Dicty News Electronic Edition Volume 17, number 2 July 14, 2001 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. =================== Postoc Position =================== Postdoctoral position (BATIIa) in Molecular Genetics at Kassel University (3 years + 2 years). A postdoctoral position is available to work on RNA mediated gene silencing (antisense RNA, RNAi) in Dictyostelium or on signal transduction in early development (see http://www.uni-kassel.de/fb19/genetics/ for detailed information). The laboratory is well equipped for molecular biology. In addition, scanning force microscopy on RNA-protein interactions is established. Cooperations with others groups in the Biology Department are encouraged. The successful applicant should have solid experience in molecular biology and/or protein biochemistry. The development of an independent reseach program along the lines of the general laboratory interests and the establishment of a small research group are encouraged. Modest participation in teaching biology students is required. Applicants should attempt the habilitation or an equivalent qualification. Interested individuals may first contact W. Nellen (nellen@hrz.uni-kassel.de) by e-mail or dirctly send applications to Prsident Universitt Kassel D-34109 Kassel Germany Kennziffer 1114 ============== Abstracts ============== Under-Agarose Folate Chemotaxis of Dictyostelium Amoebae in Permissive and Mechanically Inhibited Conditions Gary Laevsky and David A. Knecht Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269 in Press: Biotechniques ABSTRACT Under agarose chemotaxis has been used previously to assess the ability of neutrophils to respond to gradients of chemoattractant. We have adapted this assay to the chemotactic movement of Dictyostelium amoebae in response to folic acid. Troughs are used instead of wells in order to increase the area along which the cells can be visualized and to create a uniform front of moving cells. Imaging the transition zone where the cells first encounter the agarose, we find that the cells move perpendicular to the gradient and periodically manage to squeeze under the agarose and move up the gradient. As cells exit the troughs, their cross sectional area increases as the cells become flattened. Three-dimensional reconstruction of confocal optical sections through GFP labeled-cells demonstrates that the increase in cross sectional area is due to the flattening of the cells. Since the cells locally deform the agarose and become deformed by it, the concentration of the agarose and therefore its stiffness should affect the ability of the cells to migrate. Consistent with this hypothesis, cells in 0.5% agarose move faster and are less flat than cells under 2% agarose. Cells do not exit the troughs and move under 3% agarose at all. Therefore this assay can be used to compare and quantify the ability of different cells types or mutant cell lines to move in a restrictive environment. ----------------------------------------------------------------------------- Automated Real-time Measurement of Chemotactic Cell Motility. Nacima Hadjout, Gary Laevsky, David A. Knecht, and Michael A. Lynes Department of Molecular and Cell Biology, University of Connecticut Storrs, CT 06269-3125 in Press: Biotechniques ABSTRACT We have developed a novel method (ECIS/taxis) for monitoring cell movement in response to chemotactic and chemokinetic factors. In this system, cells migrate in an under-agarose environment and their position is monitored using the Electric Cell Impedance Sensor (ECIS) technology to measure the impedance change at a target electrode lithographed onto the substrate as the cells arrive at that target. In the studies reported here, Dictyostelium discoideum was used as a prototypical motile eukaryotic cell. Using the ECIS/taxis system, the arrival of cells at the target electrode was proportional to the dose of folate used to stimulate the cells and could be assessed by changes in resistance at the electrode. ECIS/taxis was readily able to distinguish between wild-type cells and a mutant that is deficient in its chemotactic response. Finally, we have shown that an agent that interferes with chemotactic motility leads to delayed arrival of cells at the target electrode. The multiwell configuration of the assay allows for simultaneous automated screening of many samples for chemotactic or anti-chemotactic activity. This assay system is compatible with measurements of mammalian cell movement and should be valuable in the assessment of both agonists and antagonists of cell movement. ----------------------------------------------------------------------------- Reduced protein diffusion rate by cytoskeleton in vegetative and polarized Dictyostelium cells Eric O. Potma, Wim P. de Boeij, Leonard Bosgraaf, Jeroen Roelofs, Peter J. M. Van Haastert and Douwe A Wiersma Ultrafast Laser and Spectroscopy Laboratory and Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands Biophysical Journal, in press Fluorescence recovery after photobleaching measurements with high spatial resolution are performed to elucidate the impact of the actin cytoskeleton on translational mobility of green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6 fold reduction of their translational mobility relative to dilute aqueous solutions. In disrupting the actin filamentous network using latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindered protein motions is ascribed to other mechanical barriers and the viscosity of the cell liquid. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore established from measurements under different osmotic conditions. In highly locomotive polarized cells the obstructing effect of the actin filamentous network is seen to decline to 0.46 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the primary mechanical barrier for protein diffusion and that any noticeable reorganization of the network is accompanied with an altered intracellular protein mobility. ----------------------------------------------------------------------------- Recruitment of Cortexillin into the Cleavage Furrow is Controlled by Rac1 and IQGAP-related Proteins Jan Faix 1, Igor Weber 2, Ursula Mintert 2, Jana Khler 2, Friedrich Lottspeich 2, and Gerard Marriott 1 1) Department of Physiology, University of Wisconsin-Madison, 1300 University Ave, WI, 53706, USA. 2) Max-Planck-Institut fr Biochemie, D-82152 Martinsried, Germany. EMBO Journal, Vol.20, pp3705-3715, 2001 Abstract Cytokinesis in eukaryotic organisms is under the control of small GTP-binding proteins, although the underlying molecular mechanisms are not fully understood. Cortexillins are actin-binding proteins whose activity is crucial for cytokinesis in Dictyostelium. Here we show that the IQGAP-related and Rac1-binding protein DGAP1 specifically interacts with the C-terminal, actin-bundling domain of cortexillin I. Like cortexillin I, DGAP1 is enriched in the cortex of interphase cells and translocates to the cleavage furrow during cytokinesis. The activated form of the small GTPase Rac1A recruits DGAP1 into a quaternary complex with cortexillin I and II. In DGAP1- mutants, a complex can still be formed with a second IQGAP-related protein, GAPA. The simultaneous elimination of DGAP1 and GAPA, however, prevents complex formation and localization of the cortexillins to the cleavage furrow. This leads to a severe defect in cytokinesis, which is similar to that found in cortexillin I/II double-null mutants. Our observations define a novel and functionally significant signaling pathway that is required for cytokinesis. ----------------------------------------------------------------------------- Sensing and responding to chemoattractants: Signaling pathways controlling cell polarity and directional cell movement Chang Y. Chung, Satoru Funamoto, and Richard A. Firtel Trends in Biochemical Sciences (TiBS) Summary Many important biological processes, including chemotaxis, or directional cell movement up a gradient of a chemoattractant, require the ability to respond to a directional signal and a clearly established cell polarity. Recent advances using Dictyostelium cells and mammalian leukocytes have provided insights into the biochemical and molecular pathways that control chemotaxis. Phosphoinositide 3-kinase (PI3K) plays a central and possibly pivotal role in establishing and maintaining cell polarity by regulating the subcellular localization and activation of downstream effectors that are essential for regulating cell polarity and proper chemotaxis. This review outlines our present understanding of these pathways. ----------------------------------------------------------------------------- Dictyostelium centrin-related protein (DdCrp), the most divergent member of the centrin family, possesses only two EF-hands and dissociates from the centrosome during mitosis. Christine Daunderer, Manfred Schliwa and Ralph Grf1) Adolf-Butenandt-Insitut/Zellbiologie, Universitt Mnchen, Germany Eur. J. Cell Biol, in press We have identified a Dictyostelium discoideum cDNA sequence with homology to centrins. The derived protein, Dictyostelium discoideum centrin-related protein (DdCrp), is the most divergent member of the centrin family. Most strikingly it lacks the first two EF-hand consensus motifs, whereas a number of other centrin-specific sequence features are conserved. Southern and Northern blot analysis and the data presently available from the Dictyostelium genome and cDNA projects suggest that DdCrp is the only centrin isoform present in Dictyostelium. Immunofluorescence with anti-DdCrp antibodies revealed that the protein is localized to the centrosome, to a second, centrosome-associated structure close to the nucleus and to the nucleus itself. Confocal microscopy resolved that the centrosomal label is confined to the corona surrounding the centrosome core. Unlike for other centrins the localization of DdCrp is cell-cycle dependent. Both the centrosomal and the centrosome associated label disappear during prometaphase, most likely in concert with the dissociation of the corona at this stage. The striking differences of DdCrp to all other centrins may be related to the distinct structure and duplication mode of the Dictyostelium centrosome. ----------------------------------------------------------------------------- [End Dicty News, volume 17, number 2]