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.     

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.     

Rab11A-Controlled Assembly of the Inner Membrane Complex Is Required for Completion of Apicomplexan Cytokinesis

The final step during cell division is the separation of daughter cells, a process that requires the coordinated delivery and assembly of new membrane to the cleavage furrow. While most eukaryotic cells replicate by binary fission, replication of apicomplexan parasites involves the assembly of daughters (merozoites/tachyzoites) within the mother cell, using the so-called Inner Membrane Complex (IMC) as a scaffold. After de novo synthesis of the IMC and biogenesis or segregation of new organelles, daughters bud out of the mother cell to invade new host cells. Here, we demonstrate that the final step in parasite cell division involves delivery of new plasma membrane to the daughter cells, in a process requiring functional Rab11A. Importantly, Rab11A can be found in association with Myosin-Tail-Interacting-Protein (MTIP), also known as Myosin Light Chain 1 (MLC1), a member of a 4-protein motor complex called the glideosome that is known to be crucial for parasite invasion of host cells. Ablation of Rab11A function results in daughter parasites having an incompletely formed IMC that leads to a block at a late stage of cell division. A similar defect is observed upon inducible expression of a myosin A tail-only mutant. We propose a model where Rab11A-mediated vesicular traffic driven by an MTIP-Myosin motor is necessary for IMC maturation and to deliver new plasma membrane to daughter cells in order to complete cell division.     

TgDrpC,an atypical dynamin‐related protein in Toxoplasma gondii,is associated with vesicular transport factors and parasite division

Dynamin‐related proteins (Drps) are involved in diverse processes such as organelle division and vesicle trafficking. The intracellular parasite Toxoplasma gondii possesses three distinct Drps. TgDrpC, whose function remains unresolved, is unusual in that it lacks a conserved GTPase Effector Domain, which is typically required for function. Here, we show that TgDrpC localizes to cytoplasmic puncta; however, in dividing parasites, TgDrpC redistributes to the growing edge of the daughter cells. By conditional knockdown, we determined that loss of TgDrpC stalls division and leads to rapid deterioration of multiple organelles and the IMC. We also show that TgDrpC interacts with proteins that exhibit homology to those involved in vesicle transport, including members of the adaptor complex 2. Two of these proteins, a homolog of the adaptor protein 2 (AP‐2) complex subunit alpha‐1 and a homolog of the ezrin–radixin–moesin (ERM) family proteins, localize to puncta and associate with the daughter cells. Consistent with the association with vesicle transport proteins, re‐distribution of TgDrpC to the IMC during division is dependent on post‐Golgi trafficking. Together, these results support that TgDrpC contributes to vesicle trafficking and is critical for stability of parasite organelles and division.     

Daughter cell assembly in the protozoan parasite Toxoplasma gondii

The phylum Apicomplexa includes thousands of species of obligate intracellular parasites, many of which are significant human and/or animal pathogens. Parasites in this phylum replicate by assembling daughters within the mother, using a cytoskeletal and membranous scaffolding termed the inner membrane complex. Most apicomplexan parasites, including Plasmodium sp. (which cause malaria), package many daughters within a single mother during mitosis, whereas Toxoplasma gondii typically packages only two. The comparatively simple pattern of T. gondii cell division, combined with its molecular genetic and cell biological accessibility, makes this an ideal system to study parasite cell division. A recombinant fusion between the fluorescent protein reporter YFP and the inner membrane complex protein IMC1 has been exploited to examine daughter scaffold formation in T. gondii. Time-lapse video microscopy permits the entire cell cycle of these parasites to be visualized in vivo. In addition to replication via endodyogeny (packaging two parasites at a time), T. gondii is also capable of forming multiple daughters, suggesting fundamental similarities between cell division in T. gondii and other apicomplexan parasites.     

The plastid of Toxoplasma gondii is divided by association with the centrosomes

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.     

Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites

Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.     

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.     

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.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Summary Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.     

Two palmitoyl acyltransferases involved sequentially in the biogenesis of the inner membrane complex of Toxoplasma gondii

The phylum Apicomplexa includes a number of significant human pathogens like Toxoplasma gondii and Plasmodium species. These obligate intracellular parasites possess a membranous structure, the inner membrane complex (IMC), composed of flattened vesicles apposed to the plasma membrane. Numerous proteins associated with the IMC are anchored via a lipid post‐translational modification termed palmitoylation. This acylation is catalysed by multi‐membrane spanning protein S‐acyl‐transferases (PATs) containing a catalytic Asp‐His‐His‐Cys (DHHC) motif, commonly referred to as DHHCs. Contrasting the redundancy observed in other organisms, several PATs are essential for T. gondii tachyzoite survival; 2 of them, TgDHHC2 and TgDHHC14 being IMC‐resident. Disruption of either of these TgDHHCs results in a rapid collapse of the IMC in the developing daughter cells leading to dramatic morphological defects of the parasites while the impact on the other organelles is limited to their localisation but not to their biogenesis. The acyl‐transferase activity of TgDHHC2 and TgDHHC14 is involved sequentially in the formation of the sub‐compartments of the IMC. Investigation of proteins known to be palmitoylated and localised to these sub‐compartments identified TgISP1/3 as well as TgIAP1/2 to lose their membrane association revealing them as likely substrates of TgDHHC2, while these proteins are not impacted by TgDHHC14 depletion.     

The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation.     

Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division.

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.     

Two novel meiotic restitution mechanisms in haploid maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Two original mechanisms of nuclear restitution related to different processes of meiotic division of pollen mother cells (PMCs) have been found in male meiosis of the lines of maize haploids no. 2903 and no. 2904. The first mechanism, which is characteristic of haploid no. 2903, consists in spindle deformation (bend) in the conventional metaphase-anaphase I. This leads to asymmetric incomplete cytokinesis with daughter cell membranes in the form of incisions on the mother cell membrane. As a result, the chromosomes of the daughter nuclei are combined into a common spindle during the second meiotic division, and a dyad of haploid microspores is formed at the tetrad stage. The frequency of this abnormality is about 50%. The second restitution mechanism, which has been observed in PMCs of haploid no. 2904, results from disturbance of the fusion of membrane vesicles (plastosomes) at the moment of formation of daughter cell membranes and completion of cytokinesis in the first meiotic division. This type of cell division yields a binuclear monad. In the second meiotic division, the chromosomes of the daughter nuclei form a common spindle, and meiosis results in a dyad of haploid microspores. The frequency of this abnormality is as high as 15%. As a result, haploid lines no. 2903 and no. 2904 partly restore fertility.     

THE EFFECT OF METHANOL ON SPORE GERMINATION AND RHIZOID DIFFERENTIATION IN ONOCLEA SENSIBILIS

In germinating spores of Onoclea sensibilis, the nucleus migrates to one end prior to an asymmetric cell division that partitions each spore into two daughter cells of unequal size. The larger cell develops into a protonema, whereas the smaller cell immediately differentiates into a rhizoid. When spores were germinated in the presence of methanol, nuclear migration was inhibited and most nuclei moved only to the raphe on the proximal side of the spores. Subsequent cell division partitioned each spore into daughter cells of equal size of which both developed into a protonema and neither into a rhizoid. Spores became sensitive to methanol at a time just prior to or coincident with nuclear migration and the effects of the alcohol were rapidly reversible as long as the spores were removed from methanol prior to the completion of cell division. Exposure to methanol prior to, but not during, nuclear migration or after mitosis had no effect upon rhizoid differentiation. The alcohol disrupted the formation of crosswalls after mitosis and they were often convoluted and irregularly branched. These results are consistent with the interpretation that methanol may disrupt a membrane site that plays an essential role in nuclear movement and rhizoid differentiation.     

Itraconazole affects Toxoplasma gondii endodyogeny

The antifungal agent itraconazole is an effective drug against systemic mycoses inhibiting cytochrome P-450-mediated ergosterol synthesis, essential for fungal survival. In this work, we show the activity of this azole as a potential agent against Toxoplasma gondii, the causative agent of toxoplasmosis. Monolayers of LLC-MK2 epithelial cells infected with tachyzoites of RH strain were incubated with different concentrations of itraconazole for 24 and 48 h. The IC(50) values obtained were 114.0 and 53.6 nM for 24 and 48 h, respectively. Transmission electron microscopy (TEM) analysis of itraconazole-treated intracellular tachyzoites showed endoplasmic reticulum and nuclear envelope swelling. The drug also caused rupture of the parasite's surface membrane and affected the parasite's division by endodyogeny. This observation was confirmed both by fluorescence microscopy of cells labeled with diamidino-2-phenylindole and by three-dimensional reconstruction of serial thin sections analyzed by TEM. The treatment with itraconazole led to the formation of a mass of daughter cells, suggesting the interruption of the scission process during the parasite's cell division.     

Roles of VRK1 as a new player in the control of biological processes required for cell division

Cell division, in addition to an accurate transmission of genetic information to daughter cells, also requires the temporal and spatial coordination of several biological processes without which cell division would not be feasible. These processes include the temporal coordination of DNA replication and chromosome segregation, regulation of nuclear envelope disassembly and assembly, chromatin condensation and Golgi fragmentation for its redistribution into daughter cells, among others. However, little is known regarding regulatory proteins and signalling pathways that might participate in the coordination of all these different biological functions. Such regulatory players should directly have a role in the processes leading to cell division. VRK1 (Vaccinia-related kinase 1) is an early response gene required for cyclin D1 expression, regulates p53 by a specific Thr18 phosphorylation, controls chromatin condensation by histone phosphorylation, nuclear envelope assembly by phosphorylation of BANF1, and participates in signalling required for Golgi fragmentation late in the G2 phase. We propose that VRK1, a Ser-Thr kinase, might be a candidate to play an important coordinator role in these cell division processes as part of a novel signalling pathway.     

Two novel meiotic restitution mechanisms in haploid maize (Zea mays L.)

Two original mechanisms of nuclear restitution related to different processes of meiotic division of pollen mother cells (PMCs) have been found in male meiosis of the lines of maize haploids no. 2903 and no. 2904. The first mechanism, which is characteristic of haploid no. 2903, consists in spindle deformation (bend) in the conventional metaphase-anaphase I. This leads to asymmetric incomplete cytokinesis with daughter cell membranes in the form of incisions on the mother cell membrane. As a result, the chromosomes of the daughter nuclei are combined into a common spindle during the second meiotic division, and a dyad of haploid microspores is formed at the tetrad stage. The frequency of this abnormality is about 50%. The second restitution mechanism, which has been observed in PMCs of haploid no. 2904, results from disturbance of the fusion of membrane vesicles (plastosomes) at the moment of formation of daughter cell membranes and completion of cytokinesis in the first meiotic division. This type of cell division yields a binuclear monad. In the second meiotic division, the chromosomes of the daughter nuclei form a common spindle, and meiosis results in a dyad of haploid microspores. The frequency of this abnormality is as high as 15%. As a result, haploid lines no. 2903 and no. 2904 partly restore fertility.