dictyNews Electronic Edition Volume 25, number 13 December 1, 2005 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Use of a Penetratin-linked peptide in Dictyostelium. J Ryves and A. Harwood Cardiff School of Biosciences, Biomedical Sciences Building, Museum Avenue, PO box 911, Cardiff, CF10 3US, Wales, UK Molecular Biotechnology, in press The plasma membrane is an effective barrier to most macromolecules and hydrophilic molecules. Remarkably, a class of positively charged cell penetrating peptides (CPPs) has been discovered that can translocate themselves and associated cargoes into the cytoplasm. These have been used to carry oligopeptide and oligonucleotide based inhibitors into mammalian cells. A recent report indicates that the same CPPs are internalized by plant protoplasts suggesting this may be a universal phenomenon. We report here that the prototypical CPP, penetratin, enters cells of the free-living amoebae Dictyostelium discoideum. To investigate the functionality of this technology we fused the penetratin sequence to PKI, a peptide inhibitor of the cAMP dependent protein kinase (PKA). Consistent with its PKA inhibitory action, Penetratin-PKI blocked aggregation in wild type cells and at appropriate concentrations rescued the phenotype of a Dictyostelium mutant which has constitutively high PKA activity. This technology offers an effective method for delivery of oligopeptides and oligonucleotides into Dictyostelium. Submitted by: Jonathan Ryves [ryveswj@cf.ac.uk] ----------------------------------------------------------------------------- PI3 Kinase Activity Controls the Chemoattractant-Mediated Activation and Adaptation of Adenylyl Cyclase Frank I. Comer 1,2 and Carole A. Parent 1 1. Laboratory of Cellular and Molecular Biology Center for Cancer Research, NCI, NIH 2. PRAT Research Fellowship Program, NIGMS, NIH Molecular Biology of the Cell, In press published November 2, 2005 as 10.1091/mbc.E05-08-0781 The binding of chemoattractants to cognate G protein-coupled receptors activates a variety of signaling cascades that provide spatial and temporal cues required for chemotaxis. When subjected to uniform stimulation, these responses are transient, showing an initial peak of activation followed by a period of adaptation, in which activity subsides even in the presence of stimulus. A tightly regulated balance between receptor-mediated stimulatory and inhibitory pathways controls the kinetics of activation and subsequent adaptation. In Dictyostelium, the adenylyl cyclase ACA, which synthesizes the chemoattractant cAMP, is essential to relay the signal to neighboring cells. Here we report that cells lacking PI3K activity are deficient in signal relay. In LY294002-treated cells this defect is due to loss of ACA activation. In contrast, in cells lacking PI3K1 and PI3K2, the signal relay defect is due to loss of ACA adaptation. We propose that residual low levels of 3-phosphoinositides in pi3k1-/2- cells is sufficient to generate the initial peak of ACA activity, yet is insufficient to sustain the inhibitory phase required for its adaptation. Thus, PI3K activity is poised to regulate both ACA activation and adaptation, thereby providing a link to ensure the proper balance of counteracting signals required to maintain optimal chemoresponsiveness. Submitted by: Frank Comer [comerf@helix.nih.gov] ----------------------------------------------------------------------------- Identification and Purification of a DNA-Binding Protein Interacting with the Promoter of 5’-nucleotidase in Dictyostelium discoideum. Natasha S. Wiles, Can M. Eristi, Bradley R. Joyce, and Charles L. Rutherford. Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, United States. Arch. Biochem. Biophys., in press The developmental management of 5’-nucleotidase (5nt) expression in Dictyostelium discoideum has provided a focal point for studies of gene regulation at the level of transcription. To identify DNA-protein interactions involved in the 5nt regulation, EMSAs were performed using short oligonucleotides, designed to span a 357 bp promoter region. A binding activity (Rf = 0.33) was identified and shown to be specific to the nucleotide sequence between -338 and -309 bp relative the 5nt ATG. Characterization of the binding activity, including the effects of salt and temperature, provided insight into the nature and stability of the protein. The protein was purified in a series of chromatographic stages, including DEAE Sephacel, Heparin Sepharose, DNA affinity and gel filtration. SDS-PAGE analysis identified a polypeptide with a molecular weight of 70 kDa. Mass spectrometry revealed that the purified protein was a putative formyltetrahydrofolate synthase. Submitted by: Charles Rutherford [rutherfo@vt.edu] ----------------------------------------------------------------------------- Functional genomics in Dictyostelium: MidA, a novel conserved protein is required for mitochondrial function and development Patricia Torija, Juan J. Vicente, Tiago B. Rodrigues, Alicia Robles, Sebastián Cerdán, Leandro Sastre, Rosa M. Calvo and Ricardo Escalante Instituto de Investigaciones Biomédicas Alberto Sols. C.S.I.C./U.A.M., Calle Arturo Duperier 4, 28029 Madrid. Spain. Journal of Cell Science, in press Genomic sequencing has revealed a large number of evolutionary conserved genes of unknown function. In the absence of characterized functional domains, the discovery of the role of these genes must rely on experimental approaches. We have selected 30 Dictyostelium genes of unknown function that showed high similarity to uncharacterized human genes and were absent in the complete proteomes from S.cerevisiae and S. pombe. No putative functional motifs were found in their predicted encoded proteins. Eighteen genes were successfully knocked out and 3 of them showed obvious phenotypes. A detailed analysis of one of them, midA, is presented in this report. Disruption of midA in Dictyostelium leads to pleiotropic defects. Cell size, growth rate, phagocytosis and macropinocytosis were affected in the mutant. During development, midA- cells showed an enhanced tendency to remain at the slug stage and spore viability was compromised. The expression of MidA fused to GFP in midA- strain rescued the phenotype and the fused protein was located in the mitochondria. Although cell oxygen consumption, mitochondrial content and mitochondrial membrane potential were similar to wild type, the amount of ATP was significantly reduced in the mutant suggesting a mitochondrial dysfunction. Metabolomic analysis by natural-abundance 13C-Nuclear Magnetic Resonance has shown the lack of glycogen accumulation during growth. During starvation, mutant cells accumulated higher levels of ammonia that inhibited normal development. We hypothesize that the lack of MidA reduces mitochondrial ATP synthetic capacity and this has an impact in some but not all energy-dependent cellular processes. This work exemplifies the potential of Dictyostelium as a model system for functional genomic studies. Submitted by: Ricardo Escalante [rescalante@iib.uam.es] ----------------------------------------------------------------------------- Contractile ring-independent localization of DdINCENP, a protein important for spindle stability and cytokinesis Qian Chen, Hui Li, and Arturo De Lozanne Institute of Cellular and Molecular Biology, and Molecular and Cellular and Developmental Biology Department, University of Texas at Austin, Austin, TX, 78712 Molecular Biology of the Cell, in press Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule depolymerizing drugs suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. While not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. Finally, we show that the localization of DdINCENP at the cleavage furrow is modulated by myosin II but it occurs by a mechanism different from that controlling the formation of the contractile ring. Submitted by: Arturo De Lozanne [a.delozanne@mail.utexas.edu] ----------------------------------------------------------------------------- Dictyostleium discoideum to human cells: Pharmacogenetic studies demonstrate a role for sphingolipids in chemoresistance Stephen Alexander, Junxia Min and Hannah Alexander, Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400 BBA-Reviews, General subjects, in press Resistance to chemotherapy is a major obstacle for the treatment of cancer and a subject of extensive research. Numerous mechanisms of drug resistance have been proposed, and they differ for different drugs. Nevertheless, it is clear that our understanding of this important problem is still incomplete, and that new targets for modulating therapy still await discovery. The attractive biology and the availability of powerful molecular techniques have made the cellular slime mold Dictyostelium discoideum a powerful non-mammalian model for drug target discovery, and the problem of drug resistance. To understand the molecular basis of chemoresistance to the widely used drug cisplatin, both genetic and pharmacological approaches have been applied to this versatile experimental system. These studies have resulted in the identification of novel molecular pathways which can be used to increase the efficacy of cisplatin, and brought attention to the role of sphingolipids in mediating the cellular response to chemotherapeutic drugs. In the following review we will describe the history and utility of D. discoideum in pharmacogenetics, and discuss recent studies which focus attention on the role of sphingolipids in chemotherapy and chemoresistance. Submitted by: Hannah Alexander [alexanderh@missouri.edu] ============================================================================== [End dictyNews, volume 25, number 13]