The Crk4-Cyc4 complex regulates G/M transition in Toxoplasma gondii.

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk).

Essential role of the conserved oligomeric Golgi complex in .

The Golgi is an essential eukaryotic organelle and a major place for protein sorting and glycosylation. Among apicomplexan parasites, retains the most developed Golgi structure and produces many glycosylated factors necessary for parasite survival. Despite its importance, Golgi function received little attention in the past.

Restriction Checkpoint Controls Bradyzoite Development in Toxoplasma gondii.

Human toxoplasmosis is a life-threatening disease caused by the apicomplexan parasite Toxoplasma gondii. Rapid replication of the tachyzoite is associated with symptomatic disease, while suppressed division of the bradyzoite is responsible for chronic disease. Here, we identified the T.

Novel CRK-Cyclin Complex Controls Spindle Assembly Checkpoint in Endodyogeny.

Opportunistic parasites of the Apicomplexa phylum use a variety of division modes built on two types of cell cycles that incorporate two distinctive mechanisms of mitosis: uncoupled from and coupled to parasite budding. Parasites have evolved novel factors to regulate such unique replication mechanisms that are poorly understood. Here, we have combined genetics, quantitative fluorescence microscopy, and global proteomics approaches to examine endodyogeny in Toxoplasma gondii dividing by mitosis coupled to cytokinesis.

The RESTRICTION checkpoint: a window of opportunity governing developmental transitions in Toxoplasma gondii.

The life cycle of Toxoplasma gondii is characterized by active replication alternating with periods of rest. Encysted dormant sporozoites and bradyzoites initiate active replication as tachyzoites and merozoites. Here we explore the role of the cell cycle with a focus on the canonical G RESTRICTION checkpoint (R-point) as the integrator governing developmental decisions in T.

Apicomplexa Cell Cycles: Something Old, Borrowed, Lost, and New.

Increased parasite burden is linked to the severity of clinical disease caused by Apicomplexa parasites such as Toxoplasma gondii, Plasmodium spp, and Cryptosporidium. Pathogenesis of apicomplexan infections is greatly affected by the growth rate of the parasite asexual stages. This review discusses recent advances in deciphering the mitotic structures and cell cycle regulatory factors required by Apicomplexa parasites to replicate.

The Centrocone Houses Cell Cycle Regulatory Factors.

Our knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel factor that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures, and we named this factor ECR1 for essential for chromosome replication 1. ECR1 was discovered by complementation of a temperature-sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40°C similar to a ts mutant carrying a defect in topoisomerase.

Checkpoints of apicomplexan cell division identified in Toxoplasma gondii.

The unusual cell cycles of Apicomplexa parasites are remarkably flexible with the ability to complete cytokinesis and karyokinesis coordinately or postpone cytokinesis for several rounds of chromosome replication, and are well recognized. Despite this surprising biology, the molecular machinery required to achieve this flexibility is largely unknown. In this study, we provide comprehensive experimental evidence that apicomplexan parasites utilize multiple Cdk-related kinases (Crks) to coordinate cell division.

Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development.

Unlabelled: Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages.

Toxoplasma gondii Arginine Methyltransferase 1 (PRMT1) Is Necessary for Centrosome Dynamics during Tachyzoite Cell Division.

Unlabelled: The arginine methyltransferase family (PRMT) has been implicated in a variety of cellular processes, including signal transduction, epigenetic regulation, and DNA repair pathways. PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. To further define the biological function of the PRMT family, we generated T.

A novel bipartite centrosome coordinates the apicomplexan cell cycle.

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.

Genetic manipulation of the Toxoplasma gondii genome by fosmid recombineering.

Unlabelled: Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting.

Transcript maturation in apicomplexan parasites.

The complex life cycles of apicomplexan parasites are associated with dynamic changes of protein repertoire. In Toxoplasma gondii, global analysis of gene expression demonstrates that dynamic changes in mRNA levels unfold in a serial cascade during asexual replication and up to 50% of encoded genes are unequally expressed in development. Recent studies indicate transcription and mRNA processing have important roles in fulfilling the 'just-in-time' delivery of proteins to parasite growth and development.

A nucleolar AAA-NTPase is required for parasite division.

Apicomplexa division involves several distinct phases shared with other eukaryote cell cycles including a gap period (G1) prior to chromosome synthesis, although how progression through the parasite cell cycle is controlled is not understood. Here we describe a cell cycle mutant that reversibly arrests in the G1 phase. The defect in this mutant was mapped by genetic complementation to a gene encoding a novel AAA-ATPase/CDC48 family member called TgNoAP1.

Discovery of a splicing regulator required for cell cycle progression.

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans.

Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii.

Apicomplexa parasites use complex cell cycles to replicate that are not well understood mechanistically. We have established a robust forward genetic strategy to identify the essential components of parasite cell division. Here we describe a novel temperature sensitive Toxoplasma strain, mutant 13-20C2, which growth arrests due to a defect in mitosis.

Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration.

Cells require ribonucleotide reductase (RNR) activity for DNA replication. In bacteria, electrons can flow from NADPH to RNR by either a thioredoxin-reductase- or a glutathione-reductase-dependent route. Yeast and plants artificially lacking thioredoxin reductases exhibit a slow-growth phenotype, suggesting glutathione-reductase-dependent routes are poor at supporting DNA replication in these organisms.

Cre activity in fetal albCre mouse hepatocytes: Utility for developmental studies.

The albCre transgene, having Cre recombinase driven by the serum albumin (alb) gene promoter, is commonly used to generate adult mice having reliable hepatocyte-specific recombination of loxP-flanked ("floxed") alleles. Based on previous studies, it has been unclear whether albCre transgenes are also reliable in fetal and juvenile mice. Perinatal liver undergoes a dynamic transition from being predominantly hematopoietic to predominantly hepatic.

Cytoprotective Nrf2 pathway is induced in chronically txnrd 1-deficient hepatocytes.

Background: Metabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins.

Principal Findings: Here we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes.

Agonist-dependent phosphorylation of the formyl peptide receptor is regulated by the membrane proximal region of the cytoplasmic tail.

Formyl peptide receptor (FPR) is a chemoattractant G protein-coupled receptor (GPCR) involved in the innate immune response against bacteria. Receptor activation is terminated by receptor phosphorylation of two serine- and threonine-rich regions located in the distal half of the cytoplasmic tail. In this study we show that introduction of an amino acid with a bulky side chain (leucine or glutamine) adjacent to a single leucine, L320, in the membrane-proximal half of the cytoplasmic tail, significantly enhanced receptor phosphorylation, beta-arrestin1/2 translocation, and receptor endocytosis, without affecting G(i)-mediated ERK1/2 activation and release of intracellular calcium.

Role of the carboxyl terminal di-leucine in phosphorylation and internalization of C5a receptor.

The carboxyl tail of G protein-coupled receptors contains motifs that regulate receptor interactions with intracellular partners. Activation of the human neutrophil complement fragment C5a receptor (C5aR) is terminated by phosphorylation of the carboxyl tail followed by receptor internalization. In this study, we demonstrated that bulky hydrophobic residues in the membrane-proximal region of the C5aR carboxyl tail play an important role in proper structure and function of the receptor: Substitution of leucine 319 with alanine (L319A) resulted in receptor retention in the endoplasmic reticulum, whereas a L318A substitution allowed receptor transport to the cell surface, but showed slow internalization upon activation, presumably due to a defect in phosphorylation by both PKC and GRK.

Different endocytosis pathways of the C5a receptor and the N-formyl peptide receptor.

Two chemoattractant receptors, C5aR (the complement fragment C5a receptor) and FPR (the N-formyl peptide receptor), are involved in neutrophil activation at sites of inflammation. In this study, we found major differences in the intracellular trafficking of the receptors in transfected Chinese hamster ovary (CHO) cells. Western blot analysis showed that FPR was stable during a 3 h stimulation with ligand, but C5aR was reduced in quantity by 50%.

The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins.

The Sec34/35 complex was identified as one of the evolutionarily conserved protein complexes that regulates a cis-Golgi step in intracellular vesicular transport. We have identified three new proteins that associate with Sec35p and Sec34p in yeast cytosol. Mutations in these Sec34/35 complex subunits result in defects in basic Golgi functions, including glycosylation of secretory proteins, protein sorting, and retention of Golgi resident proteins.