A MORN1‐associated HAD phosphatase in the basal complex is essential for Toxoplasma gondii daughter budding

Apicomplexan parasites replicate by several budding mechanisms with two well‐characterized examples being Toxoplasma endodyogeny and Plasmodium schizogony. Completion of budding requires the tapering of the nascent daughter buds toward the basal end, driven by contraction of the basal complex. This contraction is not executed by any of the known cell division associated contractile mechanisms and in order to reveal new components of the unusual basal complex we performed a yeast two‐hybrid screen with its major scaffolding protein, TgMORN1. Here we report on a conserved protein with a haloacid dehalogenase (HAD) phosphatase domain, hereafter named HAD2a, identified by yeast two‐hybrid. HAD2a has demonstrated enzyme‐activity in vitro, localizes to the nascent daughter buds, and co‐localizes with MORN1 to the basal complex during its contraction. Conditional knockout of HAD2a in Toxoplasma interferes with basal complex assembly, which leads to incomplete cytokinesis and conjoined daughters that ultimately results in disrupted proliferation. In Plasmodium, we further confirmed localization of the HAD2a ortholog to the basal complex toward the end of schizogony. In conclusion, our work highlights an essential role for this HAD phosphatase across apicomplexan budding and suggests a regulatory mechanism of differential phosphorylation on the structure and/or contractile function of the basal complex.     

A novel actin-related protein is associated with daughter cell formation in Toxoplasma gondii

Cell division in Toxoplasma gondii occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using Toxoplasma as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immunoelectron microscopy localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells prior to the arrival of proteins such as IMC-1. The overexpression of ALP1 under the control of a strong constitutive promoter disrupted the formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in the formation of daughter cell membranes during cell division in apicomplexan parasites.     

Toxoplasma gondii myosin F,an essential motor for centrosomes positioning and apicoplast inheritance

Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so‐called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin‐based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.     

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.     

Organelle segregation into Plasmodium liver stage merozoites

The liver stage of the Plasmodium parasite remains one of the most promising targets for intervention against malaria as it is clinically silent, precedes the symptomatic blood stage and represents a bottleneck in the parasite life cycle. However, many aspects of the development of the parasite during this stage are far from understood. During the liver stage, the parasite undergoes extensive replication, forming tens of thousands of infectious merozoites from each invading sporozoite. This implies a very efficient and accurate process of cytokinesis and thus also of organelle development and segregation. We have generated for the first time Plasmodium berghei double-fluorescent parasite lines, allowing visualization of the apicoplast, mitochondria and nuclei in live liver stage parasites. Using these we have seen that in parallel with nuclear division, the apicoplast and mitochondrion become two extensively branched and intertwining structures. The organelles then undergo impressive morphological and positional changes prior to cell division. To form merozoites, the parasite undergoes cytokinesis and the complex process of organelle development and segregation into the forming daughter merozoites could be analysed in detail using the newly generated transgenic parasites.     

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.     

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.     

Replication and partitioning of the apicoplast genome of Toxoplasma gondii is linked to the cell cycle and requires DNA polymerase and gyrase

Apicomplexans are the causative agents of numerous important infectious diseases including malaria and toxoplasmosis. Most of them harbour a chloroplast-like organelle called the apicoplast that is essential for the parasites’ metabolism and survival. While most apicoplast proteins are nuclear encoded, the organelle also maintains its own genome, a 35 kb circle. In this study we used Toxoplasma gondii to identify and characterise essential proteins involved in apicoplast genome replication and to understand how apicoplast genome segregation unfolds over time. We demonstrated that the DNA replication enzymes Prex, DNA gyrase and DNA single stranded binding protein localise to the apicoplast. We show in knockdown experiments that apicoplast DNA Gyrase A and B, and Prex are required for apicoplast genome replication and growth of the parasite. Analysis of apicoplast genome replication by structured illumination microscopy in T. gondii tachyzoites showed that apicoplast nucleoid division and segregation initiate at the beginning of S phase and conclude during mitosis. Thus, the replication and division of the apicoplast nucleoid is highly coordinated with nuclear genome replication and mitosis. Our observations highlight essential components of apicoplast genome maintenance and shed light on the timing of this process in the context of the overall parasite cell cycle.     

Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis

BACKGROUND: The asymmetric division of cells and unequal allocation of cell contents is essential for correct development. This process of active segregation is poorly understood but in many instances has been shown to depend on the cytoskeleton. Motor proteins moving along actin filaments and microtubules are logical candidates to provide the motive force for asymmetric sorting of cell contents. The role of myosins in such processes has been suggested, but few examples of their involvement are known. RESULTS: Analysis of a Caenorhabditis elegans class VI myosin deletion mutant reveals a role for this motor protein in the segregation of cell components during spermatogenesis. Mutant spermatocytes cannot efficiently deliver mitochondria and endoplasmic reticulum/Golgi-derived fibrous-body membranous organelle complexes to budding spermatids, and fail to remove actin filaments and microtubules from the spermatids. The segregation defects are not due to a global sorting failure as nuclear inheritance is unaffected. CONCLUSIONS: C. elegans myosin VI has an important role in the unequal partitioning of both organelles and cytoskeletal components, a novel role for this class of motor protein.     

Genetic Evidence that an Endosymbiont-derived Endoplasmic Reticulum-associated Protein Degradation (ERAD) System Functions in Import of Apicoplast Proteins

Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1_Ap results in loss of apicoplast protein import and subsequent death of the parasite.     

ZHP-3 acts at crossovers to couple meiotic recombination with synaptonemal complex disassembly and bivalent formation in C. elegans

Crossover recombination and the formation of chiasmata normally ensure the proper segregation of homologous chromosomes during the first meiotic division. zhp-3, the Caenorhabditis elegans ortholog of the budding yeast ZIP3 gene, is required for crossover recombination. We show that ZHP-3 protein localization is highly dynamic. At a key transition point in meiotic prophase, the protein shifts from along the length of the synaptonemal complex (SC) to an asymmetric localization on the SC and eventually becomes restricted to foci that mark crossover recombination events. A zhp-3::gfp transgene partially complements a null mutation and reveals a separation of function; although the fusion protein can promote nearly wild-type levels of recombination, aneuploidy among the progeny is high, indicating defects in meiotic chromosome segregation. The structure of bivalents is perturbed in this mutant, suggesting that the chromosome segregation defect results from an inability to properly remodel chromosomes in response to crossovers. smo-1 mutants exhibit phenotypes similar to zhp-3::gfp mutants at higher temperatures, and smo-1; zhp-3::gfp double mutants exhibit more severe meiotic defects than either single mutant, consistent with a role for SUMO in the process of SC disassembly and bivalent differentiation. We propose that coordination of crossover recombination with SC disassembly and bivalent formation reflects a conserved role of Zip3/ZHP-3 in coupling recombination with SC morphogenesis.     

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.     

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.     

Organizational changes of the daughter basal complex during the parasite replication of Toxoplasma gondii

The apicomplexans are a large group of parasitic protozoa, many of which are important human and animal pathogens, including Plasmodium falciparum and Toxoplasma gondii. These parasites cause disease only when they replicate, and their replication is critically dependent on the proper assembly of the parasite cytoskeletons during cell division. In addition to their importance in pathogenesis, the apicomplexan parasite cytoskeletons are spectacular structures. Therefore, understanding the cytoskeletal biogenesis of these parasites is important not only for parasitology but also of general interest to broader cell biology. Previously, we found that the basal end of T. gondii contains a novel cytoskeletal assembly, the basal complex, a cytoskeletal compartment constructed in concert with the daughter cortical cytoskeleton during cell division. This study focuses on key events during the biogenesis of the basal complex using high resolution light microscopy, and reveals that daughter basal complexes are established around the duplicated centrioles independently of the structural integrity of the daughter cortical cytoskeleton, and that they are dynamic "caps" at the growing ends of the daughters. Compartmentation and polarization of the basal complex is first revealed at a late stage of cell division upon the recruitment of an EF-hand containing calcium binding protein, TgCentrin2. This correlates with the constriction of the basal complex, a process that can be artificially induced by increasing cellular calcium concentration. The basal complex is therefore likely to be a new kind of centrin-based contractile apparatus.     

The P1 plasmid is segregated to daughter cells by a 'capture and ejection' mechanism coordinated with Escherichia coli cell division

The fate of the P1 plasmid of Escherichia coli was followed by time-lapse photomicroscopy. A GFP-ParB fusion marked the plasmid during partition (segregation) to daughter cells at slow growth rate. The process differs from that previously inferred from statistical analysis of fixed cells. A focus of plasmid copies is captured at the cell centre. Immediately before cell division, the copies eject bidirectionally along the long axis of the cell. Cell division traps one or more plasmid copies in each daughter. They are not directed to a prescribed position but are free to move, associate and disassociate. Later, they are captured to the new cell centre to restart the cycle. A null P1 par mutant associates to form a focus, but it is neither captured nor ejected. A dominant negative ParB protein forms a plasmid focus that attaches to the cell centre but never ejects. It remains captive at the centre and blocks host cell division. The cells elongate. Eventually the intact focus is pushed to one side and the cells divide simultaneously in several places at the same time. This suggests that the wild-type plasmid imposes a regulatory node on the host cell cycle, preventing cell division until its own segregation is completed.     

Ciprofloxacin Derivatives Affect Parasite Cell Division and Increase the Survival of Mice Infected with Toxoplasma gondii

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against T. gondii in vitro, compared with the original compound. Cipro derivatives are expected to target the parasite’s DNA gyrase complex in the apicoplast. The activity of these compounds in vivo, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives in vivo, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects T. gondii tachyzoites in vitro, by immunofluorescence and transmission electron microscopy (TEM). When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13–25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment). Light microscopy examination early (6 and 24h) post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition - with the appearance of ‘tethered’ parasites – malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against C. parvum, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in T. gondii tachyzoites, supporting that these compounds targets the apicoplast. Our results show that Cipro derivatives improved the survival of mice acutely infected with T. gondii and inhibited parasite replication early in the first cycle of infection in vitro, highlighting their therapeutic potential for the treatment of toxoplasmosis.     

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.     

The Toxoplasma gondii calcium‐dependent protein kinase 7 is involved in early steps of parasite division and is crucial for parasite survival

Apicomplexan parasites express various calcium‐dependent protein kinases (CDPKs), and some of them play essential roles in invasion and egress. Five of the six CDPKs conserved in most Apicomplexa have been studied at the molecular and cellular levels in Plasmodium species and/or in Toxoplasma gondii parasites, but the function of CDPK7 was so far uncharacterized. In T. gondii, during intracellular replication, two parasites are formed within a mother cell through a unique process called endodyogeny. Here we demonstrate that the knock‐down of CDPK7 protein in T. gondii results in pronounced defects in parasite division and a major growth deficiency, while it is dispensable for motility, egress and microneme exocytosis. In cdpk7‐depleted parasites, the overall DNA content was not impaired, but the polarity of daughter cells budding and the fate of several subcellular structures or proteins involved in cell division were affected, such as the centrosomes and the kinetochore. Overall, our data suggest that CDPK7 is crucial for proper maintenance of centrosome integrity required for the initiation of endodyogeny. Our findings provide a first insight into the probable role of calcium‐dependent signalling in parasite multiplication, in addition to its more widely explored role in invasion and egress.