Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics

Abstract

During eukaryotic cell division, the sister chromatids of duplicated chromosomes are pulled apart by microtubules, which connect via kinetochores. The kinetochore is a multiprotein structure that links centromeres to microtubules, and that emits molecular signals in order to safeguard the equal distribution of duplicated chromosomes over daughter cells. Although microtubule-mediated chromosome segregation is evolutionary conserved, kinetochore compositions seem to have diverged. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined orthologs of 70 kinetochore proteins in 90 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that current-day kinetochores differ substantially. These kinetochores diverged through gene loss, duplication, and, less frequently, invention and displacement. Various kinetochore components co-evolved with one another, albeit in different manners. These co-evolutionary patterns improve our understanding of kinetochore function and evolution, which we illustrated with the RZZ complex, TRIP13, the MCC, and some nuclear pore proteins. The extensive diversity of kinetochore compositions in eukaryotes poses numerous questions regarding evolutionary flexibility of essential cellular functions.

Keywords: co‐evolution; eukaryotic diversity; evolutionary cell biology; gene loss; kinetochore.

Figure 1. The kinetochore network across 90 eukaryotic lineages

Presences and absences (“phylogenetic profiles”) of 70 kinetochore proteins in 90 eukaryotic species. Top: Phylogenetic tree of the species in the proteome set, with colored areas for the eukaryotic supergroups. Left side: Kinetochore proteins clustered by average linkage based on the pairwise Pearson correlation coefficients of their phylogenetic profiles. Protein names have the same colors if they are members of the same complex. Proteins inferred to have been present in LECA are indicated (●). The orthologous sequences (including sets of APC/C subunits, NAG, RINT1, HORMAD, Nup106, Nup133, Nup160) are available as fasta files in Dataset EV1, allowing full usage of our data for further evolutionary cell biology investigations.

Figure 2. Kinetochores of model and non‐model species

1. The human kinetochore. The colors of the proteins indicate if they were inferred to be present in LECA and their occurrence frequency across eukaryotes (see Materials and Methods).

2. The predicted kinetochore of Tetrahymena thermopila projected onto the human kinetochore.

3. The budding yeast kinetochore. Similar to panel (B).

4. The predicted kinetochore of Cryptococcus neoformans projected onto the budding yeast kinetochore.

Figure EV1. Anaphase‐promoting complex/cyclosome (APC/C) subunits across 90 eukaryotic lineages

Presences and absences (“phylogenetic profiles”) of APC/C subunits in 90 eukaryotic species. Top: Phylogenetic tree of the species in the genome set, with colored areas for the eukaryotic supergroups. Left side: APC/C proteins clustered by average linkage based on the pairwise Pearson correlation coefficients of their phylogenetic profiles. The orthologous sequences are available as fasta files in Dataset EV1, allowing full usage of our data for further evolutionary cell biology investigations.

Figure EV2. Loss frequencies and sequence evolution of kinetochore and APC/C proteins

1. A, B

   Scatter plots for loss frequencies and dN/dS values (A) and percent identity (B) of human–mouse orthologs for the kinetochore and APC/C proteins that were inferred to have been present in LECA. Loss frequencies and dN/dS values positively correlate (P = 3.9e‐5, Spearman correlation), whereas loss frequencies and percent identity negatively correlate (P = 0.0005, Spearman correlation).

Figure EV3. Copy numbers of kinetochore proteins

Heatmap indicating the copy numbers of each kinetochore protein in the 90 eukaryotic lineages. Please note that these copy numbers might contain some over‐ and underestimates due to unpredicted or imperfectly predicted genes and database errors.

Figure 3. Phylogenetic profiles of the Rod–Zwilch–ZW10 (RZZ) complex, its mitotic interaction partners (Knl1, Zwint‐1, and Spindly), and ZW10's interphase interaction partners in the NRZ (NAG and RINT1) complex

Presences and absences across eukaryotes of the RZZ subunits, Spindly, Zwint‐1, and Knl1, and of the NRZ subunits, NAG and RINT1. Colored areas indicate eukaryotic supergroups as in Fig 1. Right side: Pairwise Pearson correlation coefficients (r) between the phylogenetic profiles including a heatmap. The indicated threshold t represents the value of r for which we found a sixfold enrichment of interacting protein pairs (see Appendix Fig S1). See also Appendix Fig S3 for the procedure by which homology between Zwint‐1, Sos7, and Kre28 was detected.

Figure EV4. Gene phylogeny of histone H3 homologs

To find the putative orthologs of CenpA, we first aligned candidate orthologous sequences, which were experimentally identified centromeric H3 variants in divergent species (indicated with a pink branch in this phylogeny). From this alignment, we constructed a profile HMM and performed multiple HMM searches through our local proteome database. From these searches, we selected 831 sequences (belonging to the histone H3 family), aligned these and constructed the gene phylogeny, which is presented in this figure (see also Materials and Methods). We rooted the phylogeny on the cluster that contained all of these experimentally identified centromeric H3 variants and some additional sequences that, based on best blast hits, were also likely to be orthologous to CenpA. The cluster did not contain the candidate orthologs in Toxoplasma gondii 81. We do not know whether this is due to an error in the gene phylogeny, or to parallel invention of a centromeric H3 variants in this species, which would mean that it is not orthologous to CenpA. Nevertheless, we included these sequences in the orthologous group. The candidate centromeric H3 variants that are part of the CenpA cluster include sequences from all five eukaryotic supergroups: Homo sapiens 82, Saccharomyces cerevisiae 83, Drosophila melanogaster 84, Caenorhabditis elegans 85, Schizosaccharomyes pombe 86 (Opisthokonta), Dictyostelium discoideum 87 (Amoebozoa), Arabidopsis thaliana 88 (Archaeplastida), Tetrahymena thermophila 89, Plasmodium falciparum 90 (SAR), Giardia intestinalis 91 and Trichomonas vaginalis 92 (Excavata). The original gene tree in newick format is provided (Dataset EV3).

Figure 4. The co‐evolutionary patterns of the multifunctional protein TRIP13

1. Model for the mode of action of TRIP13 as recently suggested 79. By hydrolyzing ATP, TRIP13 changes the conformation of HORMAD and Mad2 from closed to open, the latter via binding to co‐factor p31^comet, which forms a heterodimer with Mad2. TRIP13 has a C‐terminal AAA⁺ ATPase domain (AAA⁺) and an N‐terminal domain (NTD) and forms a hexamer 80.

2. Presences and absences of TRIP13 and of its interaction partners p31^comet and HORMAD. Colored areas indicate eukaryotic supergroups as in Fig 1.

3. Numbers of lineages in which TRIP13 is present or absent, compared to the presences of p31^comet, HORMAD or their joint presences. Also the Pearson correlation coefficients of the phylogenetic profiles as in (B) are given.

Figure 5. Correlations between proteins of the Nup107‐160 complex and proteins of the SAC

Heatmap indicating the pairwise Pearson correlation coefficients (r) of the phylogenetic profiles of proteins of the Nup107‐160 complex and of the SAC. The clustering (average linkage) on the left side of this heatmap was also based on these correlations. The indicated threshold t represents the Pearson correlation coefficient for which we found a sixfold enrichment of interacting protein pairs (see Appendix Fig S1).

Figure 6. Phylogenetic co‐occurrence of Mad2 with its interaction partners Mad1 and Cdc20 and their Mad2‐interacting motifs (MIMs)

1. A

   The sequence logos of the MIMs of Mad1 (upper panel) and Cdc20 (lower panel) based on the multiple sequence alignments of the motifs. Below is indicated the required amino acid sequence of the MIM (+: positive residue, Φ: hydrophobic residue, P: proline) which is restricted by the pattern [RK] [ILV](2)X(3,7)P.

2. B, C

   Left side: Numbers of presences and absences of Mad2 in 90 eukaryotic species and its interaction partners Mad1 (B) and Cdc20 (C). Right side: Frequencies of Mad2 and canonical MIM occurrences in species having Mad1 (B) or Cdc20 (C), respectively. Also the Pearson correlation coefficients (r) for the corresponding phylogenetic profiles are shown.

Figure EV5. Evolution of the Mad2‐interacting motif (MIM) in green plants and co‐occurrences of Mad2 with the MIM under a less strict motif definition

1. A

   Viridiplantae (green plants) phylogeny 93 and the occurrences of the canonical MIM or the “land plant” MIM in Mad1 orthologs of the associated species. Asterisk (*) indicates species lacking an aligned MIM, possibly caused by incomplete gene prediction of Mad1 orthologs.

2. B

   The sequence logos of the MIMs of Mad1 (upper panel) and Cdc20 (lower panel) based on the alignments of the motifs present in the right‐sided panels of (C and D). Below is indicated the required amino acid sequence of the MIM (+: positive residue, Φ: hydrophobic residue, P: proline). In contrast to Fig 6, the MIM is considered present if it agrees with the pattern [ILV](2)X(3,7)P or [RK][ILV](2), in order that the land plant motif suffices.

3. C, D

   Left side: Numbers of presences and absences of Mad2 in 90 eukaryotic species and its interaction partners Mad1 (C) and Cdc20 (D). Right side: Frequencies of Mad2 and MIM (according to definition in B) occurrences in species having Mad1 (C) or Cdc20 (D), respectively. Also the Pearson correlation coefficients (r) for the corresponding phylogenetic profiles are shown.