Dicty News Electronic Edition Volume 22, number 5 February 27, 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. ==================== Special Announcement ==================== I just want to say hello and goodbye to all colleagues now that I have retired from the fray. Thanks to all of you for friendship, help, encouragement, criticism, and competition, over the years. Hope to see you some time, though where, when and how I donāt know. I now have my home address and email on the website and would appreciate receiving news and reprints. Julian Gross ============= Abstracts ============= Dynamics of novel feet of Dictyostelium cells during migration Authors: Kazuhiko S. K. Uchida (1) and Shigehiko Yumura (Author for correspondence)(2) (1) Present address: Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan (2) Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan Journal of Cell Science, in press We observed the dynamics of actin foci in live Dictyostelium cells expressing GFP-actin. Actin foci were dynamic structures, but they were fixed on the substratum during cell migration. Interference reflection microscopy revealed that the ventral cell membrane was closer to the substratum at sites of actin foci. Furthermore, some actin foci were incorporated into the retraction fibers, ripped off from the cells, and eventually shed on the substratum after the cells moved away. The velocity of the cells was inversely proportional to the number of actin foci. Measurement of traction force using a silicone substratum demonstrated that the traction force was transmitted to the substratum through actin foci. Taken together, several lines of evidence strongly suggest that actin foci function as the active 'feet' of Dictyostelium cells. We also found evidence suggesting that these changing steps are regulated in a coordinated manner during cell migration. Possible mechanisms by which these cells migrate across substrata are discussed in this context. Submitted by: Kazuhiko Uchida [amx02193@mail2.accsnet.ne.jp] ----------------------------------------------------------------------------- Legionella effectors that promote non-lytic release from protozoa John Chen1, Karim Suwwan de Felipe 2, Margaret Clarke 3, Hao Lu, 3O. Roger Anderson4, Gil Segal5 and Howard A. Shuman1 1 Department of Microbiology and 2 Integrated Program in Cellular, Molecular & Biophysical Studies, College of Physicians & Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032 3 Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104-5046 4 Department of Biology, Lamont-Doherty Earth Observatory, Columbia University, 61 Route 9W, Palisades, NY 10964-1000 5 Department of Molecular Microbiology & Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel Science, in press Legionella pneumophila, the bacterial agent of legionnairesā disease, replicates intracellularly within a specialized vacuole of mammalian and protozoan host cells. Little is known about the specialized vacuole except that the Icm/Dot Type IV secretion system is essential for its formation and maintenance. The Legionella genome database contains two open reading frames encoding polypeptides (LepA and LepB) with predicted coiled-coil regions and weak homology to SNAREs; these are delivered to host cells by an Icm/Dot dependent mechanism. Analysis of mutant strains suggests that the Lep proteins may enable the Legionella to commandeer a protozoan exocytic pathway for dissemination of the pathogen. Submitted by: Margaret Clarke [clarkem@omrf.ouhsc.edu] ----------------------------------------------------------------------------- Cytosolic [Ca2+]-transients in Dictyostelium discoideum depend on the filling state of internal stores and on an active SERCA Ca2+-pump Christina Schlatterer, Kathrin Happle, Daniel F. Lusche and JŸrgen Sonnemann+ Faculty for Biology, University of Konstanz, 78457 Konstanz, FRG + Institute of Pharmacology, Pediatric Oncology and Hematology, University of Greifswald, 17487 Greifswald, FRG J. Biol. Chem., in press Stimulation of Dictyostelium discoideum with cAMP evokes a change of the cytosolic free Ca2+ concentration ([Ca2+]i). We analyzed the role of the filling state of Ca2+-stores for the [Ca2+]i-transient. Parameters tested were the height of the [Ca2+]i-elevation and the percentage of responding amoebae. After loading stores with Ca2+, cAMP induced a [Ca2+]i-transient in many cells. Without prior loading cAMP evoked a [Ca2+]i-change in few cells only. This indicates that the [Ca2+]i-elevation is not mediated exclusively by Ca2+-influx but also by Ca2+-release from stores. Reducing the Ca2+-content of the stores by EGTA-preincubation led to a cAMP-activated [Ca2+]i-increase even at low extracellular [Ca2+]. Moreover, addition of Ca2+ itself elicited a capacitative [Ca2+]i-elevation. This effect was not observed when stores were emptied by the standard technique of inhibiting internal Ca2+-pumps with 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ). Therefore in Dictyostelium, an active internal Ca2+-ATPase is absolutely required to allow for Ca2+-entry. No influence of the filling state of stores on Ca2+-influx characteristics was found by the Mn2+-quenching technique which monitors the rate of Ca2+-entry. Both, basal and cAMP-activated Mn2+-influx rates were similar in control cells and cells with empty stores. By contrast, determination of extracellular free Ca2+ concentration ([Ca2+]e)-changes which represent the sum of Ca2+-influx and efflux revealed a higher rate of [Ca2+]e-decrease in EGTA-treated than in control amoebae. We conclude that emptying of Ca2+-stores does not change the rate of Ca2+-entry but results in inhibition of the plasma membrane Ca2+-ATPase. Furthermore, the activities of the Ca2+-transport ATPases of the stores is of crucial importance for the regulation of [Ca2+]i-changes. Submitted by: Christina Schlatterer [Christina.Schlatterer@uni-konstanz.de] =============================================================================== [End Dicty News, volume 22, number 5]