Genetic rescue of a <i>Toxoplasma gondii</i> conditional cell cycle mutant

White, Michael; Jerome, Maria; Vaishnava, Shipra; Guerini, Michael N.; Behnke, Michael S.; Striepen, Boris

doi:10.1111/j.1365-2958.2004.04471.x

Cited by 28 publications

(27 citation statements)

References 69 publications

(97 reference statements)

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“…Toxoplasma division is a binary process called endodyogeny during which a single chromosome replication is followed by concurrent mitosis and parasite budding (65). In our study, DGLE was detected within dividing cells at the tip of the duplicated inner membrane complexes of the daughter cells (Fig.…”

Section: Discussionmentioning

confidence: 55%

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Botté

Saïdani

Mondragón

et al. 2008

Journal of Lipid Research

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Toxoplasma gondii is a unicellular parasite characterized by unique extracellular and intracellular membrane compartments. The lipid composition of subcellular membranes has not been determined, limiting our understanding of lipid homeostasis, control, and trafficking, a series of processes involved in pathogenesis. In addition to a mitochondrion, Toxoplasma contains a plastid called the apicoplast. The occurrence of a plastid raised the question of the presence of chloroplast galactolipids. Using three independent rabbit and rat antibodies against digalactosyldiacylglycerol (DGDG) from plant chloroplasts, we detected a class of Toxoplasma lipids harboring a digalactolipid-like epitope (DGLE). Immunolabeling characterization supports the notion that the DGLE polar head is similar to that of DGDG. Mass spectrometry analyses indicated that dihexosyl lipids having various hydrophobic moieties (ceramide, diacylglycerol, and acylalkylglycerol) might react with anti-DGDG, but we cannot exclude the possibility that more complex dihexosyl-terminated lipids might also be immunolabeled. DGLE localization was analyzed by immunofluorescence and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE was scattered in pellicle membrane domains in extracellular tachyzoites and was relocalized to the anterior tip of the cell upon invasion in an actin-dependent manner, providing insights on a possible role in pathogenetic processes. DGLE was detected in other Apicomplexa (i.e

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Section: Discussionmentioning

confidence: 55%

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Botté

Saïdani

Mondragón

et al. 2008

Journal of Lipid Research

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“…Linkage between nuclear division and daughter formation is observed in the mitotic mutant, ts11C9 [7,29], which is genetically complemented by a known suppressor of cyclin kinase activity [29], and in tachyzoites that undergo rare chromosome re-replication where daughter formation is paused so that viable parasites are produced at the conclusion of the second round of DNA replication [8]. Altogether, these results give a partially answer to the question of whether checkpoints are active in Toxoplasma, but the total number of checkpoints and the details of each mechanism is still uncertain.…”

Section: Discussionmentioning

confidence: 99%

“…To confirm the haploid growth arrest by PDTC, we determined that 85% of parasites in this blocked population contained a single centrosome. By contrast to the PDTC-treatment, thymidine-blocked transgenic RH TK+ parasites are stopped at the late-G1/early S phase boundary and the majority of these parasites (67%) have a duplicated centrosome [29]. Combined, these results indicate that PDTC-treatment primarily stops parasite growth primarily in the G1 phase of the cell cycle, which is remarkably similar to the reported affects of PDTC on animal cells [27], and distinct from the thymidine-block of RH TK+ parasites.…”

Section: The Primary Pdtc Block Occurs In the G1 Phase Of The Tachyzomentioning

confidence: 93%

“…However, previous experimental studies in T. gondii have demonstrated that tachyzoites can specifically arrest in G1, at the G1/S boundary, or in mitosis [12,28,29] indicating the Toxoplasma cell cycle is coordinated at multiple places. In order to determine where in the cell cycle PDTC arrests parasite growth, we compared the DNA content of asynchronous untreated populations with parasites whose growth was blocked by PDTC ( Fig.…”

Section: The Primary Pdtc Block Occurs In the G1 Phase Of The Tachyzomentioning

confidence: 98%

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Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization

Felipe

Lehmann

Jerome

et al. 2008

Molecular and Biochemical Parasitology

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Successful completion of the Toxoplasma cell cycle requires the coordination of a series of complex and ordered processes that results in the formation of two daughters by internal budding. Although we now understand the order and timing of intracellular events associated with the parasite cell cycle, the molecular details of the checkpoints that regulate each step in T. gondii division is still uncertain. In other eukaryotic cells, the use of cytostatic inhibitors that are able to arrest replication at natural checkpoints have been exploited to induce synchronization of population growth. Herein, we describe a novel method to synchronize T. gondii tachyzoites based on the reversible growth inhibition by the drug, pyrrolidine dithiocarbamate. This method is an improvement over other strategies developed for this parasite as no prior genetic manipulation of the parasite was required. RH tachyzoites blocked by pyrrolidine dithiocarbamate exhibited a near uniform haploid DNA content and single centrosome indicating that this compound arrests parasites in the G1 phase of the tachyzoite cell cycle with a minor block in late cytokinesis. Thus, these studies support the existence of a natural checkpoint that regulates passage through the G1 period of the cell cycle. Populations released from pyrrolidine dithiocarbamate inhibition completed progression through G1 and entered S phase ~2 hours postdrug release. The transit of drug-synchronized populations through S phase and mitosis followed a similar timeframe to previous studies of the tachyzoite cell cycle. Tachyzoites treated with pyrrolidine dithiocarbamate were fully viable and completed two identical division cycles post-drug release demonstrating that this is a robust method for synchronizing population growth in Toxoplasma.

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“…The centrosome also has a master function in limiting chromosome replication in the tachyzoite that appears to require physical contact. Evidence from tachyzoite growth mutants indicates that "copy once" restrictions in the budding cycle require a connection between the centrosome and new daughter cytoskeleton [26,27]. Breaking this contact by drug treatment [28] or by genetic ablation of centrosome factors [15,22,26] leads to unregulated nuclear re-duplication and abnormal budding.…”

Section: Introductionmentioning

confidence: 99%

A Novel Bipartite Centrosome Coordinates the Apicomplexan Cell Cycle

et al. 2015

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Apicomplexan parasites can change fundamental features of cell division during their life cycles, suspending cytokinesis when needed and changing proliferative scale in different hosts and tissues. The structural and molecular basis for this remarkable cell cycle flexibility is not fully understood, although the centrosome serves a key role in determining when and how much replication will occur. Here we describe the discovery of multiple replicating core complexes with distinct protein composition and function in the centrosome of Toxoplasma gondii. An outer core complex distal from the nucleus contains the TgCentrin1/TgSfi1 protein pair, along with the cartwheel protein TgSas-6 and a novel Aurora-related kinase, while an inner core closely aligned with the unique spindle pole (centrocone) holds distant orthologs of the CEP250/C-Nap protein family. This outer/inner spatial relationship of centrosome cores is maintained throughout the cell cycle. When in metaphase, the duplicated cores align to opposite sides of the kinetochores in a linear array. As parasites transition into S phase, the cores sequentially duplicate, outer core first and inner core second, ensuring that each daughter parasite inherits one copy of each type of centrosome core. A key serine/threonine kinase distantly related to the MAPK family is localized to the centrosome, where it restricts core duplication to once per cycle and ensures the proper formation of new daughter parasites. Genetic analysis of the outer core in a temperature-sensitive mutant demonstrated this core functions primarily in cytokinesis. An inhibition of ts-TgSfi1 function at high temperature caused the loss of outer cores and a severe block to budding, while at the same time the inner core amplified along with the unique spindle pole, indicating the inner core and spindle pole are independent and co-regulated. The discovery of a novel bipartite organization in the parasite centrosome that segregates the functions of karyokinesis and cytokinesis provides an explanation for how cell cycle flexibility is achieved in apicomplexan life cycles.

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Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant

Cited by 28 publications

References 69 publications

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization

A Novel Bipartite Centrosome Coordinates the Apicomplexan Cell Cycle

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