Biology subjects

Akiyoshi, B.

Publications and source records attributed to Akiyoshi, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Auxin-inducible degron-mediated protein depletion in Diplonema papillatum

Diplonemids are highly abundant unicellular flagellates that are evolutionarily divergent from traditional model eukaryotes. Diplonema papillatum is a model diplonemid for which several molecular tools have recently been established. However, no method for conditional protein depletion was available. Here, we have adapted an auxin-inducible degron (AID) system in D. papillatum. Depletion of the KIN-A motor protein (a glycomonad-specific component of the chromosomal passenger complex) causes defects in chromosome segregation and cell proliferation. The AID system also successfully depleted a divergent Mad1 protein that localizes at nuclear pores, and a flagellar component, CFAP20. Our study demonstrates the power of the AID system to perform loss-of-function studies in D. papillatum.

cell biology↗

bioRxiv2026

The centromere localization domain of kinetoplastid kinetochore protein KKT2 recognizes the free N-terminus of histone H3

Kinetochores are multiprotein complexes that drive chromosome segregation in eukaryotes. Kinetoplastids, a group of early-diverging eukaryotes that lack canonical kinetochore components, have a unique set of kinetochore proteins. How their kinetochores are assembled specifically at centromeres in the absence of a centromere-specific histone H3 variant CENP-A remains unknown. Here, we demonstrate that the centromere localization (CL) domain of KKT2 has similarities to a ZZ domain. An invariant aspartate present in histone H3-binding ZZ domains is conserved in the KKT2 CL domain. Using nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we show that Trypanosoma brucei KKT2 CL binds the N-terminus of histone H3. Strikingly, even mono-methylation of the N-terminal amino group of histone H3 abolishes the binding. Our study raises a possibility that centromere-specific kinetochore assembly in T. brucei is ensured by abundant N-terminal methylations of histone H3 in non-centromeric regions.

cell biology↗

bioRxiv2026

Identification of feeding apparatus components in a heterotrophic marine flagellate

Acquiring nutrients is a fundamental biological process of all organisms, playing crucial roles in ecological sustainability. Diplonemids are highly abundant heterotrophic unicellular flagellates that are widespread in the worlds ocean. They have a highly complex microtubule-based feeding apparatus (cytostome-cytopharynx complex) located adjacent to the deep flagellar pocket from which two flagella emerge from parallel basal bodies. The apical papilla is a tongue-shaped structure unique to diplonemids that connects the cytopharynx and the flagellar pocket, the latter of which is formed by reinforcing microtubules (MTR) and two flagellar roots called intermediate and dorsal roots. Here we report identification of 17 proteins that localize at the feeding apparatus or flagellar apparatus in Diplonema papillatum. Using ultrastructure expansion microscopy, we show that Mad2 and its interaction partner MBP65 localize at the MTR, intermediate root, and dorsal root. Homologs of proteins that associate with the flagellar apparatus in Trypanosoma brucei (PFR2, KMP11, BILBO1) localize at the feeding apparatus in D. papillatum. We also identify proteins that localize at the apical papilla, MTR, parallel microtubule loop, or cytopharynx. By discovering components of the feeding apparatus for the first time in diplonemids, this work forms the foundation to understand molecular mechanisms of the feeding apparatus in these highly abundant marine plankton.

cell biology↗

bioRxiv2025

Subcellular proteomics of Paradiplonema papillatum reveals digestive capacity of the cell membrane and the plasticity of peroxisomes across euglenozoans

Diplonemids are among the most diverse and abundant protists in the deep ocean, have extremely complex and ancient cellular systems, and exhibit unique metabolic capacities. Despite this, we know very little about this major group of eukaryotes. To establish a model organism for comprehensive investigation, we performed subcellular proteomics on Paradiplonema papillatum and localized 4,870 proteins to 22 cellular compartments. We additionally confirmed the predicted location of several proteins by epitope tagging and fluorescence microscopy. To probe the metabolic capacities of P. papillatum, we explored the proteins predicted to the cell membrane compartment in our subcellular proteomics dataset. Our data revealed an accumulation of many carbohydrate active enzymes (CAZymes). Our predictions suggest that these CAZymes are exposed to extracellular space, supporting proposals that diplonemids may specialize in breaking down carbohydrates in plant and algal cell walls. Further exploration of carbohydrate metabolism revealed an evolutionary divergence in the function of glycosomes (modified peroxisomes) in diplonemids versus kinetoplastids. Our subcellular proteome provides a resource for future investigations into the unique cell biology of diplonemids.

cell biology↗

bioRxiv2025

Beyond a Linear Structure: The Tubular Organization of the Tripartite Attachment Complex and the Functional Role of TAC53

The Tripartite Attachment Complex (TAC) is essential for mitochondrial DNA (kDNA) segregation in Trypanosoma brucei, providing a physical link between the flagellar basal body and the mitochondrial genome. Although the TACs hierarchical assembly and linear organization have been extensively studied, much remains to be discovered regarding its complete architecture and composition - for instance, our identification of a new TAC component underscores these knowledge gaps. Here, we use a combination of proteomics, RNA interference (RNAi), and Ultrastructure Expansion Microscopy (U-ExM) to characterize the TAC at high resolution and identify a novel component, TAC53 (Tb927.2.6100). Depletion of TAC53 in both procyclic and bloodstream forms results in kDNA missegregation and loss, a characteristic feature of TAC dysfunction. TAC53 localizes to the kDNA in a cell cycle-dependent manner and represents the most kDNA-proximal TAC component identified to date. U-ExM reveals a previously unrecognized tubular architecture of the TAC, with two distinct TAC structures per kDNA disc, suggesting a mechanism for precise kDNA alignment and segregation. Moreover, immunoprecipitation and imaging analyses indicate that TAC53 interacts with known TAC-associated proteins HMG44, KAP68, and KAP3, forming a network at TAC-kDNA the interface. These findings redefine our understanding of TAC architecture and function and identify TAC53 as a key structural component anchoring the mitochondrial genome in T. brucei. Significance StatementThis research identifies a new component (TAC53) of the tripartite attachment complex (TAC), a cellular machinery that anchors the mitochondrial DNA to a cytoskeletal structure, the basal body in Trypanosoma brucei. Using proteomics and high-resolution microscopy, we demonstrate that TAC53 is at the interface of the TAC and the mitochondrial DNA and likely the final piece in this structure. We also describe the overall tubular architecture of the TAC from the basal body to the mitochondrial matrix, and how the presence of two TAC structures per mitochondrial genome can explain the parasites ability to maintain its mitochondrial DNA accurately. In summary, we present a new component and the architecture of the currently best understood mitochondrial DNA segregation mechanism in biology.

cell biology↗

bioRxiv2025

Discovery of unique mitotic mechanisms in Paradiplonema papillatum

Diplonemids are highly diverse and abundant marine plankton with significant ecological importance. However, little is known about their biology, even in the model diplonemid Paradiplonema papillatum whose genome sequence is available. Examining the subcellular localization of proteins using fluorescence microscopy is a powerful approach to infer their putative function. Here we report a plasmid-based method that enables YFP-tagging of a gene at the endogenous locus. By examining the localization of proteins whose homologs are involved in chromosome segregation in other eukaryotes, we discovered several interesting features in mitotically dividing P. papillatum cells. Cohesin is enriched on condensed interphase chromatin. During mitosis, chromosomes organize into two rings (termed metaphase rings herein) that surround the elongating nucleolus. Homologs of chromosomal passenger complex components (INCENP, two Aurora kinases, and KIN-A), a CLK1 kinase, spindle checkpoint protein Mad1, and microtubule regulator XMAP215 localize in between the two metaphase rings, suggesting that kinetochores may assemble in between them. We also found that a homolog of the meiotic chromosome axis protein SYCP2L1 is enriched in between metaphase rings during mitosis. These features have some resemblance to the bivalent bridge (also known as the modified synaptonemal complex), which is thought to mediate the linkage between homologous chromosomes using axis components in Bombyx mori female meiotic metaphase I. By representing the first molecular characterization of mitotic mechanisms in P. papillatum and raising a number of questions, this study forms the foundation for dissecting mitotic mechanisms in diplonemids.

cell biology↗

bioRxiv2024

The kinetoplastid kinetochore protein KKT23 acetyltransferase is a structural homolog of GCN5 that acetylates the histone H2A C-terminal tail

The kinetochore is the macromolecular protein machine that drives chromosome segregation in eukaryotes. In an evolutionarily divergent group of organisms called kinetoplastids, kinetochores are built using a unique set of proteins (KKT1-25 and KKIP1-12). KKT23 is a constitutively localised kinetochore protein containing a C-terminal acetyltransferase domain of unknown function. Here, using X-ray crystallography and NMR spectroscopy, we have determined the structure and dynamics of the KKT23 acetyltransferase domain from Trypanosoma brucei and found that it is structurally similar to the GCN5 histone acetyltransferase domain. We find that KKT23 can acetylate the C-terminal tail of histone H2A and that knockdown of KKT23 results in decreased H2A acetylation levels in T. brucei. Finally, we have determined the crystal structure of the N-terminal region of KKT23 and shown that it interacts with KKT22. Our study provides important insights into the structure and function of the unique kinetochore acetyltransferase in trypanosomes.

cell biology↗

bioRxiv2024

On the possibility of yet a third kinetochore system in the protist phylum Euglenozoa

Transmission of genetic material from one generation to the next is a fundamental feature of all living cells. In eukaryotes, a macromolecular complex called the kinetochore plays crucial roles during chromosome segregation by linking chromosomes to spindle microtubules. Little is known about this process in evolutionarily diverse protists. Within the supergroup Discoba, Euglenozoa forms a speciose group of unicellular flagellates - kinetoplastids, euglenids, and diplonemids. Kinetoplastids have an unconventional kinetochore system, while euglenids have subunits that are conserved amongst most eukaryotes. For diplonemids, a group of extremely diverse and abundant marine flagellates, it remains unclear what kind of kinetochores are present. Here, we employed deep homology detection protocols using profile-versus-profile Hidden Markov Model searches and AlphaFold-based structural comparisons to detect homologies that might have been previously missed. Interestingly, we still could not detect orthologs for most of the kinetoplastid nor canonical kinetochore subunits with few exceptions including a putative centromere-specific histone H3 variant (cenH3/CENP-A), the spindle checkpoint protein Mad2, the chromosomal passenger complex members Aurora and INCENP, and broadly conserved proteins like CLK kinase and the meiotic synaptonemal complex proteins SYCP2/3 that also function at kinetoplastid kinetochores. We examined the localization of five candidate kinetochore-associated proteins in the model diplonemid, Paradiplonema papillatum. PpCENP-A shows discrete dots in the nucleus, implying that it is likely a kinetochore component. PpMad2, PpCLKKKT10/19, PpSYCP2L1KKT17/18 and INCENP reside in the nucleus, but no clear kinetochore localization was observed. Altogether, these results point to the possibility that diplonemids evolved a hitherto unknown type of kinetochore system. IMPORTANCEA macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplonemids except for a putative centromere-specific histone H3 variant. Our results strongly suggest that diplonemids evolved kinetochores that do not resemble previously known ones.

cell biology↗

bioRxiv2024

An unconventional regulatory circuitry involving Aurora B controls anaphase onset and error-free chromosome segregation in trypanosomes

Accurate chromosome segregation during mitosis requires that all chromosomes establish stable bi-oriented attachments with the spindle apparatus. Kinetochores form the interface between chromosomes and spindle microtubules and as such are under tight control by complex regulatory circuitry. As part of the chromosomal passenger complex (CPC), the Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint, a feedback control system that delays the onset of anaphase by inhibiting the anaphase-promoting complex/cyclosome. Intriguingly, Aurora B is conserved even in kinetoplastids, an evolutionarily divergent group of eukaryotes, whose kinetochores are composed of a unique set of structural and regulatory proteins. Kinetoplastids do not have a canonical spindle checkpoint and it remains unclear how their kinetochores are regulated to ensure the fidelity and timing of chromosome segregation. Here, we show in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness, that inhibition of Aurora B using an analogue-sensitive approach arrests cells in metaphase, with a reduction in properly bi-oriented kinetochores. Aurora B phosphorylates several kinetochore proteins in vitro, including the N-terminal region of the divergent Bub1-like protein KKT14. Depletion of KKT14 partially overrides the cell cycle arrest caused by Aurora B inhibition, while overexpression of a non-phosphorylatable KKT14 protein results in a prominent delay in the metaphase-to-anaphase transition. Finally, we demonstrate using a nanobody-based system that re-targeting the catalytic module of the CPC to the outer kinetochore is sufficient to promote mitotic exit but causes massive chromosome mis-segregation in anaphase. Our results indicate that the CPC and KKT14 are involved in an unconventional pathway controlling mitotic exit and error-free chromosome segregation in trypanosomes.

cell biology↗

bioRxiv2024

Kinetoplastid kinetochore proteins KKT14-KKT15 are divergent Bub1/BubR1-Bub3 proteins

Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1-25. Available evidence suggests that T. brucei lacks components of the spindle checkpoint and is unable to delay cell cycle progression in response to spindle defects. It therefore remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of Bub1, a key component of the spindle checkpoint in other eukaryotes. In addition, KKT14 in T. brucei and other trypanosomatids has a putative ABBA motif that is present in Bub1 and its paralog BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14-KKT15 are divergent orthologs of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.

cell biology↗

bioRxiv2023

Dynamic localization of the chromosomal passenger complex is controlled by the orphan kinesin KIN-A in the kinetoplastid parasite Trypanosoma brucei

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess a unique set of kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles or how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. By employing biochemical, structural, and cell biological approaches, we demonstrate that KIN-A and KIN-B serve as the scaffold for the assembly of the remaining CPC subunits. Kinetochore localization of the CPC depends on the KKT7 - KKT8 complex pathway, with the C-terminal unstructured tail of KIN-A serving as a key interaction partner for the KKT8 complex. Our data therefore show that, unlike other eukaryotes that take advantage of histone modifications for centromere recruitment, trypanosomes rely on kinetochore proteins to recruit the CPC onto kinetochores. Furthermore, the ATPase activity of KIN-A promotes chromosome alignment in prometaphase and CPC translocation to the central spindle upon anaphase onset. Thus, KIN-A constitutes a unique two-in-one CPC localization module in complex with KIN-B, which directs the CPC to kinetochores (from S phase until metaphase) via its C-terminal tail, and to the central spindle (in anaphase) via its N-terminal kinesin motor domain. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora B kinases in early eukaryotes.

cell biology↗

bioRxiv2022

Targeted protein degradation using degradFP in Trypanosoma brucei

Targeted protein degradation is an invaluable tool in studying the function of proteins. Such a tool was not available in Trypanosoma brucei, an evolutionarily divergent eukaryote that causes human African trypanosomiasis. Here, we have adapted degradFP (degrade Green Fluorescent Protein), a protein degradation system based on the SCF E3 ubiquitin ligase complex and anti-GFP nanobody, in T. brucei. As a proof of principle, we targeted a kinetoplastid kinetochore protein (KKT3) that constitutively localizes at kinetochores in the nucleus. Induction of degradFP in a cell line that had both alleles of KKT3 tagged with YFP caused more severe growth defect than RNAi in procyclic (insect form) cells. degradFP also worked on a cytoplasmic protein (COPII subunit, SEC31). Given the easiness in making GFP fusion cell lines in T. brucei, degradFP can serve as a powerful tool to rapidly deplete proteins of interest, especially those with low turnover rates.

cell biology↗

bioRxiv2022

Divergent polo boxes in KKT2 and KKT3 initiate the kinetochore assembly cascade in Trypanosoma brucei

Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. In this study, we have characterized two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres and promote the recruitment of other kinetochore proteins. KKT2 and KKT3 have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases. We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in the procyclic form of Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in T. brucei. Furthermore, we identify the C-terminal domain of KKT1 as a direct interaction partner for the divergent polo boxes of KKT2, but not KKT3. KKT6 is found to interact with KKT1. These results show that KKT2 and KKT3 recruit downstream kinetochore proteins by direct protein-protein interactions using their divergent polo boxes.

cell biology↗

bioRxiv2021

Repurposing of Synaptonemal Complex Proteins for Kinetochores in Kinetoplastida

Chromosome segregation in eukaryotes is driven by a macromolecular protein complex called the kinetochore that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. In contrast, unicellular flagellates of the class Kinetoplastida have a unique set of kinetochore components. The evolutionary origin and history of these kinetochores remains unknown. Here, we report evidence of homology between three kinetoplastid kinetochore proteins KKT16-18 and axial element components of the synaptonemal complex, such as the SYCP2:SYCP3 multimers found in vertebrates. The synaptonemal complex is a zipper-like structure that assembles between homologous chromosomes during meiosis to promote recombination. Using a sensitive homology detection protocol, we identify divergent orthologues of SYCP2:SYCP3 in most eukaryotic supergroups including other experimentally established axial element components, such as Red1 and Rec10 in budding and fission yeast, and the ASY3:ASY4 multimers in land plants. These searches also identify KKT16-18 as part of this rapidly evolving protein family. The widespread presence of the SYCP2-3 gene family in extant eukaryotes suggests that the synaptonemal complex was likely present in the last eukaryotic common ancestor. We found at least twelve independent duplications of the SYCP2-3 gene family throughout the eukaryotic tree of life, providing opportunities for new functional complexes to arise, including KKT16-18 in Trypanosoma brucei. We propose that kinetoplastids evolved their unique kinetochore system by repurposing meiotic components of the chromosome synapsis and homologous recombination machinery that were already present in early eukaryotes.

cell biology↗

bioRxiv2020

Analysis of a Mad2 homolog in Trypanosoma brucei provides possible hints on the origin of the spindle checkpoint

The spindle checkpoint is a surveillance mechanism that ensures accurate nuclear DNA segregation in eukaryotes. It does so by delaying the onset of anaphase until all kinetochores have established proper attachments to spindle microtubules. The evolutionary origin of the spindle checkpoint remains unclear. The flagellated kinetoplastid parasite Trypanosoma brucei has a nucleus that contains the nuclear genome and a kinetoplast that contains the mitochondrial genome. The kinetoplast is physically linked to the basal body of the flagellum and its segregation is driven by the movement of basal bodies. While there is no strong evidence that T. brucei possesses a functional spindle checkpoint, it has been suggested that initiation of cytokinesis may be linked to the completion of kinetoplast segregation or basal body separation in this organism. Interestingly, the only identifiable spindle checkpoint component TbMad2 localizes at the basal body area. Here, I report identification of proteins that co-purified with TbMad2. One protein, which I propose to call TbMBP65, localizes at the basal body area and has a putative Mad2-interacting motif. Interestingly, 26S proteasome subunits also co-purified with TbMad2, raising a possibility that TbMad2 might regulate proteasome activity to regulate or monitor the segregation of basal bodies. I speculate that such a function might represent a prototype of the spindle checkpoint. I also show that TbAUK3, one of the three Aurora kinase homologs in T. brucei, localizes at the basal body area from late G1 until the time of kinetoplast separation. Immunoprecipitation of TbAUK3 identified an uncharacterized protein (termed TbABP79) that has a similar localization pattern as TbAUK3. These findings provide a starting point to reveal the function of TbMad2 and TbAUK3 as well as the origin of the spindle checkpoint.

cell biology↗

bioRxiv2020

Structural characterisation of KKT4, an unconventional microtubule-binding kinetochore protein

The kinetochore is the macromolecular protein machinery that drives chromosome segregation by interacting with spindle microtubules. Unlike most other eukaryotes that have canonical kinetochore proteins, a group of evolutionarily divergent eukaryotes called kinetoplastids (such as Trypanosoma brucei) have a unique set of kinetochore proteins. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterise the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The crystal structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Finally, we show that KKT4 binds DNA with high affinity. Taken together, these results provide the first structural insights into the unconventional kinetoplastid kinetochore protein KKT4.

cell biology↗

bioRxiv2019

Unconventional kinetochore kinases KKT2 and KKT3 have a unique zinc finger that promotes their kinetochore localization

Chromosome segregation in eukaryotes is driven by the kinetochore, the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Cells must tightly control the number and position of kinetochores so that all chromosomes assemble a single kinetochore. A central player in this process is the centromere-specific histone H3 variant CENP-A, which localizes constitutively at centromeres and promotes kinetochore assembly. However, CENP-A is absent from several eukaryotic lineages including kinetoplastids, a group of evolutionarily divergent eukaryotes that have an unconventional set of kinetochore proteins. There are six proteins that localize constitutively at centromeres in the kinetoplastid parasite Trypanosoma brucei, among which two homologous protein kinases (KKT2 and KKT3) have limited similarity to polo-like kinases. In addition to the N-terminal kinase domain and the C-terminal divergent polo boxes, KKT2 and KKT3 have a central domain of unknown function as well as putative DNA-binding motifs. Here we show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization in T. brucei. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.

cell biology↗

bioRxiv2019

Unconventional kinetochore kinases KKT10/19 promote the metaphase to anaphase transition in Trypanosoma brucei

The kinetochore is a macromolecular protein complex that drives chromosome segregation in eukaryotes. Unlike most eukaryotes that have canonical kinetochore proteins, evolutionarily divergent kinetoplastids such as Trypanosoma brucei have unconventional kinetochore proteins. T. brucei also lacks a canonical spindle checkpoint system and it therefore remains unknown how mitotic progression is regulated in this organism. Here we characterized two paralogous kinetochore proteins with a CLK-like kinase domain, KKT10 and KKT19, which localize at kinetochores in metaphase but disappear at the onset of anaphase. We found that these proteins are functionally redundant. Double knockdown of KKT10/19 led to a significant delay in the metaphase to anaphase transition. A kinase-dead mutant of KKT10 failed to rescue the KKT10/19 depletion phenotype, suggesting that its kinase activity is essential. We also found that phosphorylation of two kinetochore proteins KKT4 and KKT7 depends on KKT10/19 in vivo. Finally, we showed that the N-terminal part of KKT7 directly interacts with KKT10 and that kinetochore localization of KKT10 depends not only on KKT7 but also on the KKT8 complex. Our results reveal that kinetochore localization of KKT10/19 is tightly controlled to regulate the metaphase to anaphase transition in T. brucei.\n\nSummaryTrypanosoma brucei has unique kinetochore proteins and lacks a canonical spindle checkpoint system. How mitotic progression is regulated in this organism remains unclear. Here we show that two redundant protein kinases KKT10/19 promote the metaphase to anaphase transition.

cell biology↗