CSM News Electronic Edition Volume 5, number 14 November 11, 1995 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmsbio.nwu.edu/dicty.html" =========== Abstracts =========== FUNCTIONAL COMPLEMENTATION OF YEAST PHOSPHOFRUCTOKINASE MUTANTS BY THE NON-ALLOSTERIC ENZYME FROM DICTYOSTELIUM DISCOIDEUM Antonio M. Estevez, Jurgen J. Heinisch and Juan J. Aragon Departamento de Bioquimica de la UAM and Instituto de Investigaciones Biomedicas del CSIC, fur Mikrobiologie, Heinrich-Heine-Universitat Dusseldorf, Germany FEBS Lett. 374: 100-104 ABSTRACT Phosphofructokinase (PFK) from yeast has been replaced by the non-allosteric enzyme from the slime mold Dictyostelium discoideum. This has been achieved by overexpression of the latter in a PFK-deficient strain of Saccharomyces cerevisiae under the control of the PFK2 promoter. Transformants complemented the glucose-negative growth phenotype exhibiting generation times on glucose-containing media similar to those of an untransformed strain being wild-type for yeast PFK genes. The PFK produced reacted with an antibody against D. discoideum PFK. It exhibited the same subunit size, quaternary structure and kinetic parameters than those of the wild-type enzyme, and was also devoid of specific regulatory properties. -------------------------------------------------------------------- A novel, phospholipase C-independent pathway of Ins(1,4,5)P3 formation in Dictyostelium and rat liver. Peter Van Dijken1, Jan-Roelof de Haas1, Andrew Craxton2, Christophe Erneux3, Stephen B. Shears2 and Peter J.M. Van Haastert1 1 Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. 2 Inositol Lipid Section, Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709, USA. 3 Institut de Recherche interdisciplinaire (IRIBHN), Universite Libre de Bruxelles, Campus Erasme, Brussels, J. Biol. Chem., in press ABSTRACT In an earlier study a mutant Dictyostelium cell-line (plc-) was constructed in which all phospholipase C activity was disrupted and non-functional, yet these cells had nearly normal Ins(1,4,5)P3 levels (Drayer, A.L., Van Der Kaay, J., Mayr, G.W, Van Haastert, P.J.M. (1990) EMBO J. 13, 1601-1609). We have now investigated if these cells have a phospholipase C-independent de novo pathway of Ins(1,4,5)P3 synthesis. We found that homogenates of plc- cells produce Ins(1,4,5)P3 from endogenous precursors. The enzyme activities that performed these reactions were located in the particulate cell fraction, whereas the endogenous substrate was soluble and could be degraded by phytase. We tested various potential inositol polyphosphate precursors and found that the most efficient were Ins(1,3,4,5,6)P5, Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4. The utilization of Ins(1,3,4,5,6)P5, which can be formed independently of phospholipase C by direct phosphorylation of inositol (Stephens, L.R. and Irvine, R.F. (1990) Nature 346, 580-582), provides Dictyostelium with an alternative and novel pathway of de novo Ins(1,4,5)P3 synthesis. We further discovered that Ins(1,3,4,5,6)P5 was converted to Ins(1,4,5)P3 via both Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4. In the absence of calcium no Ins(1,4,5)P3 formation could be observed; half maximal activity was observed at low micromolar calcium concentrations. These reaction-steps could also be performed by a single enzyme purified from rat liver, namely, the multiple inositol polyphosphate phosphatase (MIPP). These data indicate that organisms as diverse as rat and Dictyostelium possess enzyme activities capable of synthesising the second-messengers Ins(1,4,5)P3 and Ins(1,3,4,5)P4 via a novel phospholipase C- independent pathway. ------------------------------------------------------------------ DICTYOSTELIUM DISCOIDEUM CONTAINS THREE INOSITOL MONOPHOSPHATASE ACTIVITIES WITH DIFFERENT SUBSTRATE SPECIFICITY AND SENSITIVITY TO LITHIUM Peter Van Dijken, Jan C.T. Bergsma, Hoebert S. Hiemstra, Berber De Vries, Jeroen Van Der Kaay and Peter J.M. Van Haastert1 Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. Biochemical Journal, in press ABSTRACT The small ion lithium, a very effective agent in the treatment of manic depressive patients, inhibits the mammalian enzyme inositol monophosphatase which is proposed to be the biological target for the effects of lithium. In this study, Dictyostelium discoideum inositol monophosphatase activity was investigated. Partial purification of the proteins in the soluble cell fraction using anion exchange chromatography revealed the presence of at least three enzyme activities capable of degrading inositol monophosphate isomers. The first activity was similar to the monophosphatase found in mammalian cells as it degraded Ins(4)P, Ins(1)P and to a lesser extend Ins(3)P, was dependent on MgCl2 and inhibited by LiCl in an uncompetitive manner. The second enzyme activity was specific for Ins(4)P; the enzyme activity was not dependent on MgCl2 and not inhibited by LiCl. The third monophosphatase activity degraded especially Ins(3)P, but also Ins(4)P and Ins(1)P; increasing concentrations of MgCl2 inhibited this enzyme activity whereas LiCl had no effect. In vivo, LiCl induces a reduction of inositol levels by about 20%. In [3H]inositol-labeled cells LiCl causes a 6 fold increase in the radioactivity of [3H]Ins(1)P, a doubling of [3H]Ins(4)P and a slight reduction of the radioactivity in [3H]Ins(3)P. These data indicate that the biological effects of lithium in Dictyostelium are not due to depletion of the inositol pool by inhibition of inositol monophosphatase activity. ---------------------------------------------------------------------- Viscoelastic properties of F-actin solutions in the presence of normal and mutated actin-binding proteins K.-P. Janssen, L. Eichinger, P. Janmey*, A. A. Noegel , M. Schliwa, W. Witke* and M. Schleicher1 Institut fuer Zellbiologie, Ludwig-Maximilians-Universitaet, Schillerstr. 42, 80336 Muenchen, F.R.G.; *Harvard Medical School, Boston MA 02115, U.S.A.; Max-Planck-Institut fuer Biochemie, 82152 Martinsried, F.R.G. Arch. Biochem. Biophys., in press. Summary A minimal level of viscoelasticity in the cytoskeleton is an essential prerequisite of cellular motility. To determine the influence of the F-actin crosslinking proteins a-actinin and ABP120 gelation factor from Dictyostelium discoideum on the properties of actin gels we used a torsion pendulum to measure directly viscoelastic changes of the filamentous networks. Using the capping proteins severin and DS151 to control actin filament length, both crosslinkers were found to increase the elasticity and the viscosity of F-actin solutions. In the case of a-actinin, this activity was completely blocked by micromolar concentrations of Ca2+. The inhibitory functions of the two EF hands of a- actinin were further investigated by introducing point-mutations into either one or both of the Ca2+- binding regions. Mutations in the Ca2+-coordinating amino acid residues in the first or in both EF hands left the dynamic storage and loss moduli of the F-actin solution unaltered, independent of the Ca2+-concentration. However, a-actinin mutated in the second EF hand increased the viscoelasticity of actin gels like the wild-type protein. The ABP120 gelation factor exhibited only negligible differences to a-actinin in viscometry measurements, whereas its impact on the ratio G=B4=B4/G=B4 (the ratio of energy lost as compared to elastically stored during a deformation) of F-actin solutions was clearly smaller than that of a-actinin. We conclude from these data: i) a torsion pendulum is an excellent tool to determine small changes of activity in normal and mutated actin-binding proteins, ii) the first EF-hand of a-actinin is crucial for its crosslinking function, iii) the viscoelastic properties of F-actin gels crosslinked by either a-actinin or the ABP120 gelation factor are different. -------------------------------------------------------------------- The Dictyostelium MAP kinase ERK2 regulates multiple, independent developmental pathways Chris Gaskins1, Alexandra M. Clark1, Laurence Aubry1, Jeffrey E. Segall2, and Richard A. Firtel1,3 1Department of Biology Center for Molecular Genetics University of California, San Diego 9500 Gilman Dr. La Jolla, CA 92093-0634 2Department of Anatomy and Structural Biology Albert Einstein College of Medicine Bronx, NY 10461 Genes and Development, in press ABSTRACT We previously showed that the MAP kinase ERK2 is essential for aggregation. erk2 null cells lack cAMP stimulation of adenylyl cyclase and thus cannot relay the cAMP chemotactic signal, although the cells chemotaxis to cAMP (Segall et al., 1995) . In this manuscript we have examined the role of ERK2 in controlling developmental gene expression and morphogensis during the multicellular stages, making use of a temperature sensitive ERK2 mutation. Using suspension assays, we show that ERK2 is not essential for aggregation-stage, cAMP-pulse-induced gene expression, or for the expression of post-aggregative genes, which are induced at the onset of mound formation in response to cAMP in wild-type cells. In contrast, the prespore-specific gene SP60 is not induced and the prestalk-specific gene ecmA is induced but at a significantly reduced level. Chimeric organisms, comprised of wild-type and erk2 null cells expressing the prestalk-specific ecmA/lacZ reporter, show an abnormal spatial patterning, in which Erk2ts:erk2 cells are excluded from the very anterior prestalk A region. To further examine the function of ERK2 during the multicellular stages, we bypassed the requirement of ERK2 for aggregation by creating an ERK2 temperature-sensitive mutant. erk2 null cells expressing the ERK2ts mutant develop normally at 20oC and express cell-type-specific genes but do not aggregate at temperatures above 25oC. Using temperature shift experiments, we showed that ERK2 is essential for proper morphogenesis and for the induction and maintenance of prespore but not prestalk gene expression. Our results indicate that ERK2 functions at independent stages during Dictyostelium development to control distinct developmental programs: during aggregation, ERK2 is required for the activation of adenylyl cyclase and during multicellular development, ERK2 is essential for morphogenesis and cell-type-specific gene expression. Analysis of these results and others supports the conclusion that the requirement of ERK2 for cell-type differentiation is independent of its role in the activation of adenylyl cyclase. ---------------------------------------------------------------------- THE PRECISION OF REGULATION IN Dictyostelium discoideum: IMPLICATIONS FOR MODELS OF CELL-TYPE PROPORTIONING IN THE ABSENCE OF SPATIAL PATTERN Vidyanand Nanjundiah and Ashok S. Bhogle Developmental Biology and Genetics Laboratory, Indian Institute of Science, and Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 012, India Indian Journal of Biochemistry and Biophysics, in press. Abstract We have made careful counts of the exact number of spore, stalk and basal disc cells in tiny fruiting bodies of Dictyostelium discoideum (undifferentiated amoebae are found only rarely and on average their fraction is 4.96x10E-4). (i). Within aggregates of a given size, the relative apportioning of amoebae to the main cell types occurs with a remarkable degree of precision. In most cases the coefficient of variation (c.v.) in the mean fraction of cells that form spores is within 4.86%. The contribution of stalk and basal disc cells is highly variable when considered separately (c.v.'s upto 25% and 100%, respectively), but markedly less so when considered together. Calculations based on theoretical models indicate that purely cell-autonomous specification of cell fate cannot account for the observed accuracy of proportioning. Cell-autonomous determination to a prestalk or prespore condition followed by cell type interconversion, and stabilised by feedbacks, suffices to explain the measured accuracy. (ii). The fraction of amoebae that differentiates into spores increases monotonically with the total number of cells. This fraction rises from an average of 73.6% for total cell numbers below 30 and reaches 86.0% for cell numbers between 170 and 200 (it remains steady thereafter at around 86%). Correspondingly, the fraction of amoebae differentiating into stalk or basal disc decreases with total size. These trends are in accordance with evolutionary expectations and imply that a mechanism for sensing the overall size of the aggregate also plays an essential role in the determination of cell-type proportions. Therefore cell-autonomous specification of cell fate and intercellular signalling without spatial heterogeneity constitute a sufficient explanation for the observed accuracy of proportioning. Given this, we suggest that the primary role of spatial patterning as specified by morphogenetic gradients could be to coordinate other aspects of post-aggregation development in space and time. In particular, such gradients may ensure the long-term stability and proper distribution of cell types within the slug and fruiting body. ---------------------------------------------------------------------- Dictyostelium Myosin Heavy Chain Kinase A Regulates Myosin Localization During Growth and Development. Michael F. Kolman, Lidia M. Futey, and Thomas T. Egelhoff Department of Physiology and Biophysics Case Western Reserve University School of Medicine Cleveland, Ohio 44106-4970 J. Cell Biol., in press ABSTRACT Phosphorylation of the Dictyostelium myosin II heavy chain (MHC) has a key role in regulating myosin localization in vivo and drives filament disassembly in vitro. Previous molecular analysis of Dictyostelium myosin II heavy chain kinase (MHCK A) gene has demonstrated that the catalytic domain of this enzyme is extremely novel, showing no significant similarity to the known classes of protein kinases (Futey, L.M., Medley, Q.G., Cote, G.P., and Egelhoff, T.T. 1995. J.Biol.Chem. 270:523-529). To address the physiological roles of this enzyme, we have analyzed the cellular consequences of MHCK A gene disruption (mhck A- cells) and MHCK A overexpression (MHCK A++ cells). The mhck A- cells are viable and competent for tested myosin-based contractile events, but display partial defects in myosin localization. Both growth phase and developed mhck A- cells show substantially reduced MHC kinase activity in crude lysates, as well as significant overassembly of myosin into the Triton-resistant cytoskeletal fractions. MHCK A++ cells display elevated levels of MHC kinase activity in crude extracts, and show reduced assembly of myosin into Triton-resistant cytoskeletal fractions. MHCK A++ cells show reduced growth rates in suspension, becoming large and multinucleated, and arrest at the mound stage during development. These results demonstrate that MHCK A functions in vivo as a protein kinase with physiological roles in regulating myosin II localization and assembly in Dictyostelium cells during both growth and developmental stages. --------------------------------------------------------------------- Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis. Kristine D. Novak, Michelle D. Peterson, Mary C. Reedy & Margaret A. Titus Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 J. Cell Biol., in press. Summary The functional relationship between three Dictyostelium myosin Is, myoA, myoB and myoC, has been addressed through the creation of double mutants. Two double mutants, myoA-/B- and myoB-/C-, exhibit similar conditional defects in fluid-phase pinocytosis. Double mutants grown in suspension culture are significantly impaired in their ability to take in nutrients from the medium, whereas they are almost indistinguishable from wild-type and single mutant strains when grown on a surface. The double mutants are also found to internalize gp126, a 116-kD membrane protein, at a slower rate than either the wild-type or single mutant cells. Ultrastructural analysis reveals that both double mutants possess numerous small internal vesicles, in contrast to the wild-type or myosin I single mutants that exhibit several large, clear vacoules. The alterations in fluid and membrane internalization in the suspension-grown double mutants, coupled with the altered vesicle profile, suggests that these cells may be compromised during the early stages of pinocytosis, a process that has been proposed to occur via actin-based cytoskeletal rearrangements. Scanning electron microscopy and rhodamine-phalloidin staining indicates that the myosin I double mutants appear to extend a larger number of actin-filled structures, such as filopodia and crowns, than wild-type cells. Rhodamine-phalloidin staining of the F-actin cytoskeleton of these suspension-grown cells also reveals that the double mutant cells are delayed in the rearrangement of cortical actin-rich structures upon adhesion to the substrate. We propose that myoA, myoB, and myoC are play roles in controlling F-actin filled membrane projections that are required for pinosome internalization in suspension. ------------------------------------------------------------------------ Coactosin interferes with the capping of actin filaments Ursula Ruhrig, Gunther Gerisch*, Ludmilla Morozova, Michael Schleicher3 and Albrecht Wegner2 Max-Planck-Institut f=FCr Biochemie, 82152 Martinsried 2 Institut fur Physiologische Chemie, Ruhr-Universitat, Universitatsstrasse 150, 44780 Bochum 3 Institut f=FCr Zellbiologie, Ludwig-Maximilians-Universitat, Schillerstrasse 42, 80336 Munchen *Author for correspondence FEBS Letters, in press. Coactosin, a 16 kD protein associated with the actin cytoskeleton from Dictyostelium discoideum, was purified by an improved method, in which other components of the cytoskeleton were removed. The highly purified coactosin had no effect on the time course of actin polymerization, but when added to actin in presence of capping proteins, coactosin counteracted the capping activity of these proteins. The capping proteins cap32/34 and severin domain 1 retarded actin polymerization, on addition of coactosin to samples containing one of these capping proteins the time course of actin polymerization became close to controls without capping proteins. ----------------------------------------------------------------------- Fusion with the green fluorescent protein monitors the chemoattractant controlled accumulation of coronin at the leading edge of Dictyostelium cells Gunther Gerisch1, Richard Albrecht, Christina Heizer, Steve Hodgkinson* and Markus Maniak Max-Planck-Institut f=FCr Biochemie, 82152 Martinsried, Germany *MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England Correspondence to: Dr. G=FCnther Gerisch, telefax: ++49-89-8578-2326; e-mail address: gerisch@vms.biochem.mpg.de Current Biology, in press. Abstract Background: The highly motile cells of Dictyostelium discoideum rapidly restructure their actin filament system when they change their direction of locomotion either spontaneously or in response to chemoattractant. Coronin is a soluble cytoplasmic protein that accumulates at cortical sites of locomoting cells and contributes to the dynamics of the actin system. It is a member of the WD-repeat family of proteins. In coronin null mutants cell locomotion is slowed down and cytokinesis is impaired. ----------------------------------------------------------------------- Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein tag Markus Maniak, Robert Rauchenberger, Richard Albrecht, John Murphy, and Gunther Gerisch Max-Planck-Institut fur Biochemie, D 82152 Martinsried, Germany Address correspondence to: Gunther Gerisch, Max-Planck-Insitut fur Biochemie, D-82152 Martinsried Phone: ++49-89-8578-2326; Fax: ++49-89-8578-3888; e-mail: gerisch@vms.biochem.mpg.de Cell, in press. Summary Coronin is a protein involved in cell locomotion and cytokinesis of Dictyostelium discoideum. Here we show that coronin strongly accumulates in phagocytic cups formed in response to particle attachment. The dynamics of coronin accumulation has been recorded in transfected cells that produce a fusion of coronin with green fluorescent protein (GFP). Coronin-GFP accumulates in the cups within less than a minute upon attachment of a particle and is gradually released from the phagosome within one minute after engulfment is completed. Phagocytic cup formation competes with leading edge formation and can be interrupted at any stage. Accordingly, coronin accumulation in the cup is reversible; the protein gets separated when the cup regresses. TRITC-labelled yeast cells have been used to assay phagocytosis quantitatively in wild-type and coronin-null cells. In the mutant, the rate of uptake is reduced to about one third of the wild-type rate. This result shows that coronin contributes to the efficiency of phagocytosis to about the same extent as it improves the speed of cell locomotion. ------------------------------------------------------------------------ [End CSM News, volume 5, number 14]