Naegleria’s mitotic spindles are built from unique tubulins and highlight core spindle features

ABSTRACTNaegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis, and thus provides a unique system to study the evolution and functional specificity of mitotic tubulins and the resulting spindle. Previous studies showed that Naegleria amoebae express a divergent α-tubulin during mitosis and we now show that Naegleria amoebae express a second mitotic α- and two mitotic β-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and β- tubulins, contain residues that suggest distinct microtubule properties, and may represent drug targets for the “brain-eating amoeba” Naegleria fowleri. Using quantitative light microscopy, we find that Naegleria’s mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria’s spindle is twisted and its length increases during mitosis suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers, and secondary bundles function as bridging fibers.

Голі амеби в ґрунтах Харківської області (Україна)

У ґрунтах Харківської області (Україна) нами ідентифіковано 17 видів голих амеб. Це такі види: Rhizamoeba sp., Saccamoeba stagnicola Page, 1974, Hartmannella vermiformis Page, 1967, Deuteramoeba mycophaga (Pussard et al., 1980) Page, 1988, Thecamoeba striata (Penard, 1890) Schaeffer, 1926, Stenamoeba stenopodia (Page, 1969) Smirnov et al., 2007, Mayorella cantabrigiensis Page, 1983, Mayorella sp., Korotnevella sp., Vexillifera sp., Vannella sp., Ripella platypodia Smirnov, Nassonova, Chao et Cavalier-Smith, 2007, Acanthamoeba sp. (1), Cochliopodium sp., Vahlkampfia sp. (1), Vahlkampfia sp. (2), Naegleria gruberi Schardinger, 1899. Серед ідентифікованих видів найбільш поширеними виявились M. cantabrigiensis, Acanthamoeba sp. (1), Cochliopodium sp., N. gruberi, Vexillifera sp., Vahlkampfia sp. (1), Vahlkampfia sp. (2), S. stenopodia, найменш поширеними – D. mycophaga, Korotnevella sp., Rhizamoeba sp., Mayorella sp., середнє положення за частотою трапляння займають види H. vermiformis, Vannella sp., S. stagnicola, T. striata, R. platypodia. Найбільша кількість видів характерна для ґрунтів лісів (16 видів), найменша – для ґрунтів галявин (9 видів), у ґрунтах чагарників траплялось 11 видів голих амеб. Ідентифіковані нами види належать до 11 морфотипів: розгалуженого (Rhizamoeba sp.), моноподіального (S. stagnicola, H. vermiformis), політактичного (D. mycophaga), стріатного (T. striata), язикоподібного (S. stenopodia), майорельного (M. cantabrigiensis, Mayorella sp.), дактилоподіального (Korotnevella sp., Vexillifera sp.), віялоподібного (Vannella sp., R. platypodia), акантоподіального (Acanthamoeba sp. (1)), лінзоподібного (Cochliopodium sp.), еруптивного (Vahlkampfia sp. (1), Vahlkampfia sp. (2), N. gruberi). За видовим складом населення голих амеб поділяється на два комплекси: з ґрунтів лісів і ґрунтів галявин та чагарників. Таку ж подібність демонструють і морфотипи голих амеб. На формування видових комплексів амеб та їх морфотипів за результатами непараметричного багатовимiрного шкалювання (MDS) впливають в більшій мірі вологість та кислотність ґрунтів, в меншій мірі – температурний фактор.

The evolution of the huntingtin-associated protein 40 (HAP40) in conjunction with huntingtin

Background The huntingtin-associated protein 40 (HAP40) abundantly interacts with huntingtin (HTT), the protein that is altered in Huntington’s disease (HD). Therefore, we analysed the evolution of HAP40 and its interaction with HTT. Results We found that in amniotes HAP40 is encoded by a single-exon gene, whereas in all other organisms it is expressed from multi-exon genes. HAP40 co-occurs with HTT in unikonts, including filastereans such as Capsaspora owczarzaki and the amoebozoan Dictyostelium discoideum, but both proteins are absent from fungi. Outside unikonts, a few species, such as the free-living amoeboflagellate Naegleria gruberi, contain putative HTT and HAP40 orthologs. Biochemically we show that the interaction between HTT and HAP40 extends to fish, and bioinformatic analyses provide evidence for evolutionary conservation of this interaction. The closest homologue of HAP40 in current protein databases is the family of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs). Conclusion Our results indicate that the transition from a multi-exon to a single-exon gene appears to have taken place by retroposition during the divergence of amphibians and amniotes, followed by the loss of the parental multi-exon gene. Furthermore, it appears that the two proteins probably originated at the root of eukaryotes. Conservation of the interaction between HAP40 and HTT and their likely coevolution strongly indicate functional importance of this interaction.

Evolutionary history of mitochondrial genomes in Discoba, including the extreme halophile Pleurostomum flabellatum (Heterolobosea)

Data from Discoba (Heterolobosea, Euglenozoa, Tsukubamonadida, and Jakobida) are essential to understand the evolution of mitochondrial genomes (mitogenomes), since this clade includes the most primitive-looking mitogenomes known, as well some extremely divergent genome information systems. Heterolobosea encompasses more than 150 described species, many of them from extreme habitats, but only six heterolobosean mitogenomes have been fully sequenced to date. Here we complete the mitogenome of the heterolobosean Pleurostomum flabellatum, which is extremely halophilic and reportedly also lacks classical mitochondrial cristae, hinting at reduction or loss of respiratory function. The mitogenome of P. flabellatum maps as a 57,829 bp long circular molecule, including 40 CDSs (19 tRNA, two rRNA, and 19 orfs). The gene content and gene arrangement are similar to Naegleria gruberi and N. fowleri, the closest relatives with sequenced mitogenomes. The P. flabellatum mitogenome contains genes that encode components of the electron transport chain similar to those of Naegleria mitogenomes. Homology searches against a draft nuclear genome showed that P. flabellatum has two homologs of the highly conserved Mic60 subunit of the MICOS complex, and likely lost Mic19 and Mic10. However, electron microscopy showed no cristae structures. We infer that P. flabellatum, which originates from high salinity (313‰) water where the dissolved-oxygen concentration is low, possesses a mitochondrion capable of aerobic respiration, but with reduced development of cristae structure reflecting limited use of this aerobic capacity (e.g., microaerophily).

Moving simply: Naegleria crawls and feeds using an ancient Arp2/3-dependent mechanism

Arp2/3-nucleated actin filaments drive crawling motility and phagocytosis in animal cells and slime molds. In this issue, Velle and Fritz-Laylin (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202007158) now show that Naegleria gruberi, belonging to a lineage that diverged from opisthokonts around a billion years ago, uses similar mechanisms to crawl and phagocytose bacteria.

Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation

Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3′-OH RNA strands in the context of 3′-OH/5′-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl–AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.

Structural determinants of microtubule minus end preference in CAMSAP CKK domains

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.