Dicty News Electronic Edition Volume 23, number 20 December 31, 2004 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 Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Loss-of-Function Mutations Identified in the Helical Domain of the G Protein alpha- Subunit, Galpha2, of Dictyostelium discoideum. Robert E. Gundersen, Jianxin You, Steven Rauch, Kate Farnham, Christopher McCarty, Nicholas Willis and Alison Prince Biochemistry, Microbiology and Molecular Biology, University of Maine accepted: BBA - General Subjects The guanine nucleotide binding regulatory proteins (G proteins) play essential roles in a wide variety of physiological processes such as, vision, hormone responses, olfaction, immune response, and development. The heterotrimeric G proteins, consists of an alpha, beta and gamma- subunits and act as molecular switches to relay information from transmembrane receptors to intracellular effectors. The switch mechanism is a function of the inherent GTPase activity of the alpha-subunit. The alpha-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known alpha- subunit functions while little is know about the role of the Helical domain. To gain a better understanding of alpha-subunit function, we performed a screen for loss-of-function mutations, using the Galpha2- subunit of Dictyostelium. Galpha2 is essential for the developmental life cycle of Dictyostelium. It is known that loss of Galpha2 function results in a failure of cells to enter the developmental phase, producing a visibly abnormal phenotype. This allows easy identification of amino acids essential to Galpha2 function. A library of random point mutations in the galpha2 cDNA was constructed using low fidelity Polymerase Chain Reaction (PCR). The library was then expressed in a galpha2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the galpha2 insert. To date sixteen, single amino acids changes have been identified in Galpha2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the alpha-subunit. These loss-of-function mutations in the alpha-subunit Helical domain may provide important insight into its function. Submitted by: Robert Gundersen [gundersn@maine.edu] ----------------------------------------------------------------------------- An auto-regulatory circuit for long-range self-organization in Dictyostelium cell populations Satoshi Sawai, Peter A. Thomason & Edward C. Cox Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA Nature, in press Nutrient-deprived Dictyostelium amoebae aggregate to form a multicellular structure by chemotaxis, moving towards propagating waves of cyclic AMP that are relayed from cell to cell. Organizing centres are not formed by founder cells, but are dynamic entities consisting of cores of outwardly rotating spiral waves that self-organize in a homogeneous cell population. Spiral waves are ubiquitously observed in chemical reactions as well as in biological systems. Although feedback control of spiral waves in spatially extended chemical reactions has been demonstrated in recent years, the mechanism by which control is achieved in living systems is unknown. Here we show that mutants of the cyclic AMP/protein kinase A pathway show periodic signalling, but fail to organize coherent long-range wave territories, owing to the appearance of numerous spiral cores. A theoretical model suggests that autoregulation of cell excitability mediated by protein kinase A acts to optimize the number of signalling centres. Submitted by: Satoshi Sawai [ssawai@molbio.princeton.edu] ----------------------------------------------------------------------------- Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II- independent processes in Dictyostelium Shi Shu, Xiong Liu and Edward D. Korn* Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892 Proc. Natl. Acad. Sci., in press Blebbistatin, a cell-permeable inhibitor of class-II myosins, was developed to provide a tool for studying the biologic roles of myosin II. Consistent with this, we find that blebbistatin inhibits three myosin II- dependent processes in Dictyostelium: growth in suspension culture, capping of concanavalin A receptors and development to fruiting bodies and does not inhibit growth on plates, which does not require myosin II. As expected, macropinocytosis (myosin I-dependent), contractile vacuole activity (myosin V-dependent) and phagocytosis (myosin VII- dependent), none of which requires myosin II, are not inhibited by blebbistatin in myosin II-null cells but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing myosin II. Expression of catalytically inactive myosin II in myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin- inhibited myosin II and catalytically inactive myosin II form cytoplasmic aggregates, which may be why they inhibit myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in myosin II-null cells. Our results are consistent with myosin II being the only Dictyostelium myosin that is inhibited by blebbistatin but also show that blebbistatin-inactivated myosin II inhibits some myosin II- independent processes and that blebbistatin inhibits other activities in the absence of myosin II. Submitted by: Edward D. Korn [edk@nih.gov] ============================================================================== [End Dicty News, volume 23, number 20]