The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny,duplication rate and parasite virulence

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora‐related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub‐cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock‐down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3‐depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target.     

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly,Apicoplast Division and Cytokinesis

The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother''s cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation.     

A family of intermediate filament‐like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii

The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament‐like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14‐member subfamily of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatiotemporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.     

TgZFP2 is a novel zinc finger protein involved in coordinating mitosis and budding in Toxoplasma

Zinc finger proteins (ZFPs) are one of the most abundant groups of proteins with a wide range of molecular functions. We have characterised a Toxoplasma protein that we named TgZFP2, as it bears a zinc finger domain conserved in eukaryotes. However, this protein has little homology outside this region and contains no other conserved domain that could hint for a particular function. We thus investigated TgZFP2 function by generating a conditional mutant. We showed that depletion of TgZFP2 leads to a drastic arrest in the parasite cell cycle, and complementation assays demonstrated the zinc finger domain is essential for TgZFP2 function. More precisely, whereas replication of the nuclear material is initially essentially unaltered, daughter cell budding is seriously impaired: to a large extent newly formed buds fail to incorporate nuclear material. TgZFP2 is found at the basal complex in extracellular parasites and after invasion, but as the parasites progress into cell division, it relocalises to cytoplasmic punctate structures and, strikingly, accumulates in the pericentrosomal area at the onset of daughter cell elongation. Centrosomes have emerged as major coordinators of the budding and nuclear cycles in Toxoplasma, and our study identifies a novel and important component of this machinery.     

GFP‐targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites

Background information. The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo‐erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. Results. In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo‐erythrocytic schizogony in vitro, leading to impaired parasite maturation. Conclusions. Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red‐blood‐cell‐infective merozoites.     

A HT/PEXEL Motif in Toxoplasma Dense Granule Proteins is a Signal for Protein Cleavage but not Export into the Host Cell

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL‐like motif at the N‐terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin‐tagged versions of these uncharacterized proteins show co‐localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM .     

MORN1 has a conserved role in asexual and sexual development across the apicomplexa

The gene encoding the membrane occupation and recognition nexus protein MORN1 is conserved across the Apicomplexa. In Toxoplasma gondii, MORN1 is associated with the spindle poles, the anterior and posterior rings of the inner membrane complex (IMC). The present study examines the localization of MORN1 during the coccidian development of T. gondii and three Eimeria species (in the definitive host) and erythrocytic schizogony of Plasmodium falciparum. During asexual proliferation, MORN1 is associated with the posterior ring of the IMCs of the multiple daughters forming during T. gondii endopolygeny and schizogony in Eimeria and P. falciparum. Furthermore, the expression of P. falciparum MORN1 protein peaked in late schizogony. These data fit a model with a conserved role for MORN1 during IMC assembly in all variations of asexual development. An important new observation is the reactivity of MORN1 antibody with certain sexual stages in T. gondii and Eimeria species. Here MORN1 is organized as a ring-like structure where the microgametes bud from the microgametocyte while in mature microgametes it is present near the flagellar basal bodies and mitochondrion. These observations suggest a conserved role for MORN1 in both asexual and sexual development across the Apicomplexa.     

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication.     

Comparison of dinucleotide frequency and codon usage inToxoplasma andPlasmodium: Evolutionary implications

Summary The weight-averaged observed/expected dinucleotide frequencies for the sum total of the coding regions of fiveToxoplasma genes were compared with the same parameters previously determined for the coding regions of 21Plasmodium genes. In addition, codon usage in the fiveToxoplasma genes was compared with that in the 21Plasmodium genes, and the percent distribution of amino acids in theToxoplasma protein pool and thePlasmodium protein pool were compared with that in a general protein pool of 314 proteins. The results are consistent with the hypothesis that, contrary to currently held opinion, the generaToxoplasma andPlasmodium are not especially closely related.     

ATG8 localization in apicomplexan parasites: Apicoplast and more?

The ATG genes are highly conserved in eukaryotes including yeasts, plants, and mammals. However, these genes appear to be only partially present in most protists. Recent studies demonstrated that, in the apicomplexan parasites Plasmodium (malaria parasites) and Toxoplasma, ATG8 localizes to the apicoplast, a unique nonphotosynthetic plastid with 4 limiting membranes. In contrast to this established localization, it remains unclear whether these parasites can induce canonical macroautophagy and if ATG8 localizes to autophagosomes. Furthermore, the molecular function of ATG8 in its novel workplace, the apicoplast, is totally unknown. Here, we review recent studies on ATG8 in Plasmodium and Toxoplasma, summarize both consensus and controversial findings, and discuss its potential role in these parasites.     

Evolving Insights into Protein Trafficking to the Multiple Compartments of the Apicomplexan Plastid1

ABSTRACT. The apicoplast is a relict plastid found in many medically important apicomplexan parasites, such as Plasmodium and Toxoplasma. Phylogenetic analysis and the presence of four bounding membranes indicate that the apicoplast arose from a secondary endosymbiosis. Here we review what has been discovered about the complex journey proteins take to reach compartments of the apicoplast. The targeting sequences for luminal proteins are well‐defined, but those routing proteins to other compartments are only beginning to be studied. Recent work suggests that the trafficking mechanisms involve a variety of molecules of different phylogenetic origins. We highlight some remaining questions regarding protein trafficking to this divergent organelle.     

Tetrahymena Expresses More than a Hundred Proteins with Lipid‐binding MORN Motifs that can Differ in their Subcellular Localisations

Proteins with membrane occupation and recognition nexus (MORN) motifs are associated with cell fission in apicomplexan parasites, chloroplast division in Arabidopsis and the motility of sperm cells. We found that ciliates are among those that encode the largest variety of MORN proteins. Tetrahymena thermophila expresses 129 MORN protein‐encoding genes, some of which are specifically up‐regulated during conjugation. A lipid‐binding assay underpins the assumption that the predominant function of MORN motifs themselves is to confer the ability of lipid binding. The localisation of four MORN candidate proteins with similar characteristics highlights the functional diversity of this group especially in ciliates.     

Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite‐specific functions during Plasmodium falciparum intraerythrocytic development

Lysine acetylation has emerged as a major post‐translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine‐acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium‐specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.     

Growth cycle of daughter and mother cells ofSaccharomyces cerevisiae

Maximum values of specific rate of RNA synthesis, specific growth rate and a critical cell size determined by the surface to volume ratioS/V =1.0 are the factors which control the onset of budding in daughter cells. The increased rate of RNA synthesis is due not only to daughter cells but also to all buds formed on mother cells.     

Ajuba: a new microtubule-associated protein that interacts with BUBR1 and Aurora B at kinetochores in metaphase

Background information. The role of the LIM‐domain‐containing protein Ajuba was initially described in cell adhesion and migration processes and recently in mitosis as an activator of the Aurora A kinase. Results. In the present study, we show that Ajuba localizes to centrosomes and kinetochores during mitosis. This localization is microtubule‐dependent and Ajuba binds microtubules in vitro. A microtubule regrowth assay showed that Ajuba follows nascent microtubules from centrosomes to kinetochores. Owing to its contribution to mitotic commitment and its microtubule‐dependent localization, Ajuba could also play a role during the metaphase—anaphase transition. We show that Ajuba interacts with Aurora B and BUBR1 [BUB (budding uninhibited by benomyl)‐related 1], two major components of the mitotic checkpoint. Inhibition of BUBR1 by siRNA (small interfering RNA) disrupts chromosome alignment at the metaphase plate and modifies Ajuba localization due to premature mitotic exit. Conclusions. Ajuba is a microtubule‐associated protein that collaborates with Aurora B and BUBR1 at the metaphase—anaphase transition and this may be important to ensure proper chromosome segregation.     

Compartmentalized Toxoplasma EB1 bundles spindle microtubules to secure accurate chromosome segregation

Toxoplasma gondii replicates asexually by a unique internal budding process characterized by interwoven closed mitosis and cytokinesis. Although it is known that the centrosome coordinates these processes, the spatiotemporal organization of mitosis remains poorly defined. Here we demonstrate that centrosome positioning around the nucleus may signal spindle assembly: spindle microtubules (MTs) are first assembled when the centrosome moves to the basal side and become extensively acetylated after the duplicated centrosomes reposition to the apical side. We also tracked the spindle MTs using the MT plus end–binding protein TgEB1. Endowed by a C-terminal NLS, TgEB1 resides in the nucleoplasm in interphase and associates with the spindle MTs during mitosis. TgEB1 also associates with the subpellicular MTs at the growing end of daughter buds toward the completion of karyokinesis. Depletion of TgEB1 results in escalated disintegration of kinetochore clustering. Furthermore, we show that TgEB1’s MT association in Toxoplasma and in a heterologous system (Xenopus) is based on the same principles. Finally, overexpression of a high-MT-affinity TgEB1 mutant promotes the formation of overstabilized MT bundles, resulting in avulsion of otherwise tightly clustered kinetochores. Overall we conclude that centrosome position controls spindle activity and that TgEB1 is critical for mitotic integrity.     

Toxoplasma gondii Toc75 Functions in Import of Stromal but not Peripheral Apicoplast Proteins

Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist. As a result the apicoplast is surrounded by four membranes. This complex structure necessitates a system of transport signals and translocons allowing nuclear encoded proteins to find their way to specific apicoplast sub‐compartments. Previous studies identified translocons traversing two of the four apicoplast membranes. Here we provide functional support for the role of an apicomplexan Toc75 homolog in apicoplast protein transport. We identify two apicomplexan genes encoding Toc75 and Sam50, both members of the Omp85 protein family. We localize the respective proteins to the apicoplast and the mitochondrion of Toxoplasma and Plasmodium. We show that the Toxoplasma Toc75 is essential for parasite growth and that its depletion results in a rapid defect in the import of apicoplast stromal proteins while the import of proteins of the outer compartments is affected only as the secondary consequence of organelle loss. These observations along with the homology to Toc75 suggest a potential role in transport through the second innermost membrane.     

Babesia divergens and Neospora caninum apical membrane antigen 1 structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion

Host cell invasion by the obligate intracellular apicomplexan parasites, including Plasmodium (malaria) and Toxoplasma (toxoplasmosis), requires a step‐wise mechanism unique among known host–pathogen interactions. A key step is the formation of the moving junction (MJ) complex, a circumferential constriction between the apical tip of the parasite and the host cell membrane that traverses in a posterior direction to enclose the parasite in a protective vacuole essential for intracellular survival. The leading model of MJ assembly proposes that Rhoptry Neck Protein 2 (RON2) is secreted into the host cell and integrated into the membrane where it serves as the receptor for apical membrane antigen 1 (AMA1) on the parasite surface. We have previously demonstrated that the AMA1‐RON2 interaction is an effective target for inhibiting apicomplexan invasion. To better understand the AMA1‐dependant molecular recognition events that promote invasion, including the significant AMA1‐RON2 interaction, we present the structural characterization of AMA1 from the apicomplexan parasites Babesia divergens (BdAMA1) and Neospora caninum (NcAMA1) by X‐ray crystallography. These studies offer intriguing structural insight into the RON2‐binding surface groove in the AMA1 apical domain, which shows clear evidence for receptor–ligand co‐evolution, and the hyper variability of the membrane proximal domain, which in Plasmodium is responsible for direct binding to erythrocytes. By incorporating the structural analysis of BdAMA1 and NcAMA1 with existing AMA1 structures and complexes we were able to define conserved pockets in the AMA1 apical groove that could be targeted for the design of broadly reactive therapeutics.     

Non‐dormant Axillary Bud 1 regulates axillary bud outgrowth in sorghum

Tillering contributes to grain yield and plant architecture and therefore is an agronomically important trait in sorghum (Sorghum bicolor). Here, we identified and functionally characterized a mutant of the Non‐dormant Axillary Bud 1 (NAB1) gene from an ethyl methanesulfonate‐mutagenized sorghum population. The nab1 mutants have increased tillering and reduced plant height. Map‐based cloning revealed that NAB1 encodes a carotenoid‐cleavage dioxygenase 7 (CCD7) orthologous to rice (Oryza sativa) HIGH‐TILLERING DWARF1/DWARF17 and Arabidopsis thaliana MORE AXILLARY BRANCHING 3. NAB1 is primarily expressed in axillary nodes and tiller bases and NAB1 localizes to chloroplasts. The nab1 mutation causes outgrowth of basal axillary buds; removing these non‐dormant basal axillary buds restored the wild‐type phenotype. The tillering of nab1 plants was completely suppressed by exogenous application of the synthetic strigolactone analog GR24. Moreover, the nab1 plants had no detectable strigolactones and displayed stronger polar auxin transport than wild‐type plants. Finally, RNA‐seq showed that the expression of genes involved in multiple processes, including auxin‐related genes, was significantly altered in nab1. These results suggest that NAB1 functions in strigolactone biosynthesis and the regulation of shoot branching via an interaction with auxin transport.     

Effect of static magnetic fields on the budding of yeast cells

The effect of static magnetic fields on the budding of single yeast cells was investigated using a magnetic circuit that was capable of generating a strong magnetic field (2.93 T) and gradient (6100 T² m^?1). Saccharomyces cerevisiae yeast cells were grown in an aqueous YPD agar in a silica capillary under either a homogeneous or inhomogeneous static magnetic field. Although the size of budding yeast cells was only slightly affected by the magnetic fields after 4 h, the budding angle was clearly affected by the direction of the homogeneous and inhomogeneous magnetic fields. In the homogeneous magnetic field, the budding direction of daughter yeast cells was mainly oriented in the direction of magnetic field B. However, when subjected to the inhomogeneous magnetic field, the daughter yeast cells tended to bud along the axis of capillary flow in regions where the magnetic gradient, estimated by B(dB/dx), were high. Based on the present experimental results, the possible mechanism for the magnetic effect on the budding direction of daughter yeast cells is theoretically discussed. Bioelectromagnetics 31:622–629, 2010. © 2010 Wiley‐Liss, Inc.