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.

Towards unraveling the origins of eukaryotic nuclear genome organization.

With 3D genome mapping maturing over the past decade, studies exposed the differences between eukaryotic and prokaryotic genome organization. This raises the question of how the complex eukaryotic genome organization originated. Here, I explore potential pathways to answering this question, guided by our changing understanding of the origins of eukaryotes.

Increased Sampling and Intracomplex Homologies Favor Vertical Over Horizontal Inheritance of the Dam1 Complex.

Kinetochores connect chromosomes to spindle microtubules to ensure their correct segregation during cell division. Kinetochores of human and yeasts are largely homologous, their ability to track depolymerizing microtubules, however, is carried out by the nonhomologous complexes Ska1-C and Dam1-C, respectively. We previously reported the unique anti-correlating phylogenetic profiles of Dam1-C and Ska-C found among a wide variety of eukaryotes. Based on these profiles and the limited presence of Dam1-C, we speculated that horizontal gene transfer could have played a role in the evolutionary history of Dam1-C. Here, we present an expanded analysis of Dam1-C evolution, using additional genome as well as transcriptome sequences and recently published 3D structures. This analysis revealed a wider and more complete presence of Dam1-C in Cryptista, Rhizaria, Ichthyosporea, CRuMs, and Colponemidia. The fungal Dam1-C cryo-EM structure supports earlier hypothesized intracomplex homologies, which enables the reconstruction of rooted and unrooted phylogenies. The rooted tree of concatenated Dam1-C subunits is statistically consistent with the species tree of eukaryotes, suggesting that Dam1-C is ancient, and that the present-day phylogenetic distribution is best explained by multiple, independent losses and no horizontal gene transfer was involved. Furthermore, we investigated the ancient origin of Dam1-C via profile-versus-profile searches. Homology among 8 out of the 10 Dam1-C subunits suggests that the complex largely evolved from a single multimerizing subunit that diversified into a hetero-octameric core via stepwise subunit duplication and subfunctionalization of the subunits before the origin of the last eukaryotic common ancestor.

Chromosomal instability by mutations in the novel minor spliceosome component CENATAC.

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.

Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species.

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

Timing the origin of eukaryotic cellular complexity with ancient duplications.

Eukaryogenesis is one of the most enigmatic evolutionary transitions, during which simple prokaryotic cells gave rise to complex eukaryotic cells. While evolutionary intermediates are lacking, gene duplications provide information on the order of events by which eukaryotes originated. Here we use a phylogenomics approach to reconstruct successive steps during eukaryogenesis. We find that gene duplications roughly doubled the proto-eukaryotic gene repertoire, with families inherited from the Asgard archaea-related host being duplicated most. By relatively timing events using phylogenetic distances, we inferred that duplications in cytoskeletal and membrane-trafficking families were among the earliest events, whereas most other families expanded predominantly after mitochondrial endosymbiosis. Altogether, we infer that the host that engulfed the proto-mitochondrion had some eukaryote-like complexity, which drastically increased upon mitochondrial acquisition. This scenario bridges the signs of complexity observed in Asgard archaeal genomes to the proposed role of mitochondria in triggering eukaryogenesis.

Author Correction: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mosaic origin of the eukaryotic kinetochore.

The emergence of eukaryotes from ancient prokaryotic lineages embodied a remarkable increase in cellular complexity. While prokaryotes operate simple systems to connect DNA to the segregation machinery during cell division, eukaryotes use a highly complex protein assembly known as the kinetochore. Although conceptually similar, prokaryotic segregation systems and the eukaryotic kinetochore are not homologous. Here we investigate the origins of the kinetochore before the last eukaryotic common ancestor (LECA) using phylogenetic trees, sensitive profile-versus-profile homology detection, and structural comparisons of its protein components. We show that LECA's kinetochore proteins share deep evolutionary histories with proteins involved in a few prokaryotic systems and a multitude of eukaryotic processes, including ubiquitination, transcription, and flagellar and vesicular transport systems. We find that gene duplications played a major role in shaping the kinetochore; more than half of LECA's kinetochore proteins have other kinetochore proteins as closest homologs. Some of these have no detectable homology to any other eukaryotic protein, suggesting that they arose as kinetochore-specific folds before LECA. We propose that the primordial kinetochore evolved from proteins involved in various (pre)eukaryotic systems as well as evolutionarily novel folds, after which a subset duplicated to give rise to the complex kinetochore of LECA.

Inferring the Evolutionary History of Your Favorite Protein: A Guide for Molecular Biologists.

Comparative genomics has proven a fruitful approach to acquire many functional and evolutionary insights into core cellular processes. Here it is argued that in order to perform accurate and interesting comparative genomics, one first and foremost has to be able to recognize, postulate, and revise different evolutionary scenarios. After all, these studies lack a simple protocol, due to different proteins having different evolutionary dynamics and demanding different approaches. The authors here discuss this challenge from a practical (what are the observations?) and conceptual (how do these indicate a specific evolutionary scenario?) viewpoint, with the aim to guide investigators who want to analyze the evolution of their protein(s) of interest. By sharing how the authors draft, test, and update such a scenario and how it directs their investigations, the authors hope to illuminate how to execute molecular evolution studies and how to interpret them. Also see the video abstract here https://youtu.be/VCt3l2pbdbQ.

A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKK's minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends.

Unique Phylogenetic Distributions of the Ska and Dam1 Complexes Support Functional Analogy and Suggest Multiple Parallel Displacements of Ska by Dam1.

Faithful chromosome segregation relies on kinetochores, the large protein complexes that connect chromatin to spindle microtubules. Although human and yeast kinetochores are largely homologous, they track microtubules with the unrelated protein complexes Ska (Ska-C, human) and Dam1 (Dam1-C, yeast). We here uncovered that Ska-C and Dam1-C are both widespread among eukaryotes, but in an exceptionally inverse manner, supporting their functional analogy. Within the complexes, all Ska-C and various Dam1-C subunits are ancient paralogs, showing that gene duplication shaped these complexes. We examined various evolutionary scenarios to explain the nearly mutually exclusive patterns of Ska-C and Dam1-C in present-day species. We propose that Ska-C was present in the last eukaryotic common ancestor, that subsequently Dam1-C displaced Ska-C in an early fungus and was horizontally transferred to diverse non-fungal lineages, displacing Ska-C in these lineages too.

Small homologous blocks in phytophthora genomes do not point to an ancient whole-genome duplication.

Genomes of the plant-pathogenic genus Phytophthora are characterized by small duplicated blocks consisting of two consecutive genes (2HOM blocks) and by an elevated abundance of similarly aged gene duplicates. Both properties, in particular the presence of 2HOM blocks, have been attributed to a whole-genome duplication (WGD) at the last common ancestor of Phytophthora. However, large intraspecies synteny-compelling evidence for a WGD-has not been detected. Here, we revisited the WGD hypothesis by deducing the age of 2HOM blocks. Two independent timing methods reveal that the majority of 2HOM blocks arose after divergence of the Phytophthora lineages. In addition, a large proportion of the 2HOM block copies colocalize on the same scaffold. Therefore, the presence of 2HOM blocks does not support a WGD at the last common ancestor of Phytophthora. Thus, genome evolution of Phytophthora is likely driven by alternative mechanisms, such as bursts of transposon activity.