Dicty News Electronic Edition Volume 10, number 12 April 25, 1998 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" =========== Abstracts =========== A Developmentally Regulated Kinesin-Related Motor Protein from Dictyostelium discoideum Eugenio L. de Hostos1*, Gretchen McCaffrey2#, Richard Sucgang4, Daniel W. Pierce2, and Ronald D. Vale2,3 1Department of Biochemistry and Cell Biology, Rice University, Houston TX 77005 2Department of Molecular and Cellular Pharmacology and 3Howard Hughes Medical Institute , University of California, San Francisco, CA 94143 4Department of Biochemistry, Baylor College of Medicine, Houston TX 77030 Mol. Biol. Cell, in press. ABSTRACT The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six kinesin-related proteins (KRPs), several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive a-helical coiled-coil domain. The non-motor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in E. coli, supports plus-end directed microtubule motility in vitro at a speed of 0.14 µm/sec, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 hr after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line, but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the pre-stalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the pre-stalk zone or to differentiate into pre-stalk cells. ------------------------------------------------------------------------- Fatty acids induce release of Ca2+ from acidosomal stores and activate capacitative Ca2+-entry in Dictyostelium discoideum Ralph Schaloske, Jürgen Sonnemann, Dieter Malchow and Christina Schlatterer Fakultät für Biologie, Universität Konstanz, D-78457 Konstanz Biochemical Journal, in press Summary cAMP-induced Ca2+-fluxes in Dictyostelium discoideum largely depend on phospholipase A2 activity generating free fatty acids (Schaloske, R. and Malchow, D. (1997) Biochem. J. 327:233-238). In the present study the effect of fatty acids on the Ca2+-homeostasis of Dictyostelium discoideum was investigated. [Ca2+]i was analyzed by digital imaging of single fura2-dextran-loaded cells. Arachidonic and linoleic acid induced a transient change in [Ca2+]i. The concentration of arachidonic acid determined the percentage of responding cells with the mean height of the increase being dose-independent. In nominally Ca2+-free medium or in the presence of BAPTA the [Ca2+]i-transient was not detectable. In spite of this, we found that 1. arachidonic acid induced Ca2+-release from permeabilized cells and from vesicular fractions at concentrations that elicited Ca2+-influx in intact cells and 2. that Ca2+-entry was inhibited by inhibitors of Ca2+-transport ATPases and V-type H+-ATPase indicating that intracellular Ca2+-release precedes Ca2+-entry. Inhibition studies and mutant analyses point to the acidosomal Ca2+-stores to be a target of fatty acids. Although fatty acids can fully substitute for cAMP with respect to Ca2+-influx in wildtype cells, experiments with a mutant strain revealed that cAMP additionally sensitizes the Ca2+-entry mechanism: cAMP-induced Ca2+- influx was normal in a knockout mutant of phospholipase C but influx was fairly insensitive to arachidonic acid in this strain. This defect could be overcome by higher doses of arachidonic acid that cause more Ca2+ to be released from the stores sufficient to trigger extracellular Ca2+-entry. ------------------------------------------------------------------------- The Actomyosin Cytoskeleton of Amoebae of the Cellular Slime Molds Acrasis rosea and Protostelium mycophaga: Structure, Biochemical Properties, and Function. Maarit Hellstén and Urs-Peter Roos, Institut für Pflanzenbiologie, Universität Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland Fungal Genetics and Biology (Special Issue on Fungal Architecture), in press ABSTRACT Amoebae of the cellular slime molds (mycetozoans) Acrasis rosea and Protostelium mycophaga move 3-6 times faster than those of Dictyostelium discoideum and their mode of pseudopod formation is different. In both species there are bundles of F-actin that radiate from the endoplasm-ectoplasm interface into the pseudopodia, where G-actin is also located. We conclude that these actin bundles form a core scaffold driving pseudopod extension which is subsequently completed by filling with a more loosely organized meshwork of F-actin. Some bipolar, elongate amoebae of A. rosea also contained long bundles of F-actin that traverse the cells lengthwise and remotely resemble stress fibers. We also observed scattered rodlets of F-actin in the body of amoebae of A. rosea, some of which formed star-shaped or polygonal complexes near or around contractile vacuoles. These may play a role in vacuole contraction. In total extracts prepared by the TCA-acetone procedure and analyzed by SDS-PAGE and immunoblots the actins of the two slime molds migrated like the rabbit skeletal muscle control. The relative proportion of actin in total protein extracts was 7.9% for A. rosea and 34.5% for P. mycophaga. We detected 4-5 isoactins in extracts of both species and we determined that the genome of each species contains approximately six actin genes. Whether they are all expressed or if post-translational modifications occur remains to be determined. Myosin II was enriched in actomyosin extracts; its Mr was 187.8 kDa for A. rosea and 220.7 kDa for P. mycophaga. Cell models prepared as cytoskeletons insoluble in Triton X-100 ("ghosts") contained minor amounts of actin and myosin II compared to actomyosin extracts, but they were able to contract upon the addition of ATP. We conclude that ameobae of A. rosea and P. mycophaga contain the basic repertoire of molecules and mechanisms allowing pseudopod extension by actin polymerization and ATP-induced contraction of the cell cortex by an interaction between actin filaments and myosin II. ------------------------------------------------------------------------- The Role of PKA in Timing Developmental Events in Dictyostelium Cells William F. Loomis Center for Molecular Genetics, Department of Biology, University of California San Diego, La Jolla, CA 92093 Microbiology and Molecular Biology Reviews, in press Abstract The cAMP dependent protein kinase, PKA, is dispensible for growth of Dictyostelium cells but plays a variety of crucial roles in development. The catalytic subunit of PKA is inhibited when associated with its regulatory subunit but is activated when cAMP binds to the regulatory subunit. Deletion of pkaR or over-expression of the gene encoding the catalytic subunit, pkaC, results in constitutive activity. Development is independent of cAMP in strains carrying these genetic alterations and proceeds rapidly to the formation of both spores and stalk cells. However, morphogenesis is aberrant in these mutants. In the wild type, PKA activity functions in a circuit that can spontaneously generate pulses of cAMP necessary for long range aggregation. It is also essential for transcriptional activation of both prespore and prestalk genes during the slug stage. During culmination, PKA functions in both prespore and prestalk cells to regulate the relative timing of terminal differentiation. A positive feedback loop results in the rapid release of a signal peptide, SDF-2, when prestalk cells are exposed to low levels of SDF-2. The signal transduction pathway that mediates the response to SDF-2 in both prestalk and prespore cells involves the two-component system of DhkA and RegA. When the cAMP phosphodiesterase RegA is inhibited, cAMP accumulates and activates PKA leading to vacuolization of stalk cells and encapsulation of spores. These studies indicate that multiple inputs regulate PKA activity to control the relative timing of differentiations in Dictyostelium. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 12]