Dicty News Electronic Edition Volume 19, number 10 November 15, 2002 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. ============= Abstracts ============= Dynamic organization of the actin system in the motile cells of Dictyostelium Till Bretschneider +1, James Jonkman +2, Jana Khler +1, Ohad Medalia +1, Karmela Barisic +1,4, Igor Weber +3, Ernst H.K. Stelzer +2, Wolfgang Baumeister +1 and G. Gerisch +1 +1 Max-Planck-Institut fr Biochemie, D-82152 Martinsried, Germany; +2 European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany; +3 Rudjer Boskovic Institut, Bijenicka cesta 54, 10000 Zagreb, Croatia; +4 Permanent address: University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Medical Biochemistry and Hematology, Domagojeva 2, 10000 Zagreb, Croatia. Journal of Muscle Research and Cell Motility, Special Issue: Dictyostelium, Ed. Dietmar J. Manstein, in press Abstract The actin system forms a supramolecular, membrane-associated network that serves multiple functions in Dictyostelium cells, including cell motility controlled by chemoattractant, phagocytosis, macropinocytosis, and cytokinesis. In executing these functions the monomeric G-actin polymerizes reversibly, and the actin filaments are assembled into membrane-anchored networks together with other proteins involved in shaping the networks and controlling their dynamics. Most impressive is the speed at which actin- based structures are built, reorganized, or disassembled. We used GFP- tagged coronin and Arp3, an intrinsic constituent of the Arp2/3 complex, as examples of proteins that are recruited to highly dynamic actin- filament networks. By fluorescence recovery after photobleaching (FRAP), average exchange rates of cell-cortex bound coronin were estimated. A nominal value of 5 seconds for half-maximal incorporation of coronin into the cortex, and a value of 7 seconds for half-maximal dissociation from cortical binding sites has been obtained. Actin dynamics implies also flow of F-actin from sites of polymerization to sites of depolymerization, i.e. to the tail of a migrating cell, the base of a phagocytic cup, and the cleavage furrow in a mitotic cell. To monitor this flow, we expressed in Dictyostelium cells a GFP-tagged actin-binding fragment of talin. This fragment (GFP-TalC63) translocates from the front to the tail during cell migration and from the polar regions to the cleavage furrow during mitotic cell division. The intrinsic dynamics of the actin system can be manipulated in vivo by drugs or other probes that act either as inhibitors of actin polymerization or as stabilizers of filamentous actin. In order to investigate structure-function relationships in the actin system, a technique of reliably arresting transient network structures is in demand. We discuss the potential of electron tomography of vitrified cells to visualize actin networks in their native association with membranes. ----------------------------------------------------------------------------- Multiple activities of a novel substance, dictyopyrone C isolated from Dictyostelium discoideum, in cellular growth and differentiation Y. Maeda1*, H. Kikuchi2*, K. Sasaki1, A. Amagai1, J. Sekiya2, Y. Takaya3 and Y. Oshima2 1Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences , Tohoku University, Aoba, Sendai 980-8578, 2Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Sendai 980-8578, and 3Faculty of Pharmacy, Meijo University, Yagotoyama, Tempaku, Nagoya 468-8503 (Japan) *equal contribution Protoplasma (in press) Summary. We report here that a novel substance named dictyopyrone C(DPC) has remarkable effects on growth and differentiation of D. discoideum Ax-2 cells, in a dose-dependent manner. In the presence of 3-15M DPC, differentiation of starving Ax-2 (clone MS) cells was greatly enhanced under submerged conditions, when vegetative MS-cells were harvested at the mid-late exponential growth phase (3 X 106 cells/ml) and starved. In contrast, DPC above 30M markedly impaired the progression of differentiation including cell aggregation, most of starved cells being rounded in shape 3-4 h after DPC-application and then lysed during further incubation. In the presence of 30M DPC however, MS-cells that had been harvested at the early exponential growth phase (5 X 105 cells/ml) and starved were neither rounded in shape nor lysed, and exhibited rather enhanced differentiation. Essentially the same results were obtained in cultures of starved cells on nonnutrient agar. With respect to DPC-effect on MS-cells growing in axenic medium, cell lysis and growth inhibition by more than 15M DPC were realized in the mid-late exponential growth phase cells (3 X 106 cells/ml), but not in the early exponential growth phase cells (5 X 105 cells/ml). Moreover, analysis using synchronized MS-cells has demonstrated that the DPC-effect changes in a cell-cycle dependent manner. In contrast to such unique DPC-actions, the pyrone ring of DPC had no effects on growth and differentiation within a range of 3-120M tested. These findings have strongly suggested the importance of a combined structure of the pyrone ring (PDP) and the linear carbon chain in revelation of the DPC activities. submitted by: Yasuo MAEDA [ymaeda@mail.cc.tohoku.ac.jp] ----------------------------------------------------------------------------- Cytoskeleton Interactions Involved in the Assembly and Function of Glycoprotein-80 Adhesion Complexes in Dictyostelium Tony J. C. Harris*, Amir Ravandi*, Donald E. Awrey and Chi-Hung Siu* *Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada, Affinium Pharmaceuticals, 100 University Avenue, Toronto, Ontario M5J 1V6, Canada. J. Biol. Chem. (in press.) SUMMARY Adhesion complexes typically assemble from clustered receptors that link to the cytoskeleton via cytoplasmic adapter proteins. However, it is unclear how phospholipid-anchored adhesion molecules, such as the Dictyostelium receptor gp80, interact with the cytoskeleton. gp80 has been found to form adhesion complexes from raft-like membrane domains, which can be isolated as a Triton X-100-insoluble floating fraction (TIFF). We report here that the actin- binding protein ponticulin mediates TIFF-cytoskeleton interactions. Analysis of gp80-null cells revealed that these interactions were minimal in the absence of gp80. During development, gp80 was required to enhance these interactions as its adhesion complexes assembled. While ponticulin and gp80 could partition independently into TIFF, gp80 was shown to recruit ponticulin to cell-cell contacts and to increase its partitioning into TIFF. However, these proteins did not co-immunoprecipitate. Furthermore, sterol sequestration abrogated the association of ponticulin with TIFF without affecting gp80, suggesting that sterols may mediate the interactions between ponticulin and gp80. In ponticulin-null cells, large gp80 adhesion complexes assembled in the absence of ponticulin despite the lack of cytoskeleton association. We propose that such nascent gp80 adhesion complexes produce expanded raft-like domains that recruit ponticulin and thereby establish stable cytoskeleton interactions to complete the assembly process. submited by: chi.hung.siu@utoronto.ca ----------------------------------------------------------------------------- Reciprocal Raft-Receptor Interactions and the Assembly of Adhesion Complexes Tony J. C. Harris and Chi-Hung Siu* Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada. BioEssays (in press) Summary Cell adhesion complexes are critical for the physical coordination of cell- cell interactions and the morphogenesis of tissues and organs. Many adhesion receptors are anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) moiety and are thereby partitioned into membrane rafts. In this review, we focus on reciprocal interactions between rafts and adhesion molecule, leading to receptor clustering and raft expansion and stability. A model for a 3-stage adhesion complex assembly process is also proposed. First, GPI-anchored adhesion molecules are recruited into rafts, which in turn promote receptor cis-oligomerization and thereby produce precursory complexes primed for avid trans-interactions. Second, trans-interactions of the receptors cross-link and stabilize large amalgams of rafts at sites of adhesion complex assembly. Finally, the enlarged and stabilized rafts acquire enhanced abilities to recruit the cytoskeleton and induce signaling. This process exemplifies how the domain structure of the plasma membrane can impact the function of its receptors. submitted by: chi.hung.siu@utoronto.ca ----------------------------------------------------------------------------- Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium Georgios Tsiavaliaris, Setsuko Fujita-Becker, Renu Batra, Dmitrii I. Levitsky 1, F. Jon Kull 2, Michael A. Geeves 3 & Dietmar J. Manstein + Max-Planck-Institut fr medizinische Forschung, Jahnstrasse 29, D-69120 Heidelberg, Germany, 1 A.N.Bach Institute of Biochemistry, Russian Academy of Science, Moscow 119071, Russia, 2 Dartmouth College, 6128 Burke Laboratory, Hanover, NH 03755, USA, 3 Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK EMBO Reports, in press Dominant-negative inhibition is a powerful genetic tool for the characterization of gene function in vivo, based on the specific impairment of a gene product by the coexpression of a mutant version of the same gene product. Here we describe the detailed characterization of two myosin constructs containing either point mutations F487A or F506G in the relay region. Dictyostelium cells transformed with F487A or F506G myosin are unable to undergo processes that require myosin II function including fruiting-body formation, normal cytokinesis and growth in suspension. Our results show that the dominant-negative inhibition of myosin function is caused by disruption of the communication between active site and lever arm, which blocks motor activity completely, and perturbation of the communication between active site and actin-binding site, leading to an approximately 100-fold increase in the mutants affinity for actin in the presence of ATP. submitted by: Manstein.Dietmar@mh-hannover.de ----------------------------------------------------------------------------- [End Dicty News, volume 19, number 10]