Dicty News Electronic Edition Volume 13, number 3 July 31, 1999 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/dicty.html" ============= Abstracts ============= DICTYOSTELIUM AS MODEL SYSTEM FOR STUDIES OF THE ACTIN CYTOSKELETON BY MOLECULAR GENETICS Ludwig Eichinger, Soo Sim Lee, and Michael Schleicher Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, Schillerstr. 42, 80336 München, Germany Microscopy Research and Technique, in press Abstract The actin cytoskeleton is an essential structure for most movements at the cellular and intracellular level. Whereas a muscle cell requires for contraction a rather static organisation of cytoskeletal proteins, cell motility of amoeboid cells relies on a tremendously dynamic turnover of filamentous networks in a matter of seconds and at distinct regions inside the cell. The best model system for studying cell motility is Dictyostelium discoideum. The cells live as single amoebae but can also start a developmental program that leads to multicellular stages and differentiation into simple types of tissues. Thus cell motility can be studied on single cells and on cells in a tissue-like aggregate. The ability to combine protein purification and biochemistry with fairly easy molecular genetics is a unique feature for investigation of the cytoskeleton and cell motility. The actin cytoskeleton in Dictyostelium harbours essentially all classes of actin-binding proteins that have been found throughout eukaryotes. By conventional mutagenesis, gene disruption, antisense approaches, or gene replacements many genes that code for cytoskeletal proteins have been disrupted, and altered phenotypes in transformants that lacked one or more of those cytoskeletal proteins allowed solid conclusions about their in vivo function. In addition, tagging the proteins or selected domains with green fluorescent protein allows the monitoring of protein redistribution during cell movement. Gene tagging by restriction enzyme mediated integration of vectors and the ongoing international genome and cDNA sequencing projects offer the chance to understand the dynamics of the cytoskeleton by identification and functional characterisation of all proteins involved. ---------------------------------------------------------------------------- Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120) Paola Fucini (1), Bernd Koeppel (1), Michael Schleicher (2), Ariel Lustig (3), Tad A. Holak (1), Rolf Mueller (5), Murray Stewart (4) and Angelika A. Noegel (5) (1) Max-Planck-Institut f. Biochemie, Martinsried, FRG,(2) Institut f. Zellbiologie, Muenchen, FRG, (3) Biozentrum, Basel, Switzerland, (4) MRC Laboratory of Molecular Biology, Cambridge, England, (5) Institut f. Biochemie I, Koeln, FRG accepted: J. Mol. Biol. The Dictyostelium discoideum gelation factor is a two-chain actin-crosslinking protein that, in addition to an N-terminal actin-binding domain, has a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin fold. To define the architecture of the rod domain of gelation factor, we have expressed in E. coli a series of constructs corresponding to different numbers of gelation factor rod repeats and have characterised them by chemical crosslinking, ultracentrifugation, column chromatography, matrix-assisted laser desorption ionisation (MALDI) mass spectrometry and NMR spectroscopy. Fragments corresponding to repeats 1-6 and 5-6 dimerise, whereas repeats 1-5 and single repeats 3 and 4 are monomeric. Repeat 6 interacts weakly and was present as monomer and dimer when analysed by analytical ultracentrifugation. Proteolytic digestion of rod5-6 resulted in generation of two polypeptides that roughly corresponded to rod5 and part of rod6. None of these polypeptides formed dimers after chemical crosslinking. Stable dimerisation therefore appears to require repeats 5 and 6. Based on these data a model of gelation factor architecture is presented. We suggest an arrangement of the chains where only the carboxy-terminal repeats interact as was observed for filamin/ABP280, the mammalian homologue of gelation factor. ---------------------------------------------------------------------------- A model for Dictyostelium slug movement Till Bretschneider, Bakhtier Vasiev, Cornelis J. Weijer Journal of Theoretical Biology (in press) SUMMARY Dictyostelium slug movement results from the co-ordinated movement of its 10e5 constituent cells. We have shown experimentally rotational cell movement in the tip and periodic forward movement of the cells in the back of the slug. To explain this mode of movement we have put forward the hypothesis that cell movement in slugs is controlled by chemotaxis to periodic cAMP waves coming from the tip. In the tip they propagate as scroll waves and convert to twisted scroll or planar waves in the back of the slug. The co- ordinated movement of individual cells in response to these waves could then result in forward movement of the slug. To test this hypothesis we extend our model for aggregation and mound formation to include two cell types with different signalling and movement properties. In the model each cell is described as a cAMP relaying unit which interacts with neighbouring cells and moves chemotactically in response to cAMP gradients. Cells interact by adhesion, pressure and friction with neighbouring cells and the extracellular matrix. The model is capable of explaining scroll wave propagation and slug movement and allows us to understand the movement of an organism based on the properties of its constituent cells. We use the model to investigate the influence of cell type specific differences in excitability, adhesion and cell interactions on slug motion. ---------------------------------------------------------------------------- Transient expression of a mitochondorial gene cluster including rps4 is essential for the phase-shift of Dictyostelium cells from growth to differentiation Y. Inazu, S-C. Chae and Y. Maeda Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai, Japan Develop. Genetics, in press. ABSTRACT Using synchronized Dictyostelium discoideum Ax-2 cells and the differential display method, a mitochondrial gene cluster (referred to as differentiation-associated gene 3; dia3) was isolated as one of genes expressed specifically during the transition of Ax-2 cells from growth to differentiation. The dia3 gene encodes for a mitochondrial protein cluster (NADH dehydrogenase (NAD) subunit 11, 5, ribosomal protein S4 (RPS4), RPS2 and NAD4L). Northern blot analysis using non-synchronized Ax-2 cells has shown that the dia3 RNA of about 8kb is scarcely expressed during the vegetative growth phase, and the maximal expression was attained at 2 hours after starvation. To analyze the gene function of dia3, we tried inactivation of rps4 by means of homologous recombination and obtained several transformed clones showing mitochondrial DNA heteroplasmy. The transformed cells grew normally in nutrient medium, but their development after starvation was greatly impaired, thus resulting in failure of many cells to differentiate. In this connection, the cAMP receptor 1 (car1) expression, which is one of the earliest marker of differentiation, was found to be markedly reduced in the rps4 -inactivated cells. ---------------------------------------------------------------------------- Expression of prestalk and prespore proteins in minute, two-dimensional Dictyostelium slugs J. T. Bonner*, P. Fey, E. C. Cox Departments of Ecology and Developmental Biology, and Molecular Biology Princeton University, Princeton, NJ 08544 Mechanisms of Develop., in press Abstract We show that exceedingly small two-dimensional slugs of Dictyostelium differentiate normally and have an anterior prestalk zone and a posterior prespore zone. Using GFP as a marker attached to the appropriate promoter, prestalk expression is concentrated in the anterior, while prespore expression is produced in the posterior, closely resembling what is found in normal, large slugs. ---------------------------------------------------------------------------- The RdeA-RegA System: a Eukaryotic Phospho-Relay Controlling cAMP Breakdown Peter A. Thomason, David Traynor, Jeffry B. Stock1, and Robert R. Kay MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH U.K. 1Department of Molecular Biology, Princeton University, Princeton New Jersey 08544 J. Biol. Chem., in press. SUMMARY The regA and rdeA gene products of Dictyostelium are involved in the regulation of cAMP signaling. The response regulator, RegA, is composed of an N-terminal receiver domain linked to a C-terminal cAMP-phosphodiesterase domain. RdeA may be a phospho-transfer protein that supplies phosphates to RegA. We show genetically that phospho-RegA is the activated form of the enzyme in vivo, in that the predicted site of aspartate phosphorylation is required for full activity. We show biochemically that RdeA and RegA communicate, as evidenced by phospho-transfer between the two proteins in vitro. Phospho-transfer is dependent on the presumed phospho-accepting amino acids, histidine 65 of RdeA and aspartate 212 of RegA, and occurs in both directions. Phosphorylation of RegA by a heterologous phospho-donor protein activates RegA phosphodiesterase activity at least 20-fold. Our results suggest that the histidine phosphotransfer protein, RdeA, and the response regulator, RegA, constitute two essential elements in a eukaryotic His-Asp phospho-relay network that regulates Dictyostelium development and fruiting body maturation. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 3]