dictyNews Electronic Edition Volume 38, number 18 July 20, 2012 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 on twitter: http://twitter.com/dictybase ========= Abstracts ========= Myosin Heavy Chain Kinases Play Essential Roles In Ca2+, But Not cAMP, Chemotaxis And The Natural Aggregation of Dictyostelium discoideum Deborah Wessels, Daniel F. Lusche, Paul A. Steimle, Amanda Scherer, Spencer Kuhl, Kristen Wood, Brett Hanson, Thomas T. Egelhoff and David R. Soll Journal of Cell Science, in press Behavioral analyses of the deletion mutants of the four known myosin II heavy chain (Mhc) kinases of D. discoideum revealed that all played a minor role in the efficiency of basic cell motility, but none played a role in chemotaxis in a spatial gradient of cAMP generated in vitro. However, each of the two kinases MhckA and MhckC, was essential for chemotaxis in a spatial gradient of Ca2+, shear induced directed movement, and reorientation in the front of waves of cAMP during natural aggregation. The mutant phenotypes of mhckA- and mhckC- were highly similar to that of the Ca2+ channel/receptor mutant iplA- and the myosin II phosphorylation mutant 3XALA, which produces constitutively unphosphorylated myosin II. These results demonstrate that IplA, MhckA and MhckC play a selective role in chemotaxis in a spatial gradient of Ca2+, but not cAMP and suggest that Ca2+ chemotaxis plays a role in the orientation of cells in the front of cAMP waves during natural aggregation. Submitted by Deborah Wessels [deborah-wessels@uiowa.edu] -------------------------------------------------------------------------------------- The balance in the delivery of ER components and the vacuolar proton pump to the phagosome depends on myosin IK in Dictyostelium. Regis Dieckmann1#, Aurelie Gueho1, Roger Monroy1, Thomas Ruppert2, Gareth Bloomfield3 and Thierry Soldati1* Molecular & Cellular Proteomics, in press 1Department de Biochimie, Faculte des Sciences, University de Geneve, Sciences II, 30 quay Ernest Ansermet, CH-1211 Geneve-4, Switzerland 2Core Facility for Mass Spectrometry and Proteomics, Zentrum fuer Molekulare Biologie der Universitaet Heidelberg (ZMBH), Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany 3MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK #Present address: : Klinisches Institut fuer Pathologie, 1090 Wien, Austria *Corresponding author Molecular & Cellular Proteomics, in press In Dictyostelium, the cytoskeletal proteins Actin binding protein 1 (Abp1) and the class I myosin MyoK directly interact and couple actin dynamics to membrane deformation during phagocytosis. Together with the kinase PakB, they build a regulatory switch that controls the efficiency of uptake of large particles. As a basis for further functional dissection, exhaustive phagosome proteomics was performed and established that about 1300 proteins participate in phagosome biogenesis. Then, quantitative and comparative proteomic analysis of phagosome maturation was performed to investigate the impact of the absence of MyoK or Abp1. Immunoblots and two-dimensional differential gel electrophoresis (2D-DIGE) of phagosomes isolated from myoK-null and abp1-null cells were used to determine the relative abundance of proteins during the course of maturation. Immunoblot profiling showed that absence of Abp1 alters the maturation profile of its direct binding partners such as actin and the Arp2/3 complex, suggesting that Abp1 directly regulates actin dynamics at the phagosome. Comparative 2D-DIGE analysis resulted in the quantification of mutant-to-wild type abundance ratios at all stages of maturation for over one hundred identified proteins. Coordinated temporal changes in these ratio profiles determined the classification of identified proteins into functional groups. Ratio profiling revealed that the early delivery of ER proteins to the phagosome was affected by the absence of MyoK and was coupled to a reciprocal imbalance in the delivery of the vacuolar proton pump and Rab11 GTPases. As direct functional consequences, a delayed acidification and a reduced intra-phagosomal proteolysis were demonstrated in vivo in myoK-null cells. In conclusion, the absence of MyoK alters the balance of the contributions of the ER and an endo-lysosomal compartment, and slows down phagosome acidification as well as the speed and efficiency of particle degradation inside the phagosome. Submitted by Thierry Soldati [thierry.soldati@unige.ch] ------------------------------------------------------------------------------------- Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration. Jessica S Kelsey, Nathan M Fastman, Elizabeth F Noratel and Daphne D Blumberg* Molecular Biology of the Cell, In Press The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm which is described here. The Ndm protein is predicted to contain a coiled-coil BAR like domain, a domain involved in endocytosis and membrane bending. Ndm knockout and Ndm-mRFP expressing cell lines were used to establish a role for ndm in suppressing endocytosis. An increase in the rate of endocytosis and in the number of endosomes was detected in ndm- cells. During migration ndm- cells formed numerous endocytic cups instead of the broad lamellipodia structure characteristic of moving cells. A second lamellipodia based function, cell spreading, was also defective in the ndm- cells. The increase in endocytosis and the defect in lamellipodia formation were associated with reduced chemotaxis in ndm- cells. Immunofluorescence results and GST pull down assays revealed an association of Ndm with coronin and F-actin. The results establish Ndm as a gene important in regulating the balance between formation of endocytic cups and lamellipodia structures. Submitted by Daphne Blumberg [blumberg@umbc.edu] ============================================================== [End dictyNews, volume 38, number 18]