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. It 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 a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue 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 orthologues of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.

Dynamic localization of the chromosomal passenger complex in trypanosomes is controlled by the orphan kinesins KIN-A and KIN-B.

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 unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor 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 kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique 'two-in-one' CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. 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 kinases in early eukaryotes.

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.

HP1 proteins regulate nucleolar structure and function by secluding pericentromeric constitutive heterochromatin.

Nucleoli are nuclear compartments regulating ribosome biogenesis and cell growth. In embryonic stem cells (ESCs), nucleoli containing transcriptionally active ribosomal genes are spatially separated from pericentromeric satellite repeat sequences packaged in largely repressed constitutive heterochromatin (PCH). To date, mechanisms underlying such nuclear partitioning and the physiological relevance thereof are unknown. Here we show that repressive chromatin at PCH ensures structural integrity and function of nucleoli during cell cycle progression. Loss of heterochromatin proteins HP1α and HP1ß causes deformation of PCH, with reduced H3K9 trimethylation (H3K9me3) and HP1γ levels, absence of H4K20me3 and upregulated major satellites expression. Spatially, derepressed PCH aberrantly associates with nucleoli accumulating severe morphological defects during S/G2 cell cycle progression. Hp1α/ß deficiency reduces cell proliferation, ribosomal RNA biosynthesis and mobility of Nucleophosmin, a major nucleolar component. Nucleolar integrity and function require HP1α/ß proteins to be recruited to H3K9me3-marked PCH and their ability to dimerize. Correspondingly, ESCs deficient for both Suv39h1/2 H3K9 HMTs display similar nucleolar defects. In contrast, Suv4-20h1/2 mutant ESCs lacking H4K20me3 at PCH do not. Suv39h1/2 and Hp1α/ß deficiency-induced nucleolar defects are reminiscent of those defining human ribosomopathy disorders. Our results reveal a novel role for SUV39H/HP1-marked repressive constitutive heterochromatin in regulating integrity, function and physiology of nucleoli.

Successful mating and hybridisation in two closely related flatworm species despite significant differences in reproductive morphology and behaviour.

Reproductive traits are some of the fastest diverging characters and can serve as reproductive barriers. The free-living flatworm Macrostomum lignano, and its congener M. janickei are closely related, but differ substantially in their male intromittent organ (stylet) morphology. Here, we examine whether these morphological differences are accompanied by differences in behavioural traits, and whether these could represent barriers to successful mating and hybridization between the two species. Our data shows that the two species differ in many aspects of their mating behaviour. Despite these differences, the species mate readily with each other in heterospecific pairings. Although both species have similar fecundity in conspecific pairings, the heterospecific pairings revealed clear postmating barriers, as few heterospecific pairings produced F1 hybrids. These hybrids had a stylet morphology that was intermediate between that of the parental species, and they were fertile. Finally, using a mate choice experiment, we show that the nearly two-fold higher mating rate of M. lignano caused it to mate more with conspecifics, leading to assortative mating, while M. janickei ended up mating more with heterospecifics. Thus, while the two species can hybridize, the mating rate differences could possibly lead to higher fitness costs for M. janickei compared to M. lignano.