[Show abstract][Hide abstract] ABSTRACT: Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.
[Show abstract][Hide abstract] ABSTRACT: ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialised complexes with discrete functions. In the unicellular parasite Trypanosoma brucei which causes African sleeping sickness, TbISWI downregulates RNA polymerase I (Pol I) transcribed Variant Surface Glycoprotein (VSG) gene expression sites (ES) which are mono-allelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins which do not show significant homology with known ISWI associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners: Regulator of Chromosome Condensation 1-like protein (RCCP) and phenylalanine/ tyrosine rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knock-down of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent VSG ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life-cycle stages, and show similar distributions at Pol I transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution.
Journal of Biological Chemistry 09/2015; DOI:10.1074/jbc.M115.679019 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Distinct mutations in the centrosomal-cilia protein CEP290 lead to diverse clinical findings in syndromic ciliopathies. We show that CEP290 localizes to the transition zone in ciliated cells, precisely to the region of Y-linkers between central microtubules and plasma membrane. To create models of CEP290-associated ciliopathy syndromes, we generated Cep290(ko/ko) and Cep290(gt/gt) mice that produce no or a truncated CEP290 protein, respectively. Cep290(ko/ko) mice exhibit early vision loss and die from hydrocephalus. Retinal photoreceptors in Cep290(ko/ko) mice lack connecting cilia, and ciliated ventricular ependyma fails to mature. The minority of Cep290(ko/ko) mice that escape hydrocephalus demonstrate progressive kidney pathology. Cep290(gt/gt) mice die at mid-gestation, and the occasional Cep290(gt/gt) mouse that survives shows hydrocephalus and severely cystic kidneys. Partial loss of CEP290-interacting ciliopathy protein MKKS mitigates lethality and renal pathology in Cep290(gt/gt) mice. Our studies demonstrate domain-specific functions of CEP290 and provide novel therapeutic paradigms for ciliopathies.
Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
Human Molecular Genetics 04/2015; 24(13). DOI:10.1093/hmg/ddv123 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reversible protein phosphorylation regulated by kinases
and phosphatases controls many cellular processes.
Although essential functions for the malaria
parasite kinome have been reported, the roles of
most protein phosphatases (PPs) during Plasmodium
development are unknown. We report a functional
analysis of the Plasmodium berghei protein phosphatome,
which exhibits high conservation with the
P. falciparum phosphatome and comprises 30 predicted
PPs with differential and distinct expression
patterns during various stages of the life cycle. Gene
disruption analysis of P. berghei PPs reveals that
half of the genes are likely essential for asexual
blood stage development, whereas six are required
for sexual development/sporogony in mosquitoes.
Phenotypic screening coupled with transcriptome
sequencing unveiled morphological changes and
altered gene expression in deletion mutants of two
N-myristoylated PPs. These findings provide systematic
functional analyses of PPs in Plasmodium, identifyhowphosphatases
regulate parasite development
and differentiation, and can informthe identification of
drug targets for malaria.
[Show abstract][Hide abstract] ABSTRACT: Trypanosoma brucei evades the adaptive immune response through the expression of antigenically distinct Variant Surface Glycoprotein (VSG) coats. To understand the progression and mechanisms of VSG switching, and to identify the VSGs expressed in populations of trypanosomes, it is desirable to predetermine the available repertoire of VSG genes (the 'VSGnome'). To date, the catalogue of VSG genes present in any strain is far from complete and the majority of current information regarding VSGs is derived from the TREU927 strain that is not commonly used as an experimental model. We have assembled, annotated and analyzed 2,563 distinct and previously unsequenced genes encoding complete and partial VSGs of the widely used Lister 427 strain of T. brucei. Around 80% of the VSGnome consists of incomplete genes or pseudogenes. Read-depth analysis demonstrated that most VSGs exist as single copies, but 360 exist as two or more indistinguishable copies. The assembled regions include five functional metacyclic VSG expression sites. One third of minichromosome sub-telomeres contain a VSG (64-67 VSGs on ∼96 minichromosomes), of which 85% appear to be functionally competent. The minichromosomal repertoire is very dynamic, differing among clones of the same strain. Few VSGs are unique along their entire length: frequent recombination events are likely to have shaped (and to continue to shape) the repertoire. In spite of their low sequence conservation and short window of expression, VSGs show evidence of purifying selection, with ∼40% of non-synonymous mutations being removed from the population. VSGs show a strong codon-usage bias that is distinct from that of any other group of trypanosome genes. VSG sequences are generally very divergent between Lister 427 and TREU927 strains of T. brucei, but those that are highly similar are not found in 'protected' genomic environments, but may reflect genetic exchange among populations.
[Show abstract][Hide abstract] ABSTRACT: The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite.
Biology Open 11/2013; 2(11):1160-70. DOI:10.1242/bio.20136163 · 2.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trypanosomes use a microtubule focused mechanism for cell morphogenesis and cytokinesis. We used scanning electron and video microscopy of living cells to provide the first detailed description of cell morphogenesis and cytokinesis in the early-branching eukaryote Trypanosoma brucei. We outline four distinct stages of cytokinesis and show that an asymmetric division fold bisects the two daughter cells, with a cytoplasmic bridge-like structure connecting the two daughters immediately prior to abscission. Using detection of tyrosinated α-tubulin as a marker for new or growing microtubules and expression of XMAP215, a plus end binding protein, as a marker for microtubule plus ends we demonstrate spatial asymmetry in the underlying microtubule cytoskeleton throughout the cell division cycle. This leads to inheritance of different microtubule cytoskeletal patterns and demonstrates the major role of microtubules in achieving cytokinesis. RNA interference techniques have led to a large set of mutants, often with variations in phenotype between procyclic and bloodstream life cycle forms. Here, we show morphogenetic differences between these two life cycle forms of this parasite during new flagellum growth and cytokinesis. These discoveries are important tools to explain differences between bloodstream and procyclic form RNAi phenotypes involving organelle mis-positioning during cell division and cytokinesis defects.
[Show abstract][Hide abstract] ABSTRACT: Abstract Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself.
Critical Reviews in Biochemistry and Molecular Biology 07/2013; 48(4):373-396. DOI:10.3109/10409238.2013.821444 · 7.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins
that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes
that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at
the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated.
When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity
to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved
domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover,
the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.
[Show abstract][Hide abstract] ABSTRACT: Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl(-) mutants, with global phosphorylation studies of ookinete differentiation from 1.5-24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cilia/flagella are ancient organelles with motility and sensory functions. Cilia display significant ultrastructural conservation where present across the eukaryotic phylogeny; however, diversity in ciliary biology exists and the ability to produce cilia has been lost independently on a number of occasions. Land plants provide an excellent system for the investigation of cilia evolution and loss across a broad phylogeny, because early divergent land plant lineages produce cilia, whereas most seed plants do not. This review highlights the differences in cilia form and function across land plants and discusses how recent advances in genomics are providing novel insights into the evolutionary trajectory of ciliary proteins. We propose a renewed effort to adopt ciliated land plants as models to investigate the mechanisms underpinning complex ciliary processes, such as number control, the coordination of basal body placement and the regulation of beat patterns.
New Phytologist 06/2012; 195(3):526-40. DOI:10.1111/j.1469-8137.2012.04197.x · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression of transgenes is central to forward and reverse genetic analysis in Trypanosoma brucei. The inducible expression of transgenes in trypanosomes is based on the tetracycline repressor binding to a tetracycline operator to prevent transcription in the absence of tetracycline. The same inducible system is used to produce double-stranded RNA for RNAi knockdown of target genes. This study describes a new plasmid pSPR2.1 that drives consistent high-level expression of tetracycline repressor in procyclic form trypanosomes. A complementary expression plasmid, p3227, was constructed. The major difference between this and current plasmids is the separation of the inducible transgene and selectable marker promoters by the plasmid backbone. The plasmid p3227 was able to support inducible expression in cell lines containing pSPR2.1 as well as the established Lister 427 29-13 cell line. p3666, a derivative of p3227, was made for inducible expression of stem loop RNAi constructs and was effective for knockdown of DRBD3, which had proved problematic using existing RNAi plasmids with head-to-head promoters. The plasmid system was also able to support inducible transgene expression and DRBD3 RNAi knockdown in bloodstream form cells expressing tetracycline repressor from an integrated copy of the plasmid pHD1313.
PLoS ONE 04/2012; 7(4):e35167. DOI:10.1371/journal.pone.0035167 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: African trypanosomes are the only organisms known to use RNA polymerase I (pol I) to transcribe protein-coding genes. These
genes include VSG, which is essential for immune evasion and is transcribed from an extranucleolar expression site body (ESB). Several trypanosome
pol I subunits vary compared to their homologues elsewhere, and the question arises as to how these variations relate to pol
I function. A clear example is the N-terminal extension found on the second-largest subunit of pol I, RPA2. Here, we identify
an essential role for this region. RPA2 truncation leads to nuclear exclusion and a growth defect which phenocopies single-allele
knockout. The N terminus is not a general nuclear localization signal (NLS), however, and it fails to accumulate unrelated
proteins in the nucleus. An ectopic NLS is sufficient to reinstate nuclear localization of truncated RPA2, but it does not
restore function. Moreover, NLS-tagged, truncated RPA2 has a different subnuclear distribution to full-length protein and
is unable to build stable pol I complexes. We conclude that the RPA2 N-terminal extension does not have a role exclusive to
the expression of protein-coding genes, but it is essential for all pol I functions in trypanosomes because it directs trypanosomatid-specific
interactions with RPA1.
[Show abstract][Hide abstract] ABSTRACT: Dyneins are large molecular motors that hydrolyze ATP to generate a minus-end-directed force along microtubules. Each dynein consists of one to three dynein heavy chains (HCs), which encompass the ATPase activity, complexed to intermediate (IC), light-intermediate (LIC), and light chains (LC). This chapter explains the evolutionary biology of dyneins. It begins by describing the classification of the constituent parts of dynein proteins and explaining the relationship between them. It then reviews the present status of sequence-based classification and analysis of dynein components and shows how genome-based knowledge of dynein repertoires informs the understanding of both the evolution of dyneins themselves and the biology of individual organisms. In addition to this, it traces the major dynein families back to their roots in the last common eukaryotic ancestor (LCEA). Based on this, it tries to reconstruct the evolution of dyneins in the proto-eukaryote and explores the possible origins of dynein motors in the large biological gap between the prokaryotes and the last ancestor of all modern eukaryotic species. Finally, the study explores the question about the functional conditions that might have led to the evolution of this important group of molecular motors, throwing light on the structural and functional evolution of dynein proteins.
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cilia are complex, highly conserved microtubule-based organelles with a broad phylogenetic distribution. Cilia were present in the last eukaryotic common ancestor and many proteins involved in cilia function have been conserved through eukaryotic diversification. However, cilia have also been lost multiple times in different lineages, with at least two losses occurring within the land plants. Whereas all non-seed plants produce cilia for motility of male gametes, some gymnosperms and all angiosperms lack cilia. During these evolutionary losses, proteins with ancestral ciliary functions may be lost or co-opted into different functions.
Here we identify a core set of proteins with an inferred ciliary function that are conserved in ciliated eukaryotic species. We interrogate this genomic dataset to identify proteins with a predicted ancestral ciliary role that have been maintained in non-ciliated land plants. In support of our prediction, we demonstrate that several of these proteins have a flagellar localisation in protozoan trypanosomes. The phylogenetic distribution of these genes within the land plants indicates evolutionary scenarios of either sub- or neo-functionalisation and expression data analysis shows that these genes are highly expressed in Arabidopsis thaliana pollen cells.
A large number of proteins possess a phylogenetic ciliary profile indicative of ciliary function. Remarkably, many genes with an ancestral ciliary role are maintained in non-ciliated land plants. These proteins have been co-opted to perform novel functions, most likely before the loss of cilia, some of which appear related to the formation of the male gametes.
[Show abstract][Hide abstract] ABSTRACT: The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes.
Here we utilise a Bayesian approach to identify local changes in nucleotide composition in the genome of T. brucei. We show that there are several elements which are found at the starts and ends of multicopy gene arrays and that there are compositional elements that are common to all intergenic regions. We also show that there is a composition-inversion element that occurs at the position of the trans-splice site.
The nature of the elements discovered reinforces the hypothesis that context dependant RNA secondary structure has an important influence on gene expression regulation in Trypanosoma brucei.
PLoS ONE 10/2011; 6(10):e25666. DOI:10.1371/journal.pone.0025666 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytoskeleton is a system of intracellular filaments crucial for cell shape, division, and function in all three domains of life. The simple cytoskeletons of prokaryotes show surprising plasticity in composition, with none of the core filament-forming proteins conserved in all lineages. In contrast, eukaryotic cytoskeletal function has been hugely elaborated by the addition of accessory proteins and extensive gene duplication and specialization. Much of this complexity evolved before the last common ancestor of eukaryotes. The distribution of cytoskeletal filaments puts constraints on the likely prokaryotic line that made this leap of eukaryogenesis.
The Journal of Cell Biology 08/2011; 194(4):513-25. DOI:10.1083/jcb.201102065 · 9.83 Impact Factor