dictyNews Electronic Edition Volume 28, number 10 April 20, 2007 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 dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= Mutants in the Dictyostelium Arp2/3 complex and chemoattractant-induced actin polymerization Paul D. Langridge and Robert R. Kay MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK Experimental Cell Research, in press We have investigated the role of the Arp2/3 complex in Dictyostelium cell chemotaxistoward cyclic-AMP and in the actin polymerization that is triggered by thischemoattractant. We confirm that the Arp2/3 complex is recruited to the cell perimeter, orinto a pseudopod, after cyclic-AMP stimulation and that this is coincident with actinpolymerization. This recruitment is inhibited when actin polymerization is blocked usinglatrunculin suggesting that the complex binds to pre-existing actin filaments, rather thanto a membrane associated signalling complex. We show genetically that an intact Arp2/3complex is essential in Dictyostelium and have produced partially active mutants in twoof its subunits. In these mutants both phases of actin polymerization in response to cyclic-AMP are greatly reduced. One mutant projects pseudopodia more slowly than wild typeand has impaired chemotaxis, together with slower movement. The second mutantchemotaxes poorly due to an adhesion defect, suggesting that the Arp2/3 complex plays acrucial part in adhering cells to the substratum as they move. We conclude that theArp2/3 complex largely mediates the actin polymerization response to chemotacticstimulation and contributes to cell motility, pseudopod extension and adhesion inDictyostelium chemotaxis. Submitted by: Rob Kay [rrk@mrc-lmb.cam.ac.uk] -------------------------------------------------------------------------------- An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum Samuel J. Fountain, Katie Parkinson, Mark T. Young, Lishuang Cao, Christopher R.L. Thompson* and R. Alan North* *Correseponding authors Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, England, UK Nature, in press P2X receptors are membrane ion channels gated by extracellular ATP found widely in vertebrates, but not previously in microbes. We identified a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and heterologous expression in HEK cells showed that it encoded a membrane ion channel activated by ATP (30 � 100 mM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to usual P2X antagonists3 but blocked by nanomolar concentrations of copper. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation. Submitted by: Chris Thompson [christopher.thompson@man.ac.uk] -------------------------------------------------------------------------------- Essential role of PI 3-kinase and phospholipase A2 in Dictyostelium chemotaxis Peter J.M. van Haastert, Ineke Keizer-Gunnink and Arjan Kortholt Department of Molecular Cell Biology, University of Groningen, Kerklaan 30, 9751NN Haren, the Netherlands J. Cell Biology, in press Chemotaxis towards different cAMP concentrations was tested in Dicyostelium cell lines with deletion of specific genes together with drugs to inhibit one or all combinations of the second messenger systems PI3-kinase, PLC, PLA2, and cytosolic Ca2+. The results show that inhibition of either PI3-kinase or PLA2 inhibits chemotaxis in shallow cAMP gradients, while both enzymes must be inhibited to prevent chemotaxis in steep cAMP gradients, suggesting that PI3-kinase and PLA2 are two redundant mediators of chemotaxis. Mutant cells lacking PLC activity have normal chemotaxis; however, additional inhibition of PLA2 completely blocks chemotaxis, whereas inhibition of PI3-kinase has no effect, suggesting that all chemotaxis in plc-null cells is mediated by PLA2. Cells with deletion of the IP3-receptor have the opposite phenotype: chemotaxis is completely dependent on PI3-kinase, and insensitive to PLA2-inhibitors. This suggest that PI3-kinase-mediated chemotaxis is regulated by PLC, probably through controlling PIP2 levels and PTEN activity, whereas chemotaxis mediated by PLA2 appears to be controlled by intracellular Ca2+. Submitted by: Peter van Haastert [p.j.m.van.haastert@rug.nl] -------------------------------------------------------------------------------- Biased random walk by stochastic fluctuations of chemoattractant-receptor interactions at the lower limit of detection Peter J.M. van Haastert and Marten Postma Department of Molecular Cell Biology, University of Groningen, Kerklaan 30, 9751NN Haren, the Netherlands Department of Physiology, Development and Neuroscience, Cambridge University, Downing St, Cambridge CB2 3DY UK Biophysical Journal, in press Binding of ligand to its receptor is a stochastic process that exhibits fluctuations in time and space. In chemotaxis this leads to a noisy input signal. Therefore, in a gradient of chemoattractant the cell may occasionally experience a �wrong� gradient of occupied receptors. We obtained a simple equation for Ppos, the probability that half of the cell closest to the source of chemoattractant has higher receptor occupancy than the opposite half of the cell. Ppos depends on four factors, the gradient property deltaC/sqrtC, the receptor characteristic Rt/Kd , a time-averaging constant I, and non-receptor noise. We measured chemotaxis of Dictyostelium cells to known shallow gradients of cAMP and obtained direct estimates for these constants. Furthermore, we observed that in shallow gradients the measured chemotaxis index is correlated with Ppos, which suggest that chemotaxis in shallow gradients is a pure biased random walk. From the observed chemotaxis and derived time-averaging constant we deduce that the gradient transducing second messenger has a life time of 2 to 8 seconds and a diffusion rate constant of ~1 microm^2/s. Potential candidates for such second messengers are discussed. Submitted by: Peter van Haastert [p.j.m.van.haastert@rug.nl] ============================================================ [End dictyNews, volume 28, number 10]