 /M ratio over the uninduced control following tetracycline induction of cell lines in culture. Error bars represent SEM from 3 replicates. P values were calculated using a two-tailed Student's t test: ***, P , 0.001. (D) Recombinant CLK1 (rCLK1) phosphorylates recombinant KKT2 in vitro. Recombinant fragment of KKT2 including S 507-S508 (KKT2 486-536 ) was used as the substrate for rCLK1 by ADP-Glo kinase assay. The same fragment but including an S 507A-S508A mutation (blue) was used as a control. Phosphorylation of maltose binding protein (MBP) and rCLK1 autophosphorylation (no substrate) was included as a control. Error bars, SEM (n = 3); ***, P , 0.001 (two-tailed Student's t test). Conservation of amino (Continued on next page) CLK1 Regulates the Trypanosome Kinetochore ® complementation of KKT2 function when expressed in the RNAi line. In contrast, dual replacement of the KKT2 phosphorylation sites S 507 and S 508 with Ala (KKT2 S507A-S508A ) failed to complement depletion of the wild-type KKT2 with respect to parasite growth ( Fig. 2B) or cell cycle progression after 48 h of induction (Fig. 2C). The efficacy of RNAi knockdown of the endogenous KKT2 alleles was retained in these derivative cell lines (Fig. S3A, B, and C), demonstrating that the complementation effects were imparted by the recoded alleles. KKT2 S507A-S508A had good expression levels in the cell (Fig. 2B, lower) but was mislocalized (Fig. S3D), providing a possible explanation for the phenotype observed. These defects phenocopy the effect of AB1 and show the importance of the two phosphorylation sites for the function of KKT2. To assess whether protein kinase activity is essential for KKT2 function, an active-site mutant was generated in KKT2 R (KKT2 K113A ). A significant loss of function was observed after 48 h of induction, indicating protein kinase activity is essential for KKT2 function but not for regulating cell cycle progression ( Fig. 2B and C).
To address whether CLK1 phosphorylates KKT2 directly at S 507-508 residues, we expressed a recombinant peptide (amino acids [aa] 486 to 536) of KKT2, including mutations of S507 and S508 residues. We demonstrated that recombinant CLK1 could phosphorylate recombinant KKT2 in vitro at positions S507 and S508 (Fig. 2D). Given the conservation of KKT2 S 508 in kinetoplastids, we then raised a phosphospecific antibody against KKT2 S508 to monitor KKT2 phosphorylation through the T. brucei cell cycle and after treatment with AB1. The antibody specifically recognizes phosphorylation of KKT2 S508 , as phosphorylated KKT2 S508 was depleted following KKT2 or CLK1 RNAi (Fig. 2E, upper) or after treatment with AB1 in a cell line where KKT2 was endogenously tagged with Ty and mNG (Fig. 2E, lower; both endogenous KKT2 and Ty-mNG KKT2 are detected). In addition, the KKT2 phosphoantibody detects phosphorylated KKT2 in all the recoded mutants except the KKT2 S507A-S508A double mutant (Fig. S3E). KKT2 S508 phosphorylation was found to increase in S-phase after hydroxyurea synchronization and progressively decreased toward G 1 phase (Fig. 2F), in correlation with the recent demonstration of dynamic KKT2 S508 phosphorylation during the cell cycle (31). Together, these data show that KKT2 phosphorylation is downstream of CLK1 in a kinetochore-specific signaling cascade and occurs during early metaphase.
We next assessed whether KKT2 phosphorylation is required for recruitment of proteins to the trypanosome kinetochore. KKT1 and KKT9 recruitment were impaired in the KKT2 R S 507A-S508A ::KKT2-induced cell line ( Fig. 3A to C) but not the KKT2 R K 113Ainduced line (Fig. S4A), underlining the importance of KKT2 phosphorylation by CLK1 for kinetochore assembly. Individual expression of phosphomimetics S 507E and S 508E impaired KKT1 and KKT9 recruitment but also affected the timing of events during mitosis, with a notable defect in nuclear abscission (Fig. S4B).
CLK1 and AUK1 are not part of the same signaling pathway. Faithful chromosome segregation relies on the interaction between chromosomes and dynamic spindle microtubules (32). Furthermore, spindle elongation is important for correct segregation of chromosomes during anaphase (33). To further examine if CLK1 inhibition impairs microtubule spindle dynamics, we observed the expression of the mitotic spindle by staining the parasites with KMX-1 antibody and analyzing the microtubule-associated protein 103 kDa (MAP103) (Fig. S5) (34). This showed that treatment with AB1 does not affect microtubule spindle formation (Fig. 4A). Considering that CLK1 inhibition during metaphase results in an arrest in late anaphase (19,22), it is likely that the function of CLK1 during cytokinesis is related to either the control of kinetochore-spindle  microtubule attachment errors or its interactions with the chromosomal passenger complex (CPC). Of note, it has been reported that T. brucei aurora kinase B has an important role during metaphase-anaphase transition and the initiation of cytokinesis via regulation of the CPC (35)(36)(37) and nucleolar and other spindle-associated proteins (NuSAPs) (38).
In mammals, kinetochore assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein by aurora kinase B, generating kinetochores capable of binding microtubules and promoting the interaction between outer and inner kinetochore proteins (39). In T. brucei, aurora kinase B (TbAUK1) plays a crucial role in spindle assembly, chromosome segregation, and cytokinesis initiation (37). Therefore, we asked  if CLK1 and AUK1 are part of the same signaling pathway. We showed that treatment with AB1 does not affect spindle formation (Fig. 4A), in contrast to the inhibition of AUK1 (40). AUK1 is a key component of the trypanosome CPC (41). To understand if CPC dynamics are impaired by CLK1 inhibition, we monitored the localization of CPC1 throughout the cell cycle before, after AB1 treatment, and following AUK1 inhibition by Hesperadin (42). After treatment with AB1, CPC1 showed a dispersed nuclear pattern that progressively disappeared after abscission of the nucleus (Fig. 4B, middle). This was different from AUK1 inhibition by Hesperadin, which prevented translocalization of the CPC from the spindle midzone, impairing initiation of cytokinesis (Fig. 4B,  right). Finally, we confirmed that AUK1 is not involved in kinetochore assembly, since neither KKT2 S508 phosphorylation nor KKT2 localization was affected by AUK1 inhibition by Hesperadin (Fig. 4C). In addition, a cohort of divergent spindle-associated proteins has been described that is required for correct chromosome segregation in T. brucei (18). Therefore, we analyzed the subcellular localizations of NuSAP1 and NuSAP2 during the cell cycle after CLK1 inhibition. NuSAP2 expression in the central portion of the spindle after metaphase release was compromised by CLK1 inhibition, while NuSAP1 remained unaffected (Fig. 4D). NuSAP2 is a divergent ASE1/PRC1/MAP65 homolog, a family of proteins that localizes to kinetochore fibers during mitosis, playing an essential role in promoting the G 2 /M transition (43). Considering that NuSAP2 and KKT2 colocalize during interphase and metaphase (18), it is likely that KKT2 regulation by CLK1 influences posterior spindle stability and cytokinesis.

DISCUSSION
The inner kinetochore complex of T. brucei is unusual in that none of the 24 identified KKT proteins have any sequence identity with CENP proteins of the constitutive centromere-associated network (CCAN) in yeast or vertebrates (15,16). Four of the KKTs contain protein kinase domains, and here we provide the first evidence of a unique protein kinase signaling pathway that regulates inner kinetochore function in bloodstream-form T. brucei. KKT2 is a multidomain protein, constitutively associated with the centromere during the cell cycle, which contains an N-terminal protein kinase domain, a central domain with a unique zinc finger domain, and a C-terminal divergent polo box domain (PDB) (15). The PBD and the central domain are sufficient for kinetochore localization (44), but it is not clear if KKT2 binds directly to DNA or forms a protein complex at nucleosomes with other KKT proteins. In this study, we show that while KKT2 protein kinase activity is required for growth and replication of bloodstream-form trypanosomes (Fig. 2B), the localization of KKT1 and KKT9 to the kinetochore remained unaffected by the loss of KKT2 protein kinase activity (see Fig. S4A in the supplemental material). These data suggest that KKT2 protein kinase activity is required for a function of the kinetochore that is independent from assembly of its inner complex.   We also show that phosphorylation of the kinetochore, and specifically KKT2, is crucial for kinetochore assembly in bloodstream-form T. brucei. Depletion of the kinetochore protein kinase CLK1 (KKT10) by RNAi, or inhibition with the CLK1 inhibitor AB1, is lethal due to disruption of kinetochore assembly (22). Multiple phosphorylation sites have been identified in KKT2, and a number are cell cycle regulated, including S508 (31), suggesting a regulatory role. While we cannot discount phosphorylation of S507 or other sites as a requirement for kinetochore assembly, we only identified S508 to be essential, indicating that the other known phosphorylation sites cannot compensate for loss of phosphorylation on S508. S508 is located between the Cys-rich central domain and the C-terminal domain, and phosphorylation might contribute to association of KKT2 with chromatin via its DNA binding domain. Indeed, the finding that KKT2 S507A-S508A is mislocalized supports this hypothesis, and the fact that the mutant protein can localize to the kinetochore in the presence of wild-type KKT2 suggests that KKT2 is an oligomer and that the WT protein can recruit and retain the mutant protein on the kinetochore. As KKT2 protein kinase activity is not required for assembly of the kinetochore, phosphorylation of S508 seems less likely to regulate the kinase activity of KKT2.
By using chemical and molecular approaches, we demonstrate that phosphorylation of KKT2 in the bloodstream form during metaphase allows the spatial recruitment of inner kinetochore components. We provide evidence that KKT2 is phosphorylated by CLK1, but we cannot formally rule out the possibility of an intermediate kinase being involved. Recently, a study showed that in the procyclic form, CLK1 kinase activity is essential for metaphase-to-anaphase transition, although its expression was dispensable for the recruitment of kinetochore components (19). This difference may be due to cell cycle regulators having different functions in the two developmental stages of T. brucei (45,46) or because there can be protein turnover differences between life cycle stages (47). Indeed, CLK1 protein expression relative to CLK2 appears higher in the bloodstream trypanosome (22) than the procyclic form (19).
In T. brucei bloodstream forms, we show that KKT2 is a substrate for CLK1. In mammals, CLK protein kinases are found in the cytoplasm and in the nucleus, where they regulate alternative splicing through phosphorylation of serine/arginine-rich domains on splicing factors (48), as occurs with human CLK1 in association with the serine-arginine protein kinase 1 (SRPK1) (49). Human CLKs also activate the abscission checkpoint in human cells by phosphorylating aurora kinase B, most likely acting as upstream regulators (50). The role of CLKs in regulating splicing is conserved across many organisms, including Plasmodium falciparum, where inhibition of P. falciparum CLK1-3 (PfCLK1-3) is lethal to the parasite by preventing the splicing of essential genes (51). In T. brucei, most genes are constitutively transcribed as polycistronic mRNAs that are resolved through trans-splicing (52), but it remains unclear if CLK1 also has a role in that process. It has been proposed that the unique domains structure of T. brucei kinetochore proteins is consistent with the T. brucei kinetochore having a distinct evolutionary origin (15,44), and the finding of a unique CLK1/KKT2-centered regulation for kinetochore assembly supports that hypothesis.
As with most signaling networks, phosphorylation plays an essential role in the regulation of kinetochore functions, and multiple kinases have been found to regulate kinetochores (53). Key examples are aurora kinase B, MPS1, BUB1, PLK1, and CDK1 (53,54). From yeast to humans, most of the functions of aurora kinase B require its incorporation into the CPC (55) and its dynamic localization during the cell cycle (54). As a regulator of the kinetochore-microtubule attachment during mitosis, aurora kinase B contributes decisively to two feedback mechanisms, the error correction (EC) and spindle assembly checkpoint (SAC) (56). Furthermore, it promotes the inner and outer kinetochore interactions through phosphorylation of Dsn1 (39,57,58), a subunit of the Mis12 inner kinetochore complex, essential for kinetochore assembly (59). The T. brucei aurora kinase B orthologue, TbAUK1, has distinctive roles in metaphase-anaphase transition, ensuring proper spindle assembly, chromosome segregation, and cytokinesis (37,40). Alongside the parasite CPC, TbAUK1 associates with chromosomes during G 2 /M phase and with kinetochores in metaphase and finally localizes in the spindle midzone in anaphase (41), suggesting a role coordinating kinetochore recruitment and attachment. However, the potential role of this kinase in promoting kinetochore assembly has not yet been established or well separated from its regulatory function on mitosis.
In the T. brucei procyclic form, two kinetochore proteins, KKT4 and KKIP4, localize to the spindle during mitosis (17,60). Our results suggest that localization/expression of key outer kinetochore proteins remains unaffected after CLK1 inhibition, whereas KKT4, recently described as a microtubule tip-coupling protein (60), remains in anaphase, suggesting end-on interaction defects of microtubules with kinetochores. The role of aurora kinase B in the inner and outer kinetochore interaction in yeast resembles our findings of TbCLK1 functions in the recruitment of inner kinetochore during metaphase. Conversely, our results indicate that both pathways act independently in T. brucei or at least not involving inner plate recruitment through KKT2 phosphorylation; the stability of KKT2 localization further support this hypothesis. Interestingly, inhibition of CLK1 affects CPC localization at metaphase and NuSAP2 during anaphase. Understanding that centromeric localization of CPC is required to correct errors in attachment (61) and NuSAPs stabilize kinetochore microtubules during metaphase (62), it will be possible that, during anaphase onset, CLK1 and TbAUK1 coordinate different layers of regulation of kinetochore microtubule attachment and spindle stabilization. The fact that CLK1 copurifies with TbMlp2 and NuSAP1 provides further support for this (18). Interestingly, NuSAP1 to -4 partially colocalize with KKT2 (a CLK1 substrate) during the cell cycle, and knockdown of NuSAP1 destabilizes the expression of KKT1 but also triggers an unequal nuclear division without affecting spindle assembly (18), similar to our findings with KKT2 phosphomutants. Future experiments are required to determine whether the CLK1-KKT2 axis regulation of inner kinetochore assembly in T. brucei also requires a specific set of NuSAPs.
Altogether, we propose a model where CLK1 progressively phosphorylates KKT2 during S phase, allowing the timely spatial recruitment of the rest of the kinetochore proteins and posterior attachment to microtubules (Fig. 5). It is possible that KKT2 is phosphorylated by CLK1 prior to recruitment to the kinetochore, but evidence suggests this would occur during early S phase (32). Inhibition of CLK1 activity with AB1 leads to impaired inner kinetochore assembly and irreversible arrest in M phase, suggesting that this defect cannot be repaired by the parasite's checkpoint control and implying a dual function of CLK1 at different points during chromosome segregation. Considering the conservation of CLK1 between T. brucei, T. cruzi, and L. mexicana (22), the bioactivity of AB1 against the three trypanosomatids, and the conservation of the KKT2 S508 phosphorylation site in Leishmania and T. cruzi, it is quite likely that this signaling pathway is conserved across the trypanosomatids.
Plasmids. Recoded KKT2 was synthesized by Dundee Cell Products. The recoded KKT2 sequence (KKT2 R ) codes for the same amino acid sequence as KKT2 but only shares 94.23% nucleotide identity. All segments of identity between KKT2 and KKT2 R are less than 20 bp long. KKT2 R was inserted into the plasmid pGL2243 using XbaI and BamHI restriction sites, generating pGL2492. This plasmid is designed to constitutively express KKT2 from the tubulin locus, with the addition of a C-terminal 6Â HA tag. To express catalytically inactive KKT2 and phosphomutants, the active-site lysine (K 113 ) and serine (S 5 , S 8 , S 25 S 507-S508 , and S 828 ) were changed to alanine by mutating pGL2492, carrying the coding sequence for KKT2, using site-directed mutagenic PCR. A list of primers is provided in Text S1 in the supplemental material. To generate individual KKT2 recoded mutants, correspondent KKT2 R plasmids (described above) were transfected into the KKT2 RNAi cell line. Localization of endogenous KKT1 and KKT9 in KKT2 R mutants was assessed by microscopy after transfection of the correspondent mNG-KKT1 or mNG-KKT9 pPOTv6 vector into each recoded cell line. CLK1 Regulates the Trypanosome Kinetochore ® Immunofluorescence and cell cycle analysis. Cells treated for 6 h with compounds or dimethyl sulfoxide (DMSO) and were centrifuged at 1,400 Â g for 10 min before washing twice with Trypanosoma dilution buffer (TDB)-glucose at room temperature. Suspensions were centrifuged at 1,000 Â g for 5 min, pipetted into 6-well microscope slides, and dried at room temperature (RT). Cells were fixed with 25 ml of 2% paraformaldehyde diluted in phosphate-buffered saline (PBS) and incubated at room temperature for 5 min. Cells were washed in PBS to remove paraformaldehyde prior to washing twice more with PBS and permeabilized with 0.05% NP-40 for 10 min. Cells were washed twice in PBS and dried at RT. Mounting medium with 49,6-diamidino-2-phenylindole (DAPI) was added to each well with a coverslip. Slides were kept at 4°C before viewing using a Zeiss LSM 880 with Airyscan on an Axio Observer.Z1 invert confocal microscope.
Ty-NuSAP1 and Ty-NuSAP2 were detected by indirect immunofluorescence by using a mouse Imprint monoclonal anti-Ty1 antibody (clone BB2). Briefly, cells were harvested by centrifugation at 1,400 Â g for 10 min at room temperature, washed, and resuspended in TDB-glucose. A total of 2 Â 10 5 cells were dried on slides, fixed in 1% paraformaldehyde (PFA) for 1 h, washed with PBS, blocked with 50% (vol/vol) fetal bovine serum for 30 min, and then incubated with anti-TY (1:800) diluted in 0.5% blocking reagent for 1 h. Alexa-Fluor 488 (anti-mouse) was used as the secondary antibody (Invitrogen). Cells were DAPI stained and visualized using a Zeiss LSM 880 with Airyscan on an Axio Observer.Z1 inverted confocal microscope.
To study spindle formation, wild-type bloodstream forms were treated or not for 6 h with AB1 (5Â EC 50 ) or CLK1 RNAi cells were treated or not with tetracycline for 24 h. Parasites were harvested by centrifugation at 1,400 Â g for 10 min and then washed twice with TDB-glucose at room temperature. Samples were fixed for 10 min in 2%, wt/vol, formaldehyde in PBS, followed by 5 min incubation with 1 M Tris, pH 8.5, to quench the fixation. The fixed cells were washed with PBS, suspended in PBS, and adhered to SuperFrost Plus adhesion slides for 15 min. Attached parasites were then permeabilized with FIG 5 Regulation of kinetochore assembly by CLK1. This schematic diagram summarizes the recruitment defects caused by inhibition of CLK1 by AB1. In untreated cells in metaphase (top), CLK1 phosphorylates KKT2, resulting in recruitment of inner kinetochore components, allowing posterior kinetochore assembly to outer kinetochore components. When CLK1 is inhibited by AB1 (lower, arrowhead), phosphorylation of KKT2 S 508 is prevented, leading to a failure of recruitment of inner kinetochore components and consequent cell cycle arrest. We hypothesize that KKT2 binding to the centromere is compromised (KKT2?) after CLK1 inhibition (dashed circle). H3, histone H3. methanol at -20°C for 15 min and rehydrated with PBS, followed by incubation with blocking buffer (5% bovine serum albumin, 0.1% Triton X-100 in PBS) for 1 h at room temperature. Cells were immunostained at room temperature for 1 h with KMX-1 antibody to detect the mitotic spindle. After three washes (0.1% Triton X-100 in PBS), samples were incubated for 1 h with an Alexa Fluor 488-conjugated goat anti-mouse IgG (used at 1:300) secondary antibody. Finally, after three more washes, the slides were mounted in ProLong diamond antifade mountant with DAPI and examined by fluorescence microscopy. For analysis, 2K1N and 2K2N populations (n = 80) were considered, and statistical significance determined using the Holm-Sidak t test, with a = 0.05.
For cell cycle analysis, bloodstream-form T. brucei cell lines were incubated or not for 6 h with AB compounds at a final concentration of 5Â the individual EC 50 for each compound (averaged from viability assays). Control cultures were treated with 0.5 ml DMSO. Cultures were pelleted and cells were collected and washed once in TDB supplemented with 5 mM EDTA and resuspended in 70% methanol. Cells were centrifuged at 1,400 Â g for 10 min to remove methanol and washed once in TDB with 5 mM EDTA. Cells were resuspended in 1 ml 1Â TDB with 5 mM EDTA, 10 mg ml 21 propidium iodide, and 10 ml of RNase A. Cell suspensions in 1.5-ml tubes were wrapped in foil to avoid bleaching by light. Cells were incubated for 30 min at 37°C in the dark until fluorescence-activated cell sorting (FACS) analysis. Cells were analyzed for FACS using a Beckman Coulter CyAn ADP flow cytometer (excitation, 535 nm; emission, 617 nm). Cell cycle phase distribution was determined by fluorescence.
Hydroxyurea-induced synchronization of cell lines was obtained by incubating parasites in exponential growth phase with 10 mM hydroxyurea (HU) (Sigma-Aldrich) for 6 h. Removal of HU from the culture medium was achieved by centrifuging cells at 1,400 Â g for 10 min, washing twice with fresh (drug-free) medium, and resuspending cells in medium lacking HU. Subsequently, samples were collected each hour for posterior cell cycle analysis by propidium iodide staining.
Protein analysis. KKT2 and KKT3 phosphorylation profile were analyzed by using a SuperSep Phostag precast gel (29) according to the manufacturing protocol. Briefly, Ty-mNG KKT2 and Ty-mNG KKT3 were incubated with 5Â AB1 EC 50 for 18 h and collected for analysis by Western blotting in an EDTA-free radioimmunoprecipitation assay (RIPA) lysis buffer. In parallel, the expression of both proteins was analyzed after 24 h for TbCLK1 RNAi. After electrophoresis, the gel was washed 5 times with 10 mM EDTA transfer buffer to improve transference. The membrane then was transferred to a polyvinylidene difluoride (PVDF) membrane using a 0.1% SDS Tris-glycine transfer buffer at 90 mA overnight at 4°C. The membrane was blocked for 1 h with 10% bovine serum albumin (BSA) and KKT2 and KKT3 phosphorylation pattern was analyzed by using an anti-Ty1 antibody (see Text S1 for details).
Anti-phospho KKT2 S 508 was raised against a synthetic phosphopeptide antigen C-GTRVGS(pS*) LRPQRE-amide, where pS* represents phosphoserine. The peptide was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize rabbits. Phosphopeptide-reactive rabbit antiserum was first purified by protein A chromatography. Further purification was carried out using immunodepletion by nonphosphopeptide resin chromatography, after which the resulting eluate was chromatographed on a phosphopeptide resin. Anti-antigen antibodies were detected by indirect enzyme-linked immunosorbent assay with unconjugated antigens passively coated on plates, probed with anti-IgG-horseradish peroxidase conjugate, and detected with 2,29-azinobis(3-ethylbenzthiazolinesulfonic acid) substrate. Posterior antigen specificity was confirmed by Western blotting using KKT2 RNAi and endogenous tagged KKT2 cell lines. Custom antibody was produced by Thermo Fisher Scientific.
For Western blotting, parasites were washed with TDB supplemented with 20 mM glucose. After centrifugation, the samples were resuspended in the RIPA buffer (number 9806S; New England Biolabs) supplemented with protease and phosphatase inhibitors obtained from Promega and Roche Life Science, respectively. All samples were quantified by Bradford protein assay (Bio-Rad), 25 mg of protein was loaded, resolved in a 4 to 20% NuPAGE Bis-Tris gel (Invitrogen) in NuPAGE morpholinepropanesulfonic acid running buffer, and transferred onto Hybond-C nitrocellulose membranes (GE Healthcare) at 350 mA for 2 h or, for high-molecular-weight proteins, overnight at 4°C.
After transfer, membranes were washed once in 1Â TBST (Tris-buffered saline [TBS], 0.01% Tween 20 [Sigma-Aldrich]) for 10 min and then incubated for 1 h in blocking solution (1Â TBST, 5% BSA) or, if required, overnight at 4°C. Next, the membrane was rinsed for 10 min in 1Â TBST and placed in blocking buffer containing the required primary antisera for 1 h at room temperature or overnight at 4°C. The membrane was then washed 3 times with TBST and placed in blocking solution containing the appropriate fluorescent secondary antisera for 1 h. A list of antibodies is provided in Text S1.
General statistics. All statistical analyses were performed using GraphPad Prism 8 (http://www .graphpad.com/scientific-software/prism/). The appropriate tests were conducted and are detailed in the corresponding figure legends.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. TEXT S1, DOCX file, 0.03 MB.