Dicty News Electronic Edition Volume 12, number 1 January 9, 1999 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ========================= PostDoctoral Position ========================= POSTDOCTORAL POSITION in Molecular Biology of Cell Differentiation in Dictyostelium at Virginia Tech University. The project is funded by NSF and involves a phosphatase activity (5’NU) that appears during the time course of cell differentiation in Dictyostelium. The enzyme becomes restricted to a narrow band of cells forming the interface between the two differentiating cells types. We have two major goals: (1) to identify the role played by this gene in the regulation of morphogenesis by disruption of expression of the 5’NU gene, overexpression and ectopic expression. The resulting effects on morphogenesis will be determined through the analysis of cell-type specific markers and genes that are indicative of the progression of differentiation in the two major cell types; (2) to identify the regulatory mechanisms that result in cell- specific expression of the 5’NU gene. This will involve an analysis of the transcriptional regulation of the 5’NU gene, by identification of cis-acting factors that regulate the temporal and spatial expression, and identification, purification, and cloning of trans-acting factors that regulate expression of the gene. Virginia Tech University is home to 25,000 students. An interdepartmental program in Molecular and Cellular Biology offers a variety of opportunities for interaction with other laboratories on campus as well as participation in a number of semimar series in this research area. The University campus in situated in the southwestern mountains of Virginia, an area known for its clean air and an abundance of outdoor activites. For further information contact Dr. Charles L. Rutherford, 2119 Derring Hall, Virginia Tech University, Blacksburg, VA 24061; phone 540 231-5349; email rutherfo@vt.edu. ============= Abstracts ============= A Potentially Exhaustive Screening Strategy Reveals Two Novel Divergent Myosins in Dictyostelium. Eva C. Schwarz, Heidrun Geissler, and Thierry Soldati Department of Molecular Cell Reserach, Max-Planck-Institut for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany. Cell Biochemistry and Biophysics, in press Abstract In recent years, the myosin superfamily has kept expanding at an explosive rate, but the understanding of their complex functions has been lagging. Therefore, Dictyostelium discoideum, a genetically and biochemically tractable eukaryotic amoeba, appears as a powerful model organism to investigate the involvement of the acto-myosin cytoskeleton in a variety of cellular tasks. Due to the relatively high degree of functional redundancy, such studies would be greatly facilitated by the prior knowledge of the whole myosin repertoire in this organism. Here, we present a strategy based on PCR amplification using degenerate primers and followed by negative hybridization screening which led to the potentially exhaustive identification of members of the myosin family in D. discoideum. Two novel myosins were identified and their genetic loci mapped by hybridization to an ordered YAC library. Preliminary inspection of myoK and myoM sequences revealed that, despite carrying most of the hallmarks of myosin motors, both molecules harbor features surprisingly divergent from most known myosins. ---------------------------------------------------------------------------- Phosphofructokinase from Dictyostelium discoideum is a potent inhibitor of tubulin polymerization Ferenc Orosz§, Belén Santamaría||, Judit Ovádi§, and Juan J. Aragón*,|| Departamento de Bioquimica UAM and Instituto de Investigaciones Biomédicas CSIC, Facultad de Medicina de la Universidad Autónoma de Madrid, Madrid, Spain. §Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary Biochemistry, in press. Abstract We identified the non-allosteric phosphofructokinase from the slime mold Dictyostelium discoideum as a potent protein factor that inhibits the rate of polymerization of tubulin at a molar ratio of 1 molecule to about 300 tubulin dimers for half-maximal action (IC50 = 32 nM). This effect was (i) assessed by turbidity measurements, pelleting of microtubules and electron microscopy, (ii) observed when tubulin assembly was induced by taxol as well as by GTP in the presence of microtubule associated proteins or glutamate, and (iii) specific as it was not produced by the phosphofructokinase from rabbit muscle. Also in contrast to the latter, neither tubulin nor microtubules modified the catalytic activity of the slime mold isozyme. Immunoelectron microscopy provided further evidence that D. discoideum phosphofructokinase physically interacts with tubulin leading to the formation of aggregates. The process seems to be reversible since microtubules eventually formed in the presence of the inhibitor with concomitant reduction of tubulin aggregates. Limited proteolysis by subtilisin showed that the hypervariable C-termini of tubulin is not involved in the interaction with the enzyme. The possible physiological relevance of this novel function of D. discoideum phosphofructokinase different from its glycolytic action is discussed. ---------------------------------------------------------------------------- Differences in the ionic interaction of actin with the motor domains of non-muscle and muscle myosin II Juliette Van Dijk*, Marcus Furch, Jean Derancourt*, Renu Batra, Menno L.W. Knetsch, Dietmar J. Manstein & Patrick Chaussepied* *UPR 1086 du CNRS, IFR 24, 1919 route de Mende, 34293 Montpellier Cedex5, France and Max-Planck-Institut für Medizinische Forschung, Jahnstr. 29, D-69120 Heidelberg, Germany. Europ. J. Biochem., im press. Abstract Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, similar to S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast to S1, loop 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The observed differences in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin-interface of muscle and non-muscle myosins. ---------------------------------------------------------------------------- Architectural Dynamics and Gene Replacement of Coronin Suggest its Role in Cytokinesis Yoshio Fukui *1), Sarah Engler *2), Shinya Inoué *3), and Eugenio L. de Hostos *2) *1) Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, *2) Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, (Current Address of E. L. de Hostos) Tropical Disease Research Unit, University of California, San Francisco, CA 94121 *3) Marine Biological Laboratory, Woods Hole, Massachusetts 02543. Cell Motility and Cytoskeleton, in press. Abstract Coronin is a ubiquitous actin-binding protein representing a member of proteins portraying a WD-repeat sequence, including the b-subunits of trimeric G-proteins. Coronin has been suggested to participate in multiple, actin-based physiological activities such as cell movement and cell division. Although, the slow growth of coronin deletion mutants has been attributed to a defect in the fluid-phase uptake of nutrients, the exact role of coronin in cytoskeletal organization has not been elucidated. In this study, we examined a role of coronin in cytokinesis by analyzing the effect of coronin deletion on the actin cytoskeleton and its dynamic distribution using a green fluorescent protein (GFP)-coronin fusion protein. We show that GFP- coronin works similarly to natural coronin in vivo and in vitro. In live cells, GFP-coronin was found to accumulate into the cleavage furrow during cytokinesis. The fluorescence pattern suggests its association to the contractile ring throughout cytokinesis. Interestingly, a substantial amount of coronin was also bound to F-actin at the prospective posterior cortex of the daughter cells. We also show that the coronin null cells reveal irregularities in organization of actin and myosin II and divide by a process identical to the traction-mediated cytofission reported in myosin II mutants. Overall, this study suggests that coronin is essential for organizing the normal actin cytoskeleton and plays a significant role in cell division. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 1]