Summary 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 sub-family 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 spatio-temporal 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.

Heterotrimeric G proteins are components of principal signaling pathways in eukaryotes. In higher organisms, alpha subunits of G proteins have been divided into four families, Gi, Gs, Gq, and G12. We previously identified a G alpha i homologue gna-1 in the filamentous fungus Neurospora crassa. Now we report that deletion of gna-1 leads to multiple phenotypes during the vegetative and sexual cycles in N. crassa. On solid medium, delta gna-1 strains have a slower rate of hyphal apical extension than wild type, a rate that is more pronounced under hyperosmotic conditions or in the presence of a cellophane overlay. delta gna-1 mutants accumulate less mass than wild-type strains, and their mass accumulation is not affected in the same way by exposure to light. delta gna-1 strains are defective in macroconidiation, possessing aerial hyphae that are shorter, contain abnormal swellings, and differentiate adherent macroconidia. During the sexual cycle, delta gna-1 strains are fertile as males. However, the mutants are female-sterile, producing small, aberrant female reproductive structures. After fertilization, delta gna-1 female structures do not enlarge and develop normally, and no sexual spores are produced. Thus, mutation of gna-1 results in sex-specific loss of fertility.

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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 G␣ protein GNA-1 is a direct positive regulator of adenylyl cyclase. ⌬gna-1 strains are female-sterile, and ⌬gna-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 ⌬gna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. ⌬gna-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 ⌬gna-1 cr-1, defects, which is consistent with previous results demonstrating that ⌬gna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the ⌬gna-1 mutation are sterile; however, unlike cr-1 and ⌬gna-1 strains, the ⌬gna-1 cr-1 mutant does not produce protoperithecia. The ⌬gna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < ⌬gna-1 < cr-1 ‫؍‬ ⌬gna-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.Development in fungal systems frequently occurs in response to specific environmental cues and stressors. In the presence of abundant nutrients, the filamentous fungus Neurospora crassa extends hyphae that elongate and fuse to form the multicellular mycelium (for a review, see reference 51). Desiccation or nutrient deprivation causes the mycelium to differentiate aerial hyphae that give rise to conidiophores and multinucleate asexual spores, macroconidia (referred to here as conidia). Elaboration of conidiophores and production of conidia require an air-water interface; however, submerged cultures can be induced to undergo conidiation by carbon or nitrogen starvation or exposure to high temperatures (7,18,43,55). Nitrogen limitation initiates the sexual cycle by stimulating production of female reproductive structures, or protoperithecia (reviewed in reference 44). Fertilization by a conidium or hypha of the opposite mating type results in formation of the fertilized structure (perithecium), within which sexual spores (ascospores) develop. Mature ascospores are subsequently ejected from the perithecium in the direction of blue light.Heterotrimeric GTP-binding proteins, consisting of ␣, ␤, and ␥ subunits, transduce various environmental signals ...

Summary 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 re-distributes to a cytoplasmic localization pattern upon egress.