Dicty News Electronic Edition Volume 16, number 2 January 27, 2001 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.northwestern.edu/dicty" ============== Abstracts ============== Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning Anne Early(1), Marianne Gamper(2), John Moniakis(2), Eugene Kim(2), Tony Hunter(3), Jeffrey G. Williams(4), and Richard A. Firtel(2) 1 MRC Laboratory for Molecular Cell Biology and Department of Biology, University College London, Gower Street, London WC1E 6BT, UK. 2 Section of Cell and Developmental Biology and the Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA. 3 Salk Institute, 10010 North Torrey Pines Road, La Jolla CA 92037-1099, USA. 4 University of Dundee, MSI/WTB Complex, Dow Street, Dundee, DD1 5EH, UK. Developmental Biology, in press Abstract The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP display aberrant morphology. However, the signalling pathways involved have not been characterized. In re-examining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we shown that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region. ----------------------------------------------------------------------------- Transcript mapping and processing of mitochondrial RNA in Dictyostelium discoideum C. BARTH1, U. GREFERATH2, M. KOTSIFAS1, Y. TANAKA3, S. ALEXANDER4, H. ALEXANDER4 AND P. R. FISHER1 1 Department of Microbiology, La Trobe University, Bundoora 3083, Melbourne, Australia 2 Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Melbourne, Australia 3 Institute of Biological Sciences, University of Tsukuba, Ibaraki 305-8572, Japan 4 Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400 Current Genetics, in press. ABSTRACT The circular mitochondrial genome of Dictyostelium discoideum has a size of 55,564 base pairs. We present here a complete and detailed transcription map of the mitochondrial DNA. Eight major, polycistronic transcripts encoding polypeptides, ribosomal RNAs and interspersed transfer RNAs were identified in Northern hybridization studies. Most of these polycistronic transcripts are subsequently processed into smaller mono-, di- or tricistronic RNAs. In some cases the maturation involves endonucleolytic cleavage of the transcripts using transfer RNAs as excision signals. Primer extension experiments mapped the 5' ends of the transcripts, which may represent transcription initiation sites. Two of the polycistronic transcripts were found to be overlapping. Based on sequence alignments of the potential transcription start sites a short oligonucleotide consensus initiation sequence has been identified which did not reveal any significant sequence homologies to known promoter regions from other organisms. ----------------------------------------------------------------------------- An SH2-domain-containing kinase negatively regulates the phosphatidylinositol-3 kinase pathway John Moniakis, Satoru Funamoto, Masashi Fukuzawa, Jill Meisenhelder, Tsuyoshi Araki, Tomoaki Abe, Ruedi Meili, Tony Hunter, Jeffrey Williams, and Richard A. Firtel Genes and Development, in press. ABSTRACT SHK1 is a novel dual-specificity kinase that contains an SH2 domain in its C-terminal region. We demonstrate that SHK1 is required for proper chemotaxis and phagocytosis. shk1 null cells lack polarity, move very slowly, and exhibit an elevated and temporally extended chemoattractant-mediated activation of the kinase Akt/PKB. GFP fusions of the PH domain of Akt/PKB or the PH-domain-containing protein CRAC, which become transiently associated with the plasma membrane after a global stimulation with a chemoattractant, remain associated with the plasma membrane for an extended period of time in shk1 null cells. These results suggest that SHK1 is a negative regulator of the PI3 kinase pathway. Furthermore, when a chemoattractant gradient is applied to a wild-type cell, these PH-domain-containing proteins and the F-actin binding protein coronin localize to its leading edge, but in an shk1 null cell they become randomly associated with the plasma membrane and cortex, irrespective of the direction of the chemoattractant gradient, suggesting SHK1 is required for the proper spatio-temporal control of F-actin levels in chemotaxing cells. Consistent with such functions, SHK1 is localized at the plasma membrane/cortex and we show that its SH2 domain is required for this localization and the proper function of SHK1. ----------------------------------------------------------------------------- p110-related PI 3-kinases regulate phagosome-phagosome fusion and phagosomal pH through a PKB/Akt dependent pathway in Dictyostelium. Adam C. Rupper1,2, Juan M. Rodriguez-Paris2, Bryon D. Grove3 and James A. Cardelli1,2,4 1Department of Microbiology and Immunology, 2Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130; 3Department of Anatomy and Cell Biology, University of North Dakota, Grand Forks, North Dakota 58202 Journal of Cell Science (in press) ABSTRACT The Dictyostelium p110-related PI 3-kinases, PIK1 and PIK2, regulate the endosomal pathway and the actin cytoskeleton, but do not significantly regulate internalization of particles in D. discoideum. Bacteria internalized into Dddpik1/ddpik2 cells or cells treated with PI 3-kinase inhibitors remained intact as single particles in phagosomes with closely associated membranes after 2 hours of internalization, while in control cells, bacteria appeared degraded in multi-particle spacious phagosomes. Addition of LY294002 to control cells, after 60 minutes of chase, blocked formation of spacious phagosomes, suggesting PI 3-kinases acted late to regulate spacious phagosome formation. Phagosomes purified from control and drug treated cells contained equivalent levels of lysosomal proteins, including the proton pump complex, and were acidic, but in drug treated cells and Dddpik1/ddpik2 cells phagosomal pH was significantly more acidic during maturation than the pH of control phagosomes. Inhibition of phagosomal maturation by LY294002 was overcome by increasing phagosomal pH with NH4Cl, suggesting that an increase in pH might trigger homotypic phagosome fusion. A pkbA null cell line (PKB/Akt) reproduced the phenotype described for cells treated with PI 3-kinase inhibitors and Dddpik1/ddpik2 cells. We propose that PI 3-kinases, through a PKB/Akt dependent pathway, directly regulate homotypic fusion of single particle containing phagosomes to form multi-particle, spacious phagosomes, possibly through the regulation of phagosomal pH. ----------------------------------------------------------------------------- Environmental Regulation of Pathways Controlling Sporulation, Dormancy, and Germination Utilizes Bacterial-like Signaling Complexes in Dictyostelium. David A. Cottera, Dana C. Mahadeoa, David N. Cervia, Yoshiro Kishib, Keith Galec, Todd Sandsa, and Masazumi Sameshimab. aDepartment of Biological Sciences, University of Windsor, Windsor, Ontario, Canada bDepartment of Cell Biology, The Tokyo Metropolitan Institute for Medical Science, Tokyo Metropolitan Organization for Medical Research, Honkomagome, Bunkyo-ku, Tokyo, Japan cDepartment of Natural and Quantitative Sciences, Dubai, United Arab Emirates Protist Vol 151, 111-126 Summary: In the following review we have examined and compiled information regarding the later stages of development in Dictyostelium, primarily focussing on spore formation, dormancy and germination. We have given an overview on the current understanding on how these processes function based on genetic, biochemical, and physiological evidence contributed to the field. We have also compared the interaction of the control of these events compared with other similar systems such as Bacillus. Histidine kinases play a central role in the development of Dictyostelium. We have proposed a network model of interacting signals that act through these histidine kinases to achieve and maintain spore dormancy. The complex process of spore germination relies on many different factors including intrinsic and external signals. We have established a model for spore germination that incorporates our current understanding as well as proposed signaling processes that may be involved. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 2]