Dicty News Electronic Edition Volume 15, number 2 July 15, 2000 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty" ============== Abstracts ============== An F-Box/WD40 repeat-containing protein important for Dictyostelium cell-type proportioning, slug behaviour and culmination Margaret K. Nelson*, Alexandra Clark+, Tomoaki Abe^, Anson Nomura+, Negendra Yadava+, Chanin J. Funair*, Keith A. Jermyn^, Sudhasri Mohanty+, Richard A. Firtel+, and Jeffrey G. Williams^ * Department of Biology, Allegheny College, Meadville, PA 16335, USA +Department of Biology, Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla CA 92093-0634, USA ^Department of Anatomy and Physiology, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK Develop. Biol., in press. ABSTRACT FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA- cells), the spore mass fails to fully ascend the stalk. In addition, fbxA- slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA- cells is hypersensitive to ammonia, the signalling molecule that regulates the transition from the slug stage to terminal differentiation. The slug is comprised of an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further sub-divided into an anterior pstA region and a posterior pstO region. In fbxA- slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation. ---------------------------------------------------------------------------- Characterisation of a DNA sequence element that directs Dictyostelium stalk cell­specific gene expression Adriano Ceccarelli1*, Natasha Zhukovskaya2*, Takefumi Kawata2, Salvatore Bozzaro1and Jeffrey Williams2+ 1. Dipartimento di Scienze Cliniche e Biologiche, Ospedale San Luigi Gonzaga Reg. Gonzole 10 10043, Orbassano, Torino, Italy 2. Department of Anatomy and Physiology, University of Dundee, MSI/WTB Complex,, Dow Street, Dundee DD1 5EH, UK *Both these authors contributed equally to this work DIFFERENTIATION IN PRESS ABSTRACT The ecmB gene of Dictyostelium is expressed at culmination, both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap site proximal part of the promoter, the Stalk Tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We characterise an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer), within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identify a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest that, in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence. ---------------------------------------------------------------------------- A myosin I is involved in membrane recycling from early endosomes Eva M. Neuhaus and Thierry Soldati# Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany #Corresponding author: Tel: +49-6221-486407, Fax: +49-6221-486325, E-mail: soldati@mpimf-heidelberg.mpg.de Journal of Cell Biology, in press Abstract Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin-myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA-/B-, and myoB- cells) and wild-type cells treated with the myosin inhibitor BDM accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryo-EM method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA-/B- cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment. ---------------------------------------------------------------------------- Rapid patterning in 2D-cultures of Dictyostelium cells and its relation to zonal differentiation Takashi Hirano1, Satoshi Sawai2, Yasuji Sawada3 and Yasuo Maeda1* 1 Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578, 2Graduate School of Information Sciences, and 3Research Institute of Electrical Communication, Tohoku University, Katahira, Sendai 980-8577, Japan. *Author to whom all correspondence should be addressed. E-mail: ymaeda@mail.cc.tohoku.ac.jp Develop. Growth Differ., in press Summary Rapid patterning has been realized in confined two-dimensional (2-D) cultures of Dictyostelium discoideum Ax-2 cells, as the outer dark zone and inner light zone, the width of outer zone being usually about 100 µm, irrespective of the size of cell masses under the atomospheric condition. The width of outer zone, however, changed depending on external oxygen (O2) concentrations and reached to 250 µm at 100 % O2 . A clear regional difference in TMRM ( tetramethyl rhodamine methyl ester ) staining was noticed between the outer zone and the inner zone: the inner zone being more strongly stained with TMRM rather than the outer zone facing the air. Using the inhibitors ( DNP or NaN3 ) of oxidative phosphorylation and the specific inhibitor ( BHAM ) of CN-resistant respiration,it has benn demonstrated that the outer zone is basically formed by the O2 -threshold for oxidative phosphorylation, while that the inner cells mainly perform CN-resistant respiration. When cells around the early mound stage ( just before prestalk and prespore differentiation ) were cultured as 2D-cell masses, ecmA-expressing cells ( pstA cells ), ecmB-expressing cells ( pstB cells ), and D19-expressing cells ( prespore; psp cells ), there arized in a position-dependent manner in the outer zone. In the inner zone, cell motility seemed to be markedly impaired, and also neither prestalk nor prespore differentiation occurred. In addition, once-differentiated prespore cells were found to dedifferentiate rapidly in the inner zone. The reason for failure of cells to dedifferentiate as well as to differentiate in the inner zone is discussed with reference to O2-radicals. ---------------------------------------------------------------------------- A cell type-specific effect of calcium on pattern formation and differentiation in Dictyostelium discoideum. R. Baskar, Preeti Chhabra, Priya Mascarenhas and Vidyanand Nanjundiah Indian Institute of Science, Bangalore 560012, India. Int. J. Develop. Biol., in press. Spatial gradients of sequestered and free calcium exist in the slug of Dictyostelium discoideum (Maeda and Maeda, 1973; Tirlapur et al., 1991; Azhar et al., 1995; Cubitt et al., 1995). When we vary intracellular Ca2+ with the help of calcium buffers and the ionophore Br-A23187, there are striking effects on slug morphology, patterning and cell differentiation. In the presence of a calcium ionophore, high external Ca2+ levels lead to an increase of intracellular sequestered and free calcium, the formation of long slugs, a decrease in the fraction of genetically defined prespore cells and stalky fruiting bodies. Conversely, a lowering of external Ca2+ levels results in a decrease of intracellular sequestered and free calcium, the formation of short slugs, an increase in the prespore fraction and spory fruiting bodies. We infer that Ca2+ plays a significant morphogenetic role in D. discoideum development by selectively promoting the prestalk pathway relative to the prespore pathway. ---------------------------------------------------------------------------- [End Dicty News, volume 15, number 2]