Eukaryotic cell proliferation requires chromosome replication and precise segregation to ensure daughter cells have identical genomic copies. Species of the genus
, the causative agents of malaria, ...display remarkable aspects of nuclear division throughout their life cycle to meet some peculiar and unique challenges to DNA replication and chromosome segregation. The parasite undergoes atypical endomitosis and endoreduplication with an intact nuclear membrane and intranuclear mitotic spindle. To understand these diverse modes of
cell division, we have studied the behaviour and composition of the outer kinetochore NDC80 complex, a key part of the mitotic apparatus that attaches the centromere of chromosomes to microtubules of the mitotic spindle. Using NDC80-GFP live-cell imaging in
, we observe dynamic spatiotemporal changes during proliferation, including highly unusual kinetochore arrangements during sexual stages. We identify a very divergent candidate for the SPC24 subunit of the NDC80 complex, previously thought to be missing in
, which completes a canonical, albeit unusual, NDC80 complex structure. Altogether, our studies reveal the kinetochore to be an ideal tool to investigate the non-canonical modes of chromosome segregation and cell division in
.
Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of ...nonphotosynthetic plastids in many eukaryotes—notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium —highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite’s host. Hematodinium sp. thus represents a further dimension of endosymbiosis—life after the organelle.
Significance Endosymbiotic organelles are a defining feature of eukaryotes—the last common ancestor and all extant eukaryotes possess at least a mitochondrial derivative. Although mitochondria and plastids are identified with aerobic ATP synthesis and photosynthesis, respectively, their retention by their host cells requires the merging and integration of many, often redundant, metabolic pathways. As a result, complex metabolic interdependencies arise between these formerly independent cells. Complete loss of endosymbiotic organelles, even where aerobic respiration or photosynthesis is lost, is exceedingly difficult, as demonstrated by persistence of organelles throughout secondary anaerobes and parasites. Here, we identify a rare but clear case of plastid loss in a parasitic alga and detail the metabolic disentanglement that was required to achieve this exceptional evolutionary event.
The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating stages and actively motile extracellular forms that move through tissue. ...Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not understood. Here, we show that the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that PKAc1 is sequestered to the parasite periphery by dual acylation of PKA regulatory subunit 1 (PKAr1). Upon genetic depletion of PKAc1 we show that newly invaded parasites exit host cells shortly thereafter, in a perforin-like protein 1 (PLP-1)-dependent fashion. Furthermore, we demonstrate that loss of PKAc1 prevents rapid down-regulation of cytosolic Ca2+ levels shortly after invasion. We also provide evidence that loss of PKAc1 sensitises parasites to cyclic GMP (cGMP)-induced Ca2+ signalling, thus demonstrating a functional link between cAMP and these other signalling modalities. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.
Apicomplexan parasites have immense impacts on humanity, but their basic cellular processes are often poorly understood. Where endocytosis occurs in these cells, how conserved this process is with ...other eukaryotes, and what the functions of endocytosis are across this phylum are major unanswered questions. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. We identify that these endocytic structures correspond to the 'micropore' that has been observed throughout the Apicomplexa. Moreover, conserved molecular adaptation of this structure is seen in apicomplexans including the kelch-domain protein K13 that is central to malarial drug-resistance. We determine that a dominant function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.
Cryptosporidium is a leading cause of diarrheal disease in children and an important contributor to early childhood mortality. The parasite invades and extensively remodels intestinal epithelial ...cells, building an elaborate interface structure. How this occurs at the molecular level and the contributing parasite factors are largely unknown. Here, we generated a whole-cell spatial proteome of the Cryptosporidium sporozoite and used genetic and cell biological experimentation to discover the Cryptosporidium-secreted effector proteome. These findings reveal multiple organelles, including an original secretory organelle, and generate numerous compartment markers by tagging native gene loci. We show that secreted proteins are delivered to the parasite-host interface, where they assemble into different structures including a ring that anchors the parasite into its unique epicellular niche. Cryptosporidium thus uses a complex set of secretion systems during and following invasion that act in concert to subjugate its host cell.
Display omitted
•1,107 Cryptosporidium proteins were assigned to their cellular locations through the use of hyperLOPIT•Four distinct secretory organelles were shown to be discharged during host interaction•More than 150 putative Cryptosporidium effectors were identified
Cryptosporidium is a leading cause of diarrheal disease, yet how the parasite modifies host enterocytes is unknown. Guérin et al. established and validated the spatial proteome of Cryptosporidium sporozoites and identified the content of multiple secretory organelles, providing a comprehensive set of putative virulence factors to understand host-parasite interaction.
Mitochondrial genomes represent relict bacterial genomes derived from a progenitor α-proteobacterium that gave rise to all mitochondria through an ancient endosymbiosis. Evolution has massively ...reduced these genomes, yet despite relative simplicity their organization and expression has developed considerable novelty throughout eukaryotic evolution. Few organisms have reengineered their mitochondrial genomes as thoroughly as the protist lineage of dinoflagellates. Recent work reveals dinoflagellate mitochondrial genomes as likely the most gene-impoverished of any free-living eukaryote, encoding only two to three proteins. The organization and expression of these genomes, however, is far from the simplicity their gene content would suggest. Gene duplication, fragmentation, and scrambling have resulted in an inflated and complex genome organization. Extensive RNA editing then recodes gene transcripts, and trans-splicing is required to assemble full-length transcripts for at least one fragmented gene. Even after these processes, messenger RNAs (mRNAs) lack canonical start codons and most transcripts have abandoned stop codons altogether.
Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular complex 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. By contrast, unicellular flagellates of the class Kinetoplastida have a unique set of 36 kinetochore components. The evolutionary origin and history of these kinetochores remain unknown. Here, we report evidence of homology between axial element components of the synaptonemal complex and three kinetoplastid kinetochore proteins KKT16-18. The synaptonemal complex is a zipper-like structure that assembles between homologous chromosomes during meiosis to promote recombination. By using sensitive homology detection protocols, we identify divergent orthologues of KKT16-18 in most eukaryotic supergroups, including experimentally established chromosomal axis components, such as Red1 and Rec10 in budding and fission yeast, ASY3-4 in plants and SYCP2-3 in vertebrates. Furthermore, we found 12 recurrent duplications within this ancient eukaryotic SYCP
gene family, providing opportunities for new functional complexes to arise, including KKT16-18 in the kinetoplastid parasite
. We propose the kinetoplastid kinetochore system evolved by repurposing meiotic components of the chromosome synapsis and homologous recombination machinery that were already present in early eukaryotes.
Some dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Our understanding of dinoflagellate toxin biosynthesis ...has been hampered by their unusually large genomes. To overcome this challenge, for the first time, we sequenced the genome, microRNAs, and mRNA isoforms of a basal dinoflagellate, Amphidinium gibbosum, and employed an integrated omics approach to understand its secondary metabolite biosynthesis.
We assembled the ~ 6.4-Gb A. gibbosum genome, and by probing decoded dinoflagellate genomes and transcriptomes, we identified the non-ribosomal peptide synthetase adenylation domain as essential for generation of specialized metabolites. Upon starving the cells of phosphate and nitrogen, we observed pronounced shifts in metabolite biosynthesis, suggestive of post-transcriptional regulation by microRNAs. Using Iso-Seq and RNA-seq data, we found that alternative splicing and polycistronic expression generate different transcripts for secondary metabolism.
Our genomic findings suggest intricate integration of various metabolic enzymes that function iteratively to synthesize metabolites, providing mechanistic insights into how dinoflagellates synthesize secondary metabolites, depending upon nutrient availability. This study provides insights into toxin production associated with dinoflagellate blooms. The genome of this basal dinoflagellate provides important clues about dinoflagellate evolution and overcomes the large genome size, which has been a challenge previously.
Our current understanding of biology is heavily based on a small number of genetically tractable model organisms. Most eukaryotic phyla lack such experimental models, and this limits our ability to ...explore the molecular mechanisms that ultimately define their biology, ecology, and diversity. In particular, marine protists suffer from a paucity of model organisms despite playing critical roles in global nutrient cycles, food webs, and climate. To address this deficit, an initiative was launched in 2015 to foster the development of ecologically and taxonomically diverse marine protist genetic models. The development of new models faces many barriers, some technical and others institutional, and this often discourages the risky, long-term effort that may be required. To lower these barriers and tackle the complexity of this effort, a highly collaborative community-based approach was taken. Herein, we describe this approach, the advances achieved, and the lessons learned by participants in this novel community-based model for research.
PDF
