Our laboratory is investigating molecular processes critical for normal growth and development. We are using molecular, genetic, and biochemical techniques and the model eukaryotic system Dictyostelium discoideum to dissect molecular pathways that are essential for universal multicellular function. Dictyostelium has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions and we have been pursuing technical aspects to further optimize its genetic utility. The relatively small (approximately 34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We have described a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying four separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers. Presenilin (PS) was first identified by genetic linkage in humans to familial Alzheimer?s disease and was subsequently shown to interact biochemically with GSK3 and ?-catenin, a substrate of GSK3. PS was, thus, postulated to function as a scaffolding protein for GSK3 signaling. We identified two PS genes in Dictysotelium and knock-out analyses indicates an essential role for PS in development, but the phenotypes of the single and double PS mutants suggest a pathway distinct from that of GSK3. In other systems, PS, Nicastrin (Nct), and Aph1 function together to regulate ?-secretase processing of APP and notch proteins. We have also isolated and mutated (singly and doubly) the Dictyostelium Nct and Aph1 genes and shown that their phenotypes ressemble those of the ps-nulls. Our recent data indicate that complete ablation of the ?-secretase pathway in Nct-null/Aph1-null disrupts a specific pathway for cell differentiation of Dictyostelium, prespore/spore development. Since Dictyostelium lacks both APP and notch, the PS/Nct/Aph1 pathway in Dictyostelium must regulate other pathways. Indeed, we have identified a potential novel target for ?-secretase processing and are assessing its role during prespore/spore development. Inactivating mutations in either Tuberin (TSC2) or Hamartin (TSC1) cause tuberous sclerosis in humans, a multi-organ tumor syndrome characterized by cellular overgrowth. Tuberin and Hamartin are modeled as tumor suppressors and function as a heterodimeric GTPase activating (GAP) complex that targets the Ras-like, small GTPase Rheb. Consistent with this model, GTP-bound, active Rheb positively regulates cell size and growth by activating a rapamycin-sensitive TOR complex (TORC1) containing Raptor and Lst8. A second TOR complex (TORC2) that contains Pia (Rictor), RIP3, and Lst8 has been identified in several systems; TORC2 is rapamycin-insensitive and regulates the actin cytoskeleton. Dictyostelium provides a unique system to separate the functions of TORC1 and TORC2 in terms of growth and cell migration/development. In the presence of nutrients, growth is continuous and developmental processes are restricted; upon nutrient depletion, growth arrests and starving Dictyostelium chemotactically aggregate and differentiate as multi-cellular structures. In Dictyostelium, we suggest roles for Tuberin and Rheb upstream of TORC2 in controlling cell migration during development. Pia and RIP3 are known to be required for chemotaxis/aggregation during development. We now show that Lst8 is also required for aggregation and that Rheb positively regulates aggregation and chemotaxis. Furthermore, expression of a dominant-negative Rheb in wild-type cells phenocopies the impaired aggregation of rheb-null cells. Conversely, Tuberin null cells exhibit enhanced aggregation and chemotaxis compared to wildtype supporting the role of Tuberin as a negative regulator of Rheb. Collectively, these data support a link for Tuberin and Rheb in the control of chemotaxis, cell migration, and development via TORC2. Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism The ERK/MAP kinases have been implicated in the regulation of chemotactic signaling in mammalian cells and Dictyostelium. Chemotaxing Dictyostelium periodically synthesize and secrete cAMP, which in addition to its intracellular role, acts as a chemoattractant. The extracellular oscillatory cAMP signal is perceived by the seven-transmembrane receptor CAR1, which activates downstream networks by both G protein-dependent and -independent mechanisms. These downstream pathways respond transiently, adapting (de-sensitizing) to a non-fluctuating signal. It has been suggested that the activation/de-activation of ERK2 in Dictyostelium is required to regulate oscillatory cAMP production, and that deactivation of ERK2 is mediated by a PKA-dependent intracellular feedback mechanism. We have now tested this model by separately analyzing the activation and de-activation of ERK2 under controlled conditions that manipulate the level of the extracellular cAMP stimulus. We now show that the process of ERK2 activation is completely adaptive, but that ERK2 de-activation is non-adaptive and inhibited by the continuous presence of the cAMP ligand. In addition, we found that both activation and de-activation occur independently of G proteins and of ligand induced production of intracellular cAMP. These data suggest that ERK2 activity is not controlled by a simple intracellular feedback mechanism through PKA and underscore the complexity of ERK2 regulation in control of chemotactic signaling and chemotaxis.
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