F. Douglas Ivey

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.

Shared and Independent Roles for a Gαi Protein and Adenylyl Cyclase in Regulating Development and Stress Responses in Neurospora crassa

ABSTRACT Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in Neurospora crassa. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric Gα protein GNA-1 is a direct positive regulator of adenylyl cyclase. Δgna-1 strains are female-sterile, and Δgna-1 strains have reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a Δgna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. Δgna-1 cr-1 mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues cr-1, but not Δgna-1 cr-1, defects, which is consistent with previous results demonstrating that Δgna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the Δgna-1 mutation are sterile; however, unlike cr-1 and Δgna-1 strains, the Δgna-1 cr-1 mutant does not produce protoperithecia. The Δgna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < Δgna-1 < cr-1 = Δgna-1 cr-1, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate N. crassa growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.

Direct activation of fission yeast adenylate cyclase by the Gpa2 Gα of the glucose signaling pathway

G protein-mediated signaling is implicated in yeast and fungal cAMP pathways. By two-hybrid screens and pull-down experiments, we show that the fission yeast Gpa2 Gα binds an N-terminal domain of adenylate cyclase, comprising a moderately conserved sequence within a region otherwise poorly related to other fungal adenylate cyclases. Overexpressing this domain in yeast perturbs cAMP signaling, which is restored by Gpa2 coexpression. Mutations affecting this domain, over 1,100 residues from the catalytic domain, alter glucose-triggered cAMP signaling. This is evidence for direct activation of adenylate cyclase by a fungal G protein and suggests a distinct activation mechanism from that of mammals.

Schizosaccharomyces pombe Adenylate Cyclase Suppressor Mutations Suggest a Role for cAMP Phosphodiesterase Regulation in Feedback Control of Glucose/cAMP Signaling

Mutations affecting the Schizosaccharomyces pombe cAMP phosphodiesterase (PDE) gene cgs2+ were identified in a screen for suppressors of mutant alleles of the adenylate cyclase gene (git2+/cyr1+), which encode catalytically active forms of the enzyme that cannot be stimulated by extracellular glucose signaling. These mutations suppress both the git2− mutant alleles used in the suppressor selection and mutations in git1+, git3+, git5+, git7+, git10+, and git11+, which are all required for adenylate cyclase activation. Notably, these cgs2 mutant alleles fail to suppress mutations in gpa2+, which encodes the Gα subunit of a heterotrimeric G protein required for adenylate cyclase activation, although the previously identified cgs2-2 allele does suppress loss of gpa2+. Further analysis of the cgs2-s1 allele reveals a synthetic interaction with the gpa2R176H-activated allele, with respect to derepression of fbp1-lacZ transcription in glucose-starved cells. In addition, direct measurements of cAMP levels show that cgs2-s1 cells maintain normal basal cAMP levels, but are severely defective in feedback regulation upon glucose detection. These results suggest that PDE activity in S. pombe may be coordinately regulated with adenylate cyclase activity as part of the feedback regulation mechanism to limit the cAMP response to glucose detection.

Development of a Fission Yeast-Based High-Throughput Screen to Identify Chemical Regulators of cAMP Phosphodiesterases

Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3′,5′-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens. (Journal of Biomolecular Screening 2008:62-71)

New classes of PDE7 inhibitors identified by a fission yeast-based HTS

Studies of the phosphodiesterase PDE7 family are impeded by there being only one commercially available PDE7 inhibitor, BRL50481. The authors have employed a high-throughput screen of commercial chemical libraries, using a fission yeast-based assay, to identify PDE7 inhibitors that include steroids, podocarpanes, and an unusual heterocyclic compound, BC30. In vitro enzyme assays measuring the potency of BC30 and 2 podocarpanes, in comparison with BRL50481, produce data consistent with those from yeast-based assays. In other enzyme assays, BC30 stimulates the PDE4D catalytic domain but not full-length PDE4D2, suggesting an allosteric site of action. BC30 significantly enhances the anti-inflammatory effect of the PDE4 inhibitor rolipram as measured by release of tumor necrosis factor α from activated monocytes. These studies introduce several new PDE7 inhibitors that may be excellent candidates for medicinal chemistry because of the requirements for drug-like characteristics placed on them by the nature of the yeast-based screen.

Targeted proteomic dissection of Toxoplasma cytoskeleton sub-compartments using MORN1.

The basal complex in Toxoplasma functions as the contractile ring in the cell division process. Basal complex contraction tapers the daughter cytoskeleton toward the basal end and is required for daughter segregation. We have previously shown that the protein MORN1 is essential for basal complex assembly and likely acts as a scaffolding protein. To further our understanding of the basal complex, we combined subcellular fractionation with an affinity purification of the MORN1 complex and identified its protein composition. We identified two new components of the basal complex, one of which uniquely associated with the basal complex in mature parasites, the first of its kind. In addition, we identified several other novel cytoskeleton proteins with different spatiotemporal dynamics throughout cell division. Since many of these proteins are unique to Apicomplexa this study significantly contributes to the annotation of their unique cytoskeleton. Furthermore, we show that G‐actin binding protein TgCAP is localized at the apical cap region in intracellular parasites, but quickly redistributes to a cytoplasmic localization pattern upon egress.

Schizosaccharomyces pombe Git1 Is a C2-Domain Protein Required for Glucose Activation of Adenylate Cyclase

Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway, activating cAMP-dependent protein kinase A (PKA). This requires nine git (glucose insensitive transcription) genes that encode adenylate cyclase, the PKA catalytic subunit, and seven “upstream” proteins required for glucose-triggered adenylate cyclase activation, including three heterotrimeric G-protein subunits and its associated receptor. We describe here the cloning and characterization of the git1+ gene. Git1 is distantly related to a small group of uncharacterized fungal proteins, including a second S. pombe protein that is not functionally redundant with Git1, as well as to members of the UNC-13/Munc13 protein family. Mutations in git1+ demonstrate functional roles for the two most highly conserved regions of the protein, the C2 domain and the MHD2 Munc homology domain. Cells lacking Git1 are viable, but display phenotypes associated with cAMP-signaling defects, even in strains expressing a mutationally activated Gα-subunit, which activates adenylate cyclase. These cells possess reduced basal cAMP levels and fail to mount a cAMP response to glucose. In addition, Git1 and adenylate cyclase physically interact and partially colocalize in the cell. Thus, Git1 is a critical component of the S. pombe glucose/cAMP pathway.

Activated Alleles of the Schizosaccharomyces pombe gpa2+ Gα Gene Identify Residues Involved in GDP-GTP Exchange

ABSTRACT The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Gα subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gβγ dimer to repress transcription of the glucose-regulated fbp1+ gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2+ chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Gα function, thus extending our understanding of Gα protein structure-function relationships.

Sck1 Negatively Regulates Gpa2-Mediated Glucose Signaling in Schizosaccharomyces pombe

ABSTRACT Schizosaccharomyces pombe detects extracellular glucose via a G protein-mediated cyclic AMP (cAMP)-signaling pathway activating protein kinase A (PKA) and regulating transcription of genes involved in metabolism and sexual development. In this pathway, Gpa2 Gα binds to and activates adenylyl cyclase in response to glucose detection by the Git3 G protein-coupled receptor. Using a two-hybrid screen to identify extrinsic regulators of Gpa2, we isolated a clone that expresses codons 471 to 696 of the Sck1 kinase, which appears to display a higher affinity for Gpa2K270E-activated Gα relative to Gpa2+ Gα. Deletion of sck1+ or mutational inactivation of the Sck1 kinase produces phenotypes reflecting increased PKA activity in strains expressing Gpa2+ or Gpa2K270E, suggesting that Sck1 negatively regulates PKA activation through Gpa2. In contrast to the Gpa2K270E GDP-GTP exchange rate mutant, GTPase-defective Gpa2R176H weakly binds Sck1 in the two-hybrid screen and a deletion of sck1+ in a Gpa2R176H strain confers phenotypes consistent with a slight reduction in PKA activity. Finally, deleting sck1+ in a gpa2Δ strain results in phenotypes consistent with a second role for Sck1 acting in parallel with PKA. In addition to this parallel role with PKA, our data suggest that Sck1 negatively regulates Gpa2, possibly targeting the nucleotide-free form of the protein that may expose the one and only AKT/PKB consensus site in Gpa2 for Sck1 to bind. This dual role for Sck1 may allow S. pombe to produce distinct biological responses to glucose and nitrogen starvation signals that both activate the Wis1-Spc1/StyI stress-activated protein kinase (SAPK) pathway.