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Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals.

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei. Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.

Trypanosoma brucei, an important human parasite, has a flagellum that controls cell motility, morphogenesis, proliferation, and cell-cell communication. Inheritance of the newly assembled flagellum during the cell cycle requires the Polo-like kinase homolog TbPLK and the kinetoplastid-specific protein phosphatase KPP1, although whether TbPLK acts on KPP1 or vice versa has been unclear. Here, we showed that dephosphorylation of TbPLK on Thr125 by KPP1 maintained low TbPLK activity in the flagellum-associated hook complex structure, thereby ensuring proper flagellum positioning and attachment. This dephosphorylation event required the recognition of phosphorylated Thr198 in the activation loop of TbPLK by the N-terminal Plus3 domain of KPP1 and the dephosphorylation of phosphorylated Thr125 in TbPLK by the C-terminal catalytic domain of KPP1. Dephosphorylation of TbPLK by KPP1 prevented hyperphosphorylation of the hook complex protein TbCentrin2, thereby allowing timely dephosphorylation of phosphorylated TbCentrin2 for hook complex duplication and flagellum positioning and attachment. Thus, KPP1 attenuates TbPLK activity by dephosphorylating TbPLK to facilitate flagellum inheritance.

Background Faithful DNA replication is essential to maintain genomic stability in all living organisms, and the regulatory pathway for DNA replication initiation is conserved from yeast to humans. The evolutionarily ancient human parasite Trypanosoma brucei, however, lacks many of the conserved DNA replication factors and may employ unusual mechanisms for DNA replication. Neither the S-phase cyclin-dependent kinase (CDK) nor the regulatory pathway governing DNA replication has been previously identified in T. brucei. Results Here we report that CRK2 (Cdc2-related kinase 2) complexes with CYC13 (Cyclin13) and functions as an S-phase CDK to promote DNA replication in T. brucei. We further show that CRK2 phosphorylates Mcm3, a subunit of the Mcm2–7 sub-complex of the Cdc45-Mcm2–7-GINS complex, and demonstrate that Mcm3 phosphorylation by CRK2 facilitates interaction with Sld5, a subunit of the GINS sub-complex of the Cdc45-Mcm2–7-GINS complex. Conclusions These results identify the CRK2-CYC13 complex as an S-phase regulator in T. brucei and reveal its role in regulating DNA replication through promoting the assembly of the Cdc45-Mcm2–7-GINS complex.

Inheritance of the newly assembled flagellum in the human parasite Trypanosoma brucei depends on the faithful duplication and segregation of multiple flagellum-associated cytoskeletal structures, including the hook complex and its associated centrin arm. The biological functions of this unique hook complex–centrin arm assembly remain poorly understood. Here, we report a hook complex–associated protein named BOH2 that plays an essential role in promoting flagellum inheritance. BOH2 localizes to the hooked part of the hook complex by bridging the hook complex, the centrin arm, and the flagellum attachment zone filament. Depletion of BOH2 caused the loss of the shank part of the hook complex and its associated protein TbSmee1, disrupted the assembly of the centrin arm and the recruitment of centrin arm–associated protein CAAP1, inhibited the assembly of the flagellum attachment zone, and caused flagellum mispositioning and detachment. These results demonstrate crucial roles of BOH2 in maintaining hook complex integrity and promoting centrin arm formation and suggest that proper assembly of the hook complex–centrin arm structure facilitates flagellum inheritance.

The human parasite Trypanosoma brucei transitions from the trypomastigote form to the epimastigote form in the insect vector by repositioning its mitochondrial genome and flagellum-associated cytoskeleton. The molecular mechanisms underlying such changes in cell morphology remain elusive, but recent works demonstrated the involvement of three flagellar proteins, FLAM3, ClpGM6 and KIN-E, in this process by controlling the elongation of the flagellum attachment zone (FAZ). In this report, we identified a FAZ flagellum domain-localizing protein named FAZ27 and characterized its role in cell morphogenesis. Depletion of FAZ27 in the trypomastigote form caused major morphological changes and repositioning of the mitochondrial genome and flagellum-associated cytoskeleton, generating epimastigote-like cells. Furthermore, proximity biotinylation and co-immunoprecipitation identified FLAM3 and ClpGM6 as FAZ27-interacting proteins, and analyses of their functional interplay revealed an interdependency for assembly into the FAZ flagellum domain. Finally, we showed that assembly of FAZ27 occurred proximally, identical to the assembly pattern of other FAZ sub-domain proteins. Taken together, these results demonstrate a crucial role for the FAZ flagellum domain in controlling cell morphogenesis and suggest a coordinated assembly of all the FAZ sub-domains at the proximal end of the FAZ.

Faithful chromosome segregation depends on correct spindle microtubule-kinetochore attachment and requires certain spindle-associated proteins (SAPs) involved in regulating spindle dynamics and chromosome segregation. Little is known about the spindle-associated proteome in the early divergent Trypanosoma brucei and its roles in chromosome segregation. Here we report the identification of a cohort of divergent SAPs through localization-based screening and proximity-dependent biotin identification. We identified seven new SAPs and seventeen new nucleolar proteins that associate with the spindle, and demonstrated that the kinetochore protein KKIP4 also associates with the spindle. These SAPs localize to distinct subdomains of the spindle during mitosis, and all but one localize to nucleus during interphase and post-mitotic phases. Functional analyses of three nucleus- and spindle-associated proteins (NuSAPs) revealed distinct functions in chromosome segregation. NuSAP1 is a kinetoplastid-specific protein required for equal chromosome segregation and for maintaining the stability of the kinetochore proteins KKIP1 and KKT1. NuSAP2 is a highly divergent ASE1/PRC1/MAP65 homolog playing an essential role in promoting the G2/M transition. NuSAP3 is a kinetoplastid-specific Kif13-1-binding protein maintaining Kif13-1 protein stability and regulating the G2/M transition. Together, our work suggests that chromosome segregation in T. brucei requires a cohort of kinetoplastid-specific and divergent SAPs with distinct functions.

Cytokinesis, the terminal step in cell division, in the protist human pathogen Trypanosoma brucei occurs along the longitudinal axis from the anterior tip of the new flagellum attachment zone (FAZ) toward the posterior cell tip. This process is regulated by a signaling cascade composed of the Polo-like kinase homolog TbPLK, the Aurora B kinase homolog TbAUK1, and the trypanosome-specific CIF1–CIF2 protein complex. However, the regulatory mechanism and the signaling pathway for this unusual mode of cytokinesis remain poorly understood. Here, we report another trypanosome-specific protein assembly, the CIF1–CIF3 complex, and its essential role in cytokinesis initiation. Through biochemical and genetic approaches, we demonstrate that CIF3 interacts with CIF1 in a TbPLK-dependent manner and maintains CIF1 localization at the new FAZ tip. Conversely, CIF1 maintains CIF3 stability at the new FAZ tip. We further show that TbPLK is required for CIF3 localization and that CIF3 is necessary for targeting TbAUK1 to the new FAZ tip during anaphase. These results suggest that two trypanosome-specific CIF1-containing protein complexes cooperate with the evolutionarily conserved Polo-like kinase and Aurora B kinase to promote cytokinesis in T. brucei.

The flagellated eukaryote Trypanosoma brucei alternates between the insect vector and the mammalian host and proliferates through an unusual mode of cell division. Cell division requires flagellum motility-generated forces, but flagellum motility exerts distinct effects between different life cycle forms. Motility is required for the final cell abscission of the procyclic form in the insect vector, but is necessary for the initiation of cell division of the bloodstream form in the mammalian host. The underlying mechanisms remain elusive. Here we carried out functional analyses of a flagellar axonemal inner-arm dynein complex in the bloodstream form and investigated its mechanistic role in cytokinesis initiation. We showed that the axonemal inner-arm dynein heavy chain TbIAD5-1 and TbCentrin3 form a complex, localize to the flagellum, and are required for viability in the bloodstream form. We further demonstrated the interdependence between TbIAD5-1 and TbCentrin3 for maintenance of protein stability. Finally, we showed that depletion of TbIAD5-1 and TbCentrin3 arrested cytokinesis initiation and disrupted the localization of multiple cytokinesis initiation regulators. These findings identified the essential role of an axonemal inner-arm dynein complex in cell division, and provided molecular insights into the flagellum motility-mediated cytokinesis initiation in the bloodstream form of T. brucei.

The evolutionarily early divergent human parasite Trypanosoma brucei proliferates through binary cell fission in both its tsetse fly vector and mammalian host. The parasite divides unidirectionally along the longitudinal cell axis from the anterior cell tip toward the posterior cell tip through a mechanism distinct from that in the cells of its human host. Initiation of cytokinesis in T. brucei is regulated by two evolutionarily conserved protein kinases, the Polo-like kinase TbPLK and the Aurora B kinase TbAUK1, and a cohort of trypanosome-specific proteins, including the three cytokinesis initiation factors CIF1, CIF2, and CIF3. Here, using RNAi, in situ epitope tagging of proteins, GST pulldown, and coimmunoprecipitation assays, and immunofluorescence and scanning electron microscopy analyses, we report the identification and functional characterization of two trypanosome-specific proteins, flagellum attachment zone tip–localizing protein required for cytokinesis (FPRC) and CIF4. We found that the two proteins colocalize to the distal tips of the new and the old flagellum attachment zones and are required for cytokinesis initiation. Knockdown of FPRC or CIF4 disrupted the localization of CIF1, suggesting that they function upstream of CIF1. Moreover, depletion of CIF4 abolished FPRC localization, indicating that CIF4 acts upstream of FPRC. Together, these results identify two new cytokinesis regulators in T. brucei and integrate them into the CIF1-mediated cytokinesis regulatory pathway. These findings highlight the existence of a cytokinesis pathway in T. brucei that is different from that of its mammalian host and therefore suggest that cytokinesis in T. brucei could potentially be exploited as a new drug target.

Protein posttranslational modifications such as neddylation play crucial roles in regulating protein function. Only a few neddylated substrates have been validated to date, and the role of neddylation remains poorly understood. Here, using Trypanosoma brucei as the model organism, we investigated the function and substrates of TbNedd8. TbNedd8 is distributed throughout the cytosol but enriched in the nucleus and the flagellum. Depletion of TbNedd8 by RNAi abolished global protein ubiquitination, caused DNA re-replication in postmitotic cells, impaired spindle assembly, and compromised the flagellum attachment zone filament, leading to flagellum detachment. Through affinity purification and mass spectrometry, we identified 70 TbNedd8-conjugated and -associated proteins, including known Nedd8-conjugated and -associated proteins, putative TbNedd8 conjugation system enzymes, proteins of diverse biological functions, and proteins of unknown function. Finally, we validated six Cullins as bona fide TbNedd8 substrates and identified the TbNedd8 conjugation site in three Cullins. This work lays the foundation for understanding the roles of protein neddylation in this early divergent parasitic protozoan.

Trypanosoma brucei possesses a motile flagellum that determines cell morphology and the cell division plane. Inheritance of the newly assembled flagellum during the cell cycle is controlled by the Polo-like kinase homolog TbPLK, which also regulates cytokinesis initiation. How TbPLK is targeted to its subcellular locations remains poorly understood. Here we report a trypanosome-specific protein named BOH1 which cooperates with TbPLK to regulate flagellum inheritance and cytokinesis initiation in the procyclic form of T. brucei. BOH1 localizes to an unusual sub-domain in the flagellum-associated hook complex, bridging the hook complex, the centrin arm and the flagellum attachment zone. Depletion of BOH1 disrupts hook complex morphology, inhibits centrin arm elongation and abolishes flagellum attachment zone assembly, leading to flagellum mis-positioning and detachment. Further, BOH1 deficiency impairs the localization of TbPLK and the cytokinesis regulator CIF1 to the cytokinesis initiation site, providing a molecular mechanism for its role in cytokinesis initiation. These findings reveal the roles of BOH1 in maintaining hook complex morphology and regulating flagellum inheritance, and establish BOH1 as an upstream regulator of the TbPLK-mediated cytokinesis regulatory pathway.

The early divergent protozoan parasite Trypanosoma brucei alternates between the insect vector and the mammalian hosts during its life cycle and proliferates through binary cell fission. The cell cycle control system in T. brucei differs substantially from that in its mammalian hosts and possesses distinct mitosis-cytokinesis checkpoint controls between two life cycle stages, the procyclic form and the bloodstream form. T. brucei undergoes an unusual mode of cytokinesis, which is controlled by a novel signaling cascade consisting of evolutionarily conserved protein kinases and trypanosome-specific regulatory proteins in the procyclic form. However, given the distinct mitosis-cytokinesis checkpoints between the two forms, it is unclear whether the cytokinesis regulatory pathway discovered in the procyclic form also operates in a similar manner in the bloodstream form. Here, we showed that the three regulators of cytokinesis initiation, cytokinesis initiation factor 1 (CIF1), CIF2, and CIF3, are interdependent for subcellular localization but not for protein stability as in the procyclic form. Further, we demonstrated that KLIF, a regulator of cytokinesis completion in the procyclic form, plays limited roles in cytokinesis in the bloodstream form. Finally, we showed that the cleavage furrow-localizing protein FRW1 is required for cytokinesis initiation in the bloodstream form but is nonessential for cytokinesis in the procyclic form. Together, these results identify conserved and life cycle-specific functions of cytokinesis regulators, highlighting the distinction in the regulation of cytokinesis between different life cycle stages of T. brucei. IMPORTANCE The early divergent protozoan parasite Trypanosoma brucei is the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. This parasite has a complex life cycle by alternating between the insect vector and the mammalian hosts and proliferates by binary cell fission. The control of cell division in trypanosomes appears to be distinct from that in its human host and differs substantially between two life cycle stages, the procyclic (insect) form and the bloodstream form. Cytokinesis, the final step of binary cell fission, is regulated by a novel signaling cascade consisting of two evolutionarily conserved protein kinases and a cohort of trypanosome-specific regulators in the procyclic form, but whether this signaling pathway operates in a similar manner in the bloodstream form is unclear. In this report, we performed a functional analysis of multiple cytokinesis regulators and discovered their distinct functions and regulations in the bloodstream form.

Trypanosoma (Herpetosoma) lewisi is a globally distributed rat trypanosome, currently considered as a zoonotic pathogen; however, a detailed understanding of the morphological events occurring during the cell cycle is lacking. This study aimed to investigate the cell cycle morphology and cleavage events of Trypanosoma lewisi (T. lewisi) during in vitro cultivation. By establishing in vitro cultivation of T. lewisi at 37°C, various cell morphologies and stages could be observed. We have provided a quantitative analysis of the morphological events during T. lewisi proliferation. We confirmed a generation time of 12.14 ± 0.79 hours, which is similar to that in vivo (12.21 ± 0.14 hours). We also found that there are two distinct cell cycles, with a two-way transformation connection in the developmental status of this parasite, which was contrasted with the previous model of multiple division patterns seen in T. lewisi. We quantified the timing of cell cycle phases (G1n, 0.56 U; Sn, 0.14 U; G2n, 0.16 U; M, 0.06 U; C, 0.08 U; G1k, 0.65 U; Sk, 0.10 U; G2k, 0.17 U; D, 0.03 U; A, 0.05 U) and their morphological characteristics, particularly with respect to the position of kinetoplast(s) and nucleus/nuclei. Interestingly, we found that nuclear replication in T. lewisi occurred prior to kinetoplast replication, different to the order of replication found in Trypanosoma brucei and Trypanosoma cruzi, implicating a distinct cell cycle control mechanism in T. lewisi. We characterized the morphological events during the T. lewisi cell cycle and presented evidence to support the existence of two distinct cell cycles with two-way transformation between them. These results provide insights into the differentiation and evolution of this parasite and its related species. Keywords: Trypanosoma lewisi, Cell cycle, In vitro, Multiplication, Division, Zoonotic pathogen

The Polo-like kinase (PLK) in Trypanosoma brucei plays multiple roles in basal body segregation, flagellum attachment, and cytokinesis. However, the mechanistic role of TbPLK remains elusive, mainly because most of its substrates are not known. Here, we report a new substrate of TbPLK, SPBB1, and its essential roles in T. brucei. SPBB1 was identified through yeast two-hybrid screening with the kinase-dead TbPLK as the bait. It interacts with TbPLK in vitro and in vivo, and is phosphorylated by TbPLK in vitro. SPBB1 localizes to both the mature basal body and the probasal body throughout the cell cycle, and co-localizes with TbPLK at the basal body during early cell cycle stages. RNAi against SPBB1 in procyclic trypanosomes inhibited basal body segregation, disrupted the new flagellum attachment zone filament, detached the new flagellum, and caused defective cytokinesis. Moreover, RNAi of SPBB1 confined TbPLK at the basal body and the bilobe structure, resulting in constitutive phosphorylation of TbCentrin2 at the bilobe. Altogether, these results identified a basal body protein as a TbPLK substrate and its essential role in promoting basal body segregation and flagellum attachment zone filament assembly for flagellum adhesion and cytokinesis initiation.

Summary γ-Tubulin complex constitutes a key component of the microtubule-organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ-tubulin small complex (γTuSC) composed of γ-tubulin, gamma-tubulin complex protein (GCP)2 and GCP3, whereas animals contain the γ-tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ-tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ-tubulin complex in T. brucei is composed of γ-tubulin and three GCP proteins, GCP2-GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ-tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex.

The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei , an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei .

Trypanosoma brucei causes sleeping sickness in humans and nagana in cattle in sub-Saharan Africa and alternates between its mammalian hosts and its insect vector, the tsetse fly. T. brucei uses a flagellum for motility, cell division, and cell–cell communication. Proper positioning and attachment of the newly assembled flagellum rely on the faithful duplication and segregation of flagellum-associated cytoskeletal structures. These processes are regulated by the polo-like kinase homolog TbPLK, whose activity and abundance are under stringent control to ensure spatiotemporally regulated phosphorylation of its substrates. However, it remains unclear whether a protein phosphatase that counteracts TbPLK activity is also involved in this regulation. Here, we report that a putative kinetoplastid-specific protein phosphatase, named KPP1, has essential roles in regulating flagellum positioning and attachment in T. brucei. KPP1 localized to multiple flagellum-associated cytoskeletal structures and co-localized with TbPLK in several cytoskeletal structures at different cell-cycle stages. KPP1 depletion abolished basal body segregation, inhibited the duplication of the centrin arm and the hook complex of the bilobe structure, and disrupted the elongation of the flagellum attachment zone, leading to flagellum misplacement and detachment and cytokinesis arrest. Importantly, KPP1-depleted cells lacked dephosphorylation of TbCentrin2, a TbPLK substrate, at late cell-cycle stages. Together, these results suggest that KPP1-mediated protein dephosphorylation regulates the duplication and segregation of flagellum-associated cytoskeletal structures, thereby promoting flagellum positioning and attachment. These findings highlight the requirement of reversible protein phosphorylation, mediated by TbPLK and KPP1, in regulating flagellum inheritance in T. brucei.

Trypanosoma brucei undergoes life cycle form transitions from trypomastigotes to epimastigotes in the insect vector by re-positioning the mitochondrial genome and re-locating the flagellum and flagellum-associated cytoskeletal structures. The mechanism underlying these dramatic morphology transitions remains poorly understood. Here we report the regulatory role of the orphan kinesin KIN-E in controlling trypanosome morphology transitions. KIN-E localizes to the flagellum and is enriched at the flagellar tip, and this localization depends on the C-terminal m-calpain domain III-like domains. Depletion of KIN-E in the trypomastigote form of T. brucei causes major morphology changes and a gradual increase in the level of EP procyclin, generating epimastigote-like cells. Mechanistically, through its C-terminal importin α-like domain, KIN-E targets FLAM3, a flagellar protein involved in morphology transitions, to the flagellum to promote elongation of the flagellum attachment zone and positioning of the flagellum and flagellum-associated cytoskeletal structure, thereby maintaining trypomastigote cell morphology. Our findings suggest that morphology transitions in trypanosomes require KIN-E-mediated transport of FLAM3 to the flagellum., Author summary Trypanosoma brucei, the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa, has a complex life cycle by alternating between the tsetse fly vector and the mammalian hosts. In the gut of tsetse flies, trypanosomes undergo life cycle transitions from the trypomastigote form to the epimastigote form by re-positioning the mitochondrial genome and re-locating the flagellum and flagellum-associated cytoskeletal structures. Previous work demonstrated that elongation of the flagellum attachment zone plays an important role in controlling morphology transitions, but how it is regulated remains poorly understood. This work discovered that an orphan kinesin plays an essential role in regulating trypanosome morphology transitions. This novel kinesin localizes to the flagellum and targets FLAM3, one of the two flagellar proteins involved in morphology transitions, to the flagellum. This work suggests that trypanosome morphology transitions require kinesin-mediated transport of FLAM3 to the flagellum to promote the elongation of the flagellum attachment zone, thereby maintaining flagellum-cell body attachment and positioning the flagellum and flagellum-associated cytoskeletal structures to assume trypomastigote cell morphology.

Cytokinesis in trypanosome occurs uni-directionally along the longitudinal axis from the cell anterior towards the cell posterior and requires a trypanosome-specific CIF1-CIF2 protein complex. However, little is known about the contribution of the structural motifs in CIF1 and CIF2 to complex assembly and cytokinesis. Here, we demonstrated that the two zinc-finger motifs but not the coiled-coil motif in CIF1 are required for interaction with the EF-hand motifs in CIF2. We further showed that localization of CIF1 depends on the coiled-coil motif and the first zinc-finger motif and that localization of CIF2 depends on the EF-hand motifs. Deletion of the coiled-coil motif and mutation of either zinc-finger motifs in CIF1 disrupted cytokinesis. Further, mutation of either zinc-finger motif in CIF1 mis-localized CIF2 to the cytosol and destabilized CIF2, whereas deletion of the coiled-coil motif in CIF1 spread CIF2 over to the new flagellum attachment zone and stabilized CIF2. Together, these results uncovered the requirement of the coiled-coil motif and zinc-finger motifs for CIF1 function in cytokinesis and for CIF2 localization and stability, providing structural insights into the functional interplay between the two cytokinesis regulators.

The basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei. Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote., IMPORTANCE The basal body in the early-branching protozoan Trypanosoma brucei nucleates flagellum assembly and also regulates organelle segregation, cell morphogenesis, and cell division. However, the molecular composition and the assembly process of the basal body remain poorly understood. Here, we identify 14 conserved basal body proteins and 25 trypanosome-specific basal body proteins via bioinformatics, localization-based screening, and proximity-dependent biotin identification. We further localized these proteins to distinct subdomains of the basal body by using fluorescence microscopy and superresolution microscopy, discovered novel regulators of basal body duplication and separation, and uncovered new functions of conserved basal body proteins in basal body duplication and separation. This work lays the foundation for dissecting the mechanisms underlying basal body biogenesis and inheritance in T. brucei.

Mitogen-activated protein kinase (MAPK) modules are evolutionarily conserved signaling cascades that function in response to the environment and play crucial roles in intracellular signal transduction in eukaryotes. The involvement of a MAP kinase in regulating cytokinesis in yeast, animals, and plants has been reported, but the requirement for a MAP kinase for cytokinesis in the early-branching protozoa is not documented. Here, we show that a MAP kinase homolog (TbMAPK6) from Trypanosoma brucei plays distinct roles in cytokinesis in two life cycle forms of T. brucei . TbMAPK6 is distributed throughout the cytosol in the procyclic form but is localized in both the cytosol and the nucleus in the bloodstream form. RNA interference (RNAi) of TbMAPK6 results in moderate growth inhibition in the procyclic form but severe growth defects and rapid cell death in the bloodstream form. Moreover, TbMAPK6 appears to be implicated in furrow ingression and cytokinesis completion in the procyclic form but is essential for cytokinesis initiation in the bloodstream form. Despite the distinct defects in cytokinesis in the two forms, RNAi of TbMAPK6 also caused defective basal body duplication/segregation in a small cell population in both life cycle forms. Altogether, our results demonstrate the involvement of the TbMAPK6-mediated pathway in regulating cytokinesis in trypanosomes and suggest distinct roles of TbMAPK6 in cytokinesis between different life cycle stages of T. brucei .

The Polo-like kinase homolog in Trypanosoma brucei, TbPLK, plays essential roles in basal body segregation, flagellum attachment and cytokinesis. The level of TbPLK protein is tightly controlled, but the underlying mechanism remains elusive. Here, we report a Cullin-RING ubiquitin ligase composed of Cullin4, the DNA damage-binding protein 1 homolog TbDDB1 and a WD40-repeat protein WDR1 that controls TbPLK abundance in the basal body and the bilobe. WDR1, through its C-terminal domain, interacts with the PEST motif in TbPLK and, through its N-terminal WD40 motif, binds to TbDDB1. Depletion of WDR1 inhibits bilobe duplication and basal body segregation, disrupts the assembly of the new flagellum attachment zone filament and detaches the new flagellum. Consistent with its role in TbPLK degradation, depletion of WDR1 causes excessive accumulation of TbPLK in the basal body and the bilobe, leading to continuous phosphorylation of TbCentrin2 in the bilobe at late cell cycle stages. Together, these results identify a novel WD40-repeat protein as a TbPLK receptor in the Cullin4-DDB1 ubiquitin ligase complex for degrading TbPLK in the basal body and the bilobe after the G1/S cell cycle transition, thereby promoting bilobe duplication, basal body separation and flagellum-cell body adhesion., Author Summary African trypanosomes are the causative agents of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. These unicellular parasites possess a flagellum, which is a long, lash-like appendage that is protruded from the cell body and serves for locomotion of the parasites. The flagellum is attached to the cell body via a specialized structure termed flagellum attachment zone (FAZ) filament. Assembly of the FAZ filament is necessary to maintain flagellum-cell body attachment, which is required for maintaining normal cell shape and for ensuring faithful cell division. A key regulator of flagellum-cell body attachment is the Polo-like kinase (PLK), an evolutionarily conserved protein kinase that regulates many downstream effectors. The level of PLK appears to be tightly controlled, but the underlying mechanism is still elusive. Here we report a novel protein that controls PLK abundance by targeting PLK for degradation. Depletion of this novel protein causes excessive accumulation of PLK and disrupts FAZ filament assembly, leading to flagellum detachment and defective cell division. These results discover the molecular mechanism underlying the control of PLK protein abundance in trypanosomes.

The chromosomal passenger complex (CPC) in animals, consisting of Aurora B kinase and three evolutionarily conserved proteins, plays crucial roles in mitosis and cytokinesis. However, Trypanosoma brucei expresses an unusual CPC consisting of an Aurora-like kinase, TbAUK1, and two kinetoplastid-specific proteins, TbCPC1 and TbCPC2. Despite their essential functions, little is known about the regulation of TbAUK1 and the roles of TbCPC1 and TbCPC2. Here, we investigate the effect of post-translational modification on the activity and spatiotemporal control of TbAUK1, and demonstrate that phosphorylation of two conserved threonine residues in the activation loop of the kinase domain contributes to TbAUK1 activation and function. TbAUK1 is SUMOylated in vivo, and mutation of the SUMO-conjugation site compromises TbAUK1 function. Degradation of TbAUK1 requires two destruction boxes and is mediated by the anaphase-promoting complex/cyclosome (APC/C), whereas degradation of TbCPC1 and TbCPC2 is not dependent on the predicted destruction boxes and is APC/C-independent. Moreover, we determine the domains in CPC subunits that mediate the pairwise interactions, and show that disruption of the interaction impairs the localization of TbAUK1 and TbCPC2 but not TbCPC1. Our results demonstrate the requirement of post-translational modifications for TbAUK1 function and a crucial role of TbCPC1 in mediating TbAUK1 localization.

Cytokinesis in Trypanosoma brucei, an early branching protozoan, occurs along its longitudinal axis uni-directionally from the anterior tip of the new flagellum attachment zone filament toward the cell's posterior end. However, the underlying mechanisms remain elusive. Here we report that cytokinesis in T. brucei is regulated by a concerted action of Polo-like kinase, Aurora B kinase, and a trypanosome- specific protein CIF1. Phosphorylation of CIF1 by Polo-like kinase targets it to the anterior tip of the new flagellum attachment zone filament, where it subsequently recruits Aurora B kinase to initiate cytokinesis. Consistent with its role, CIF1 depletion inhibits cytokinesis initiation from the anterior end of the cell, but, surprisingly, triggers cytokinesis initiation from the posterior end of the cell, suggesting the activation of an alternative cytokinesis from the opposite cell end. Our results reveal the mechanistic roles of CIF1 and Polo-like kinase in cytokinesis initiation and elucidate the mechanism underlying the recruitment of Aurora B kinase to the cytokinesis initiation site at late anaphase. These findings also delineate a signaling cascade controlling cytokinesis initiation from the anterior end of the cell and uncover a backup cytokinesis that is initiated from the posterior end of the cell when the typical anterior-to-posterior cytokinesis is compromised.

The evolutionarily conserved centriole/basal body protein SAS-4 regulates centriole duplication in metazoa and basal body duplication in flagellated and ciliated organisms. Here, we report that the SAS-4 homolog in the flagellated protozoan Trypanosoma brucei, TbSAS-4, plays an unusual role in controlling life cycle transitions by regulating the length of the flagellum attachment zone (FAZ) filament, a specialized cytoskeletal structure required for flagellum adhesion and cell morphogenesis. TbSAS-4 is concentrated at the distal tip of the FAZ filament, and depletion of TbSAS-4 in the trypomastigote form disrupts the elongation of the new FAZ filament, generating cells with a shorter FAZ associated with a longer unattached flagellum and repositioned kinetoplast and basal body, reminiscent of epimastigote-like morphology. Further, we show that TbSAS-4 associates with six additional FAZ tip proteins, and depletion of TbSAS-4 disrupts the enrichment of these FAZ tip proteins at the new FAZ tip, suggesting a role of TbSAS-4 in maintaining the integrity of this FAZ tip protein complex. Together, these results uncover a novel function of TbSAS-4 in regulating the length of the FAZ filament to control basal body positioning and life cycle transitions in T. brucei.

Polo-like kinase in Trypanosoma brucei (TbPLK) is confined to the flagellum attachment zone (FAZ) and regulates only cytokinetic initiation. However, it apparently diffuses into the cytoplasm before the trans -localization of chromosomal passenger complex (CPC) from the midzone of central spindle to FAZ, which is known to be required for initiating cytokinesis. Synchronized T. brucei procyclic cells treated with a TbPLK inhibitor, GW843682X (GW), in late S phase were found to go through a full cell cycle at a normal pace before being arrested at cytokinetic initiation in the second cycle. However, synchronized cells treated with GW in G 1 phase were arrested at cytokinetic initiation within the first cell cycle, suggesting that inhibition of TbPLK at its emergence blocks cytokinesis within the same cell cycle. To rule out potential off-target effects from GW, TbPLK RNA interference (RNAi) was induced to deplete TbPLK, and the progression of synchronized cells from late S phase was also found to be arrested at cytokinetic initiation within the first cell cycle. Apparently, TbPLK has accomplished its role in guiding cytokinesis before the late S phase, presumably by phosphorylating a certain substrate(s) during S phase, which may play a critical role in initiating the subsequent cytokinesis.

Kinetoplastid membrane protein 11 (KMP-11) has been identified as a flagellar protein and is conserved among kinetoplastid parasites, but its potential function remains unknown. In a recent study, we identified KMP-11 as a microtubule-bound protein localizing to the flagellum as well as the basal body in both procyclic and bloodstream forms of Trypanosoma brucei (Z. Li, J. H. Lee, F. Chu, A. L. Burlingame, A. Gunzl, and C. C. Wang, PLoS One 3:e2354, 2008). Silencing of KMP-11 by RNA interference inhibited basal body segregation and cytokinesis in both forms and resulted in multiple nuclei of various sizes, indicating a continuous, albeit somewhat defective, nuclear division while cell division was blocked. KMP-11 knockdown in the procyclic form led to severely compromised formation of the new flagellum attachment zone (FAZ) and detachment of the newly synthesized flagellum. However, a similar phenotype was not observed in the bloodstream form depleted of KMP-11. Thus, KMP-11 is a flagellar protein playing critical roles in regulating cytokinesis in both forms of the trypanosomes. Its distinct roles in regulating FAZ formation in the two forms may provide a clue to the different mechanisms of cytokinetic initiation in procyclic and bloodstream trypanosomes.

Adhesion of motile flagella to the cell body in Trypanosoma brucei requires a filamentous cytoskeletal structure termed the flagellum attachment zone (FAZ). Despite its essentiality, the complete molecular composition of the FAZ filament and its roles in FAZ filament assembly remain poorly understood. By localization-based screening, we here identified a new FAZ protein, which we called FAZ2. Knockdown of FAZ2 disrupted the FAZ filament, destabilized multiple FAZ filament proteins and caused a cytokinesis defect. We also showed that FAZ2 depletion destabilized another new FAZ filament protein and several flagellum and cytoskeleton proteins. Furthermore, we identified CC2D and KMP11 as FAZ2 partners through affinity purification, and showed that they are each required for maintaining a stable complex. Finally, we demonstrated that FAZ filament proteins are incorporated into the FAZ filament from the proximal region, in contrast to the flagellum components, which are incorporated from the distal tip. In summary, we identified three new FAZ filament proteins and a FAZ filament protein complex, and our results suggest that assembly of the FAZ filament occurs at the proximal region and is essential to maintain the stability of FAZ filament proteins.

Mitosis and cytokinesis are highly coordinated in eukaryotic cells. But procyclic-form Trypanosoma brucei under G1 or mitotic arrest is still capable of dividing, resulting in anucleate daughter cells (zoids). Okadaic acid (OKA), an inhibitor of protein phosphatases PP1 and PP2A, is known to inhibit kinetoplast replication and cell division yielding multinucleate cells with single kinetoplasts. However, when OKA was applied to cells arrested in G1 or G2/M phase via RNAi knockdown of specific cdc2-related kinases (CRKs), DNA synthesis and nuclear division were resumed without kinetoplast replication or cell division, resulting in multinucleate cells as in the wild type. Cells arrested in G2/M via depleting the mitotic cyclin CycB2 or an aurora B kinase homologue TbAUK1 were, however, not released by OKA treatment. The phenomenon is thus similar to the OKA activation of Cdc2 in Xenopus oocyte by inhibiting PP2A [Maton, et al., Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A. J. Cell Sci. 118 (2005) 2485–2494]. A simultaneous knockdown of the seven PP1s or the PP2A catalytic subunit in T. brucei by RNA interference did not, however, result in multinucleate cells. This could be explained by assuming a negative regulation, either directly or indirectly, of CRK by an OKA-sensitive phosphatase, which could be a PP2A as in the Xenopus oocyte and a positive regulation of kinetoplast replication by an OKA-susceptible protein(s). Test of a PP2A-specific inhibitor, fostriecin, on cells arrested in G2/M via CRK depletion or a knockdown of the PP2A catalytic subunit from the CRK-depleted cells both showed a partial lift of the G2/M block without forming multinucleate cells. These observations support the abovementioned assumption and suggest the presence of a novel OKA-sensitive protein(s) regulating kinetoplast replication that still remains to be identified.

Aurora-B kinase is a chromosomal passenger protein essential for chromosome segregation and cytokinesis. In the procyclic form of Trypanosoma brucei , depletion of an aurora-B kinase homologue TbAUK1 inhibited spindle formation, mitosis, cytokinesis, and organelle replication without altering cell morphology. In the present study, an RNA interference knockdown of TbAUK1 or overexpression of inactive mutant TbAUK1-K58R in the bloodstream form also resulted in defects in spindle formation, chromosome segregation, and cytokinesis but allowed multiple rounds of nuclear DNA synthesis, nucleolus multiplication, and continuous replication of kinetoplast, basal body, and flagellum. The typical trypanosome morphology was lost to an enlarged round shape filled with microtubules. It is thus apparent that there are distinctive mechanisms of action of TbAUK1 in regulating cell division between the two developmental stages of trypanosome. While it exerts a tight control on mitosis, organelle replication, and cytokinesis in the procyclic form, it regulates cytokinesis without rigid control over either nuclear DNA synthesis or organelle replication in the bloodstream form. The molecular basis underlining these discrepancies remains to be explored.

Summary Ubiquitination and proteasomal degradation of cell cycle regulatory proteins are known to play a pivotal role in controlling the progression of the eukaryotic cell cycle. Using the technique of RNA interference (RNAi) on the bloodstream form of Trypanosoma brucei , we were able to knock down expression of each of the 11 non-ATPase regulatory subunit proteins (Rpns) in the 19S regulatory complex of the 26S proteasome. In each case, the knock-down led to arrest of cells within the G1 and G2 phases, suggesting blockage of cell cycle progression at both G1/S and G2/M boundaries. This finding differs from that observed previously in the procyclic form of T. brucei , in which loss of individual Rpns blocks only passage across the G2/M boundary. Thus, proteasomal degradation of additional regulatory protein(s) may be required for exiting from G1 phase in the bloodstream form. In vitro differentiation of each of the 11 Rpn-depleted bloodstream form cell lines into the procyclic form was monitored. Each cell line proceeded to completion of the differentiation process like the wild-type cells with the total percentage of differentiated cells about equivalent to the sum of G1 and G2 cells. Thus, cells trapped in either G1 or G2 phase can apparently still enter and complete the process of differentiation, which is probably neither stage specific nor dependent on the progression of the T. brucei cell cycle. The process is probably a simple pattern change of gene expression in the trypanosome induced by a temperature decrease from 37 ∞ C to 26 ∞ C in the presence of citrate and cis- aconitate.

The ubiquitin-proteasome pathway is responsible for selective degradation of short-lived and dysfunctional proteins in eukaryotes. The recently demonstrated presence of a functional 26 S proteasome in Trypanosoma brucei led to the identification and isolation of genes encoding all 11 non-ATPase (Rpn) subunit proteins in the trypanosome 19 S regulatory complex. Using the technique of RNA interference, expression of individual RPN genes was disrupted in the procyclic form of T. brucei, resulting, in each case, in intracellular accumulation of polyubiquitinated protein, cell arrest at the G2/M phase, and eventual cell death. With the exception of Rpn10, depletion of individual Rpn proteins disrupted also trypanosome 19 S complex formation, with the complex virtually depleted in the cell lysate. This functional and structural essentiality of 10 of the 11 Rpn proteins in T. brucei differs significantly from that observed in other organisms. When Rpn10 was deficient in trypanosomes, a 19 S complex without Rpn10 was still formed, whereas cell growth was arrested. This structural dispensability but functional indispensability of Rpn10 may constitute another unique aspect of the proteasomes in T. brucei.

Centrin is a conserved component of centrioles in animals and basal bodies in flagellated organisms. It also associates with axonemal inner-arm dyneins and regulates cell motility, but the underlying mechanism remains elusive. In Trypanosoma brucei, three of the five centrins associate with the flagellar basal body, but no centrin has been found to regulate flagellar motility. Here we show that TbCentrin3 is a flagellar protein and knockdown of TbCentrin3 compromises cell motility. Tandem affinity purification followed by mass spectrometry identifies an inner-arm dynein, TbIAD5-1, as the TbCentrin3 partner, and knockdown of TbIAD5-1 causes similar cell motility defect. Further, we demonstrate the interdependence of TbCentrin3 and TbIAD5-1 for maintaining a stable complex in the flagellar axoneme. Together, these results identify the essential role of TbCentrin3 in cell motility by maintaining the stability of an inner-arm dynein in the flagellum, which may be shared by all the centrin-containing flagellated and ciliated organisms.

Summary Cyclins and cyclin-dependent kinases (CDKs) represent the fundamental, crucial regulators of the cell division cycle in eukaryotes. Trypanosoma brucei expresses a large number of cyclins and Cdc2-related kinases (CRKs). However, how these cyclins and CRKs cooperate to regulate cell cycle progression remains poorly understood. Here, we carry out directional yeast two-hybrid assays to identify the interactions between the 10 cyclins and the 11 CRKs and detect a total of 26 cyclin–CRK pairs, among which 20 pairs are new. Our current efforts are focused on four PHO80-like cyclins, CYC2, CYC4, CYC5 and CYC7, and their physical and functional interactions with CRK1. Silencing of the four cyclins and CRK1 leads to the increase of G1 cells and defective DNA replication, suggesting their important roles in promoting the G1/S transition. Additionally, CYC2-, CYC7- and CRK1-deficient cells possess an elongated posterior that is filled with newly assembled microtubules. Further, we show that the four cyclins display distinct subcellular localizations and half-lives, suggesting that they likely undergo distinct regulation. Altogether, our results demonstrate the involvement of four CRK1-associated cyclins, CYC2, CYC4, CYC5 and CYC7, in promoting the G1/S transition and the requirement of CYC2 and CYC7 in maintaining posterior cytoskeletal morphogenesis during the G1/S transition.

The cytoskeleton of Trypanosoma brucei, a unicellular eukaryote and a parasitic protozoan, is defined by the subpellicular microtubule corset that is arranged underneath the plasma membrane. We recently identified two orphan kinesins, TbKIN-C and TbKIN-D, that cooperate to regulate the organization of the subpellicular microtubule corset and thereby maintain cell morphology in the procyclic form of T. brucei. In this report, we characterize the function of TbKIN-C and TbKIN-D in the bloodstream form of T. brucei and investigate their functional cooperation in both the bloodstream and procyclic forms. TbKIN-C and TbKIN-D form a tight complex in vivo in the bloodstream form. TbKIN-C is strongly enriched at the posterior tip of the cell, whereas TbKIN-D is distributed throughout the cell body at all cell cycle stages. RNAi of TbKIN-C or TbKIN-D in the bloodstream form inhibits cell proliferation and leads to cell death, due to cytokinesis defects. RNAi of TbKIN-C and TbKIN-D also results in defects in basal body segregation, but does not affect the synthesis and segregation of the flagellum and the flagellum attachment zone (FAZ) filament. Knockdown of TbKIN-C and TbKIN-D does not disrupt the organization of the subpellicular microtubule corset, but produces multinucleated cells with an enlarged flagellar pocket and misplaced flagella. Interestingly, depletion of TbKIN-C results in rapid degradation of TbKIN-D and, similarly, knockdown of TbKIN-C destabilizes TbKIN-D, suggesting that formation of TbKIN-C/TbKIN-D complex stabilizes both kinesins and is required for the two kinesins to execute their essential cellular functions. Altogether, our results demonstrate the essential role of the two kinesins in cell morphogenesis and cytokinesis in the bloodstream form and the requirement of heteromeric complex formation for maintaining the stability of the two kinesins.

Polo-like kinases (Plks) play multiple roles in mitosis and cytokinesis in eukaryotes and are characterized by the C-terminal Polo-box domain (PBD) implicated in binding to Plk substrates, targeting Plk, and regulating Plk activity. The Plk homolog in Trypanosoma brucei possesses a similar architecture, but it lacks the crucial residues involved in substrate binding and regulates cytokinesis but not mitosis. Despite these, little is known about the regulation of TbPLK and the role of the PBD in TbPLK localization and function. Here, we addressed the requirement of the kinase activity and the PBD for TbPLK localization and function through coupling RNAi of endogenous TbPLK with ectopic expression of TbPLK mutants. We demonstrate that the kinase activity and phosphorylation of two threonine residues, Thr198 and Thr202, in the activation loop (T-loop) of the kinase domain are essential for TbPLK function but not for TbPLK localization. Deletion of the PBD abolishes TbPLK localization, but the PBD itself is not correctly targeted, indicating that TbPLK localization requires both the PBD and the kinase domain. Surprisingly, the kinase domain of TbPLK, but not the PBD, binds to its substrates, TbCentrin2 and p110, suggesting that TbPLK may interact with its substrate through different mechanisms. Finally, the PBD interacts with the kinase domain of TbPLK and inhibits its activity, and this inhibition is relieved when Thr198 is phosphorylated. Together, these results suggest an essential role of T-loop phosphorylation in TbPLK activation and crucial roles of the PBD in regulating TbPLK activity and localization.

ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA). RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms., Author Summary ATP-dependent protein-hydrolyzing enzyme complexes are present in all living organisms, including the 26S proteasome in eukaryotes and the HslVU complex in bacteria. A simultaneous presence of both complexes in an organism was originally deemed unlikely until some HslVU homologs were found in certain ancient eukaryotes, though their potential function in these organisms remains unclear. We characterized an HslVU complex in Trypanosoma brucei, a protozoan parasite that causes human sleeping sickness in Africa. The complex is an active enzyme localized to the mitochondria of the parasite and closely associated with the mitochondrial DNA complex, which consists of several thousand small circular DNAs and a few dozen mitochondrial genomic DNAs. Depletion of this HslVU from the parasite resulted in a continuous synthesis of the small circular DNA, which led to aberrant segregation and incessant growth of the mitochondrial DNA complex to an enormous size that eventually blocks cell division. This novel HslVU function, which has not been observed in other organisms previously, could be a potential target for anti-sleeping sickness chemotherapy.

Cyclins bind and activate cyclin-dependent kinases that regulate cell cycle progression in eukaryotes. Cell cycle control in Trypanosoma brucei was analyzed in the present study. Genes encoding four PHO80 cyclin homologues and three B-type cyclin homologues but no G1 cyclin homologues were identified in this organism. Through knocking down expression of the seven cyclin genes with the RNA interference technique in the procyclic form of T. brucei, we demonstrated that one PHO80 homologue (CycE1/CYC2) and a B-type cyclin homologue (CycB2) are the essential cyclins regulating G1/S and G2/M transitions, respectively. This lack of overlapping cyclin function differs significantly from that observed in the other eukaryotes. Also, PHO80 cyclin is known for its involvement only in phosphate signaling in yeast with no known function in cell cycle control. Both observations thus suggest the presence of simple and novel cell cycle regulators in trypanosomes. T. brucei cells deficient in CycE1/CYC2 displayed a long slender morphology, whereas those lacking CycB2 assumed a fat stumpy form. These cells apparently still can undergo cytokinesis generating small numbers of anucleated daughter cells, each containing a single kinetoplast known as a zoid. Two different types of zoids were identified, the slender zoid derived from reduced CycE1/CYC2 expression and the stumpy zoid from CycB2 deficiency. This observation indicates an uncoupling between the kinetoplast and the nuclear cycle, resulting in cell division driven by kinetoplast segregation with neither a priori S phase nor mitosis in the trypanosome.