dictyNews Electronic Edition Volume 33, number 10 October 16, 2009 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. Follow dictyBase now on twitter: http://twitter.com/dictybase ========= Abstracts ========= Roles of an Unconventional Protein Kinase and Myosin II in Amoeba Osmotic Shock Responses Venkaiah Betapudi and Thomas T. Egelhoff* Traffic, in press The contractile vacuole (CV) is a dynamic organelle that enables Dictyostelium amoeba and other protist to maintain osmotic homeostasis by expelling excess water. In the present study, we have uncovered a mechanism that coordinates the mechanics of the CV with myosin II, regulated by VwkA, an unconventional protein kinase that is conserved in an array of protozoa. GFP-VwkA fusion proteins localize persistently to the CV during both filling and expulsion phases of water. In vwkA null cells, the established CV marker dajumin still localizes to the CV, but these structures are large, spherical, and severely impaired for discharge. Furthermore, myosin II cortical localization and assembly are abnormal in vwkA null cells. Parallel analysis of wild type cells treated with myosin II inhibitors or of myosin II null cells also results in enlarged CVs with impaired dynamics. We suggest that the myosin II cortical cytoskeleton, regulated by VwkA, serves a critical conserved role in the periodic contractions of the CV, as part of the osmotic protective mechanism of protozoa. Submitted by Venkaiah Betapudi & Thomas Eegelhoff [betapuv@ccf.org] -------------------------------------------------------------------------------- Functional characterisation of intracellular Dictyostelium discoideum P2X receptors Melanie J. Ludlow, Latha Durai and Steven J. Ennion University of Leicester, United Kingdom JBC (http://www.jbc.org/content/early/2009/10/15/jbc.M109.045674) Indicative of cell surface P2X ion channel activation, extracellular ATP evokes a rapid and transient calcium influx in the model eukaryote Dictyostelium discoideum. Five P2X-like proteins (dP2XA-E) are present in this organism. However, their roles in purinergic signalling are unclear since dP2XA proved to have an intracellular localisation on the contractile vacuole where it is thought to be required for osmoregulation. To determine functional properties of the remaining four dP2X-like proteins and to assess their cellular roles, we recorded membrane currents from expressed cloned receptors and generated a quintuple knockout Dictyostelium strain devoid of dP2X receptors. ATP evoked inward currents at dP2XB and dP2XE receptors but not at dP2XC or dP2XD. β,γ-imido-ATP was more potent than ATP at dP2XB but a weak partial agonist at dP2XE. Currents in dP2XB and dP2XE were strongly inhibited by Na+ but insensitive to copper and the P2 receptor antagonists PPADS and suramin. Unusual for P2X channels, dP2XA and dP2XB were also Cl- permeable. The extracellular purinergic response to ATP persisted in p2xA/B/C/D/E quintuple knockout Dictyostelium demonstrating that dP2X channels are not responsible. dP2XB,C,D, and E were found to be intracellular localised to the contractile vacuole with the ligand binding domain facing the lumen. However, quintuple p2xA/B/C/D/E null cells were still capable of regulating cell volume in water demonstrating that, contrary to previous findings, dP2X receptors are not required for osmoregulation. Responses to the calmodulin antagonist calmidazolium however were reduced in p2xA/B/C/D/E null cells suggesting that dP2X receptors play a role in intracellular calcium signalling. Submitted by Steven Ennion [se15@leicester.ac.uk ============================================================== [End dictyNews, volume 33, number 10]