Dicty News Electronic Edition Volume 22, number 3 February 6, 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 ============= The necessity of mitochondrial genome DNA for normal development of Dictyostelium cells Junji Chida, Hitomi Yamaguchi, Aiko Amagai and Yasuo Maeda* Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan J. Cell Sci. (in press) Summary Most unexpectedly, there is now increasing evidence that mitochondria have novel and crucial functions in the regulatory machinery of the growth/differentiation transition, cell-type determination, cellular movement and pattern formation. Here we created rho delta cells with a reducedamount (about 1/4) of mitochondrial DNA (mtDNA) from Dictyostelium discoideum Ax-2 cells, by means of exposure of Ax-2 cells to ca. 30 mg/ml of ethidium bromide (EtBr) in axenic growth medium. Importantly, the rho delta cellsexhibited a series of fascinating behaviors: when they were starved, theyexhibited a marked delay of differentiation and stopped their development atthe slug stage, thus failing to construct fruiting bodies. Moreover, cellpatterning and cell-type proportioning were found to be greatly modified inslugs (referred to as rho delta slugs) derived from rho delta cells. That is,prestalk differentiation was significantly enhanced in rho delta slugs, whileprespore differentiation was markedly inhibited. In addition, the clear anterior prestalk/posterior prespore pattern was considerably disturbed in rho delta slugs, presumably because of incomplete sorting between the two typesof differentiated cells. After the assay of phototaxis, rho delta slugs alsoexhibited highly disordered movement toward the light source. Taken togetherthese results suggest that mtDNA might have important multiple functions ina variety of cellular processes during Dictyostelium development. Submitted by: Yasuo Maeda [ymaeda@mail.cc.tohoku.ac.jp] ----------------------------------------------------------------------------- Structure of an activated Dictyostelium STAT in its DNA-unbound form Montserrat Soler-Lopez1,3, Carlo Petosa1,3, Masashi Fukuzawa2, Raimond Ravelli1, Jeffrey G. Williams2, Christoph W. Mueller1 Molecular Cell, in press 1 European Molecular Biology Laboratory, Grenoble Outstation, B.P. 181, 38042 Grenoble Cedex 9, France 2 School of Biological Sciences, University of Dundee, MSI/WTB Complex Dow Street, Dundee, DD1 5EH, U.K. 3 These authors contributed equally to this work. Summary Dd-STATa is a STAT protein which transcriptionally regulates cellular differentiation in Dictyostelium discoideum, the only non-metazoan known to employ SH2 domain signalling. The 2.7 Angstrom crystal structure of a tyrosine phosphorylated Dd-STATa homodimer reveals a four-domain architecture similar to that of mammalian STATs 1 and 3, but with an inverted orientation for the coiled coil domain. Dimerization is mediated by SH2 domain:phosphopeptide interactions and by a novel, direct interaction between SH2 domains. The unliganded Dd-STATa dimer adopts a fully extended conformation remarkably different from that of the DNA-bound mammalian STATs, implying a large conformational change upon target site recognition. Buried hydrophilic residues predicted to destabilize the coiled coil domain suggest how hydrophobic residues may become exposed and mediate nuclear export. Functional and evolutionary implications for metazoan STAT proteins are discussed. Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk] ----------------------------------------------------------------------------- THE STAT PROTEINS OF DICTYOSTELIUM JEFFREY G. WILLIAMS - N. B. This is a review article, written some time ago, so some aspects are out of date, e.g. a paper on Dd-STATb is now published and the complete genome sequence reveals no additional STATs. In 'Signal Transducers and Activators of Transcription (STATs)' edited by Pravin B. Sehgal, M.D., Ph.D., David E. Levy, Ph.D., Toshio Hirano, M.D.Kluwer, Acad. Publishers School of Life Sciences University of Dundee MSI/WTB Complex Dow Street Dundee DD1 5EH, U.K. Overview Dictyostelium possesses at least four STATs. They all contain the three defining STAT domains: a DNA binding domain, an SH2 domain and a site of tyrosine phosphorylation. However, their N terminal halves are highly diverged from those of metazoan STATs and they also appear to lack C terminal transcriptional trans-activation domains; in all four proteins the site of tyrosine phosphorylation is just a few amino acids from the C terminus. Only two of the proteins, Dd-STATa and Dd-STATc, have been characterised and they function, in different contexts, as either activators or repressor of gene expression. Dd-STATa and Dd-STATc both regulate the transcription of prestalk and stalk cell-specific genes and Dd-STATc also activates specific gene trancription in response to a variety of environmental stresses. Dd-STATa and Dd-STATc have additional, less well-characterised roles; disruption of the Dd-STATa gene impairs cellular chemotaxis to cAMP and retards early development, while inactivation of the Dd-STATc gene reduces the cellular growth rate and accelerates early development. Upon binding of extracellular cAMP to its serpentine receptor Dd-STATa is very rapidly tyrosine phosphorylated and translocates to the nucleus Activation and nuclear accumulation do not require G protein function. Over a slower time scale, cAMP signalling also activates GSK-3 to phosphorylate Dd-STATa and this facilitates its export from the nucleus. During development Dd-STATc is activated by DIF-1, a chlorinated hexaphenone. DIF-1 induces the nuclear accumulation of Dd-STATc by reducing its rate of export from the nucleus. The signalling steps that activate Dd-STATa and Dd-STATc are unknown and, although the Dictyostelium genome project is now 95% complete, there is as yet no obvious candidate for a JAK or a receptor tyrosine kinase. Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk] ----------------------------------------------------------------------------- Sensitisation of Dictyostelium chemotaxis by PI3-kinase mediated self-organising signalling patches Marten Postma, Jeroen Roelofs, Joachim Goedhart, Harri‘t M. Loovers, Antonie J.W.G. Visser, and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands J Cell Science, in press The leading edge of Dictyostelium cells in chemoattractant gradients can be visualised using GFP tagged to the PH domain of CRAC, which presumable binds PI(3,4,5)P3. Uniform cAMP induces persistent translocation PHCrac-GFP from the cytosol to multiple patches, which are similar to the single patch of PHCrac-GFP at the leading edge in a cAMP gradient. We show that cAMP determines the probability of patch formation (half-maximal effect at 0.5 nM cAMP), but not size, lifetime or intensity of patches, indicating that patches are self-organising structures. A pseudopod is extended from the area of the cell with a PHCrac-GFP patch at about 10 s after patch formation. Cells treated with the F-actin inhibitor latrunculin A are round without pseudopodia; uniform cAMP still induces localised patches of PHCrac-GFP. Inhibition of PI3-kinase activity with LY294002 inhibits PHCrac-GFP patches and inhibits chemotaxis towards nanomolar cAMP, but has no effect at higher cAMP concentrations. Thus, very low cAMP concentrations induce self-organising PHCrac-GFP patches that serve as a spatial cue for pseudopod formation, which enhances the sensitivity and amplitude of chemotactic movement. Submitted by: Peter van Haastert [P.J.M.van.Haastert@chem.rug.nl] =============================================================================== [End Dicty News, volume 22, number 3]