Dicty News Electronic Edition Volume 13, number 7 September 18, 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 Available =================================== The Manstein laboratory at the Max-Planck-Institute for Medical Research in Heidelberg is searching for a Postdoctoral Fellow to work on the identification and characterization of novel gene products involved in cell motility and membrane trafficking. Our integrative approach involves the use of molecular biological, biochemical, and biophysical techniques. The technical capabilities required are experience in biochemical kinetic measurements and/or optical physics. A working knowledge of cell and molecular biology would be helpful. Total internal reflection fluorescence microscopy (TIRFM) will be used for the identification of gene products of interest and the kinetic characterization of myosin and dynamin family proteins on the single molecule level. TIRFM, in combination with surface immobilization of recombinant proteins via engineered high-affinity tags, will also provide the basis for the development of new in vitro assays for the functional characterization of motor proteins and proteins involved in vesicle trafficking. Applicants should provide a letter of intent explaining qualifications relevant to our system and possible areas of interest in our research program, a CV, and names and contact information for at least three individuals capable of commenting on your scientific ability. Please indicate when you expect to be available to join us. Applications may be submitted by e-mail to: manstein@mpimf-heidelberg.mpg.de, by FAX to (+49-6221) 486437 or by post to: Dr. Dietmar J. Manstein Dept. of Biophysics Max-Planck-Institute for Medical Research Jahnstr. 29 D-69120 Heidelberg ============= Abstracts ============= Migration Forces in Dictyostelium Measured by Centrifuge DIC Microscopy Yoshio Fukui 1, Taro Q. P. Uyeda2, Chikako Kitayama 2, and Shinya Inoué3 1 Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611-3008. 2 Biomolecular Research Group, National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki 305-8562, Japan. 3 Marine Biological Laboratory, Woods Hole, MA 02543-1015. Biological Bulletin (accepted for publication) Abstract Amoeboid locomotion represents an important biological activity involved in cell growth and development (1). Forces that underlie movement of the giant amoeba, Chaos chaos, have been estimated to be 1.5 x 102 pN/mm2 as measured by Kamiya’s double chamber method (2). For a slime mold, Dictyostelium discoideum, the forces of cell locomotion have been unknown, but the cortex resists poking with a microneedle (cortical tension) at 1.4 x 103 pN/mm2 (3). By micropipette aspiration, the cortical tension of D. discoideum has been measured as 1.55 x 103 pN/mm2 (4). In the present study, we have determined the migration stalling forces of D. discoideum by using a Centrifuge Polarizing Microscope (CPM) equipped with DIC optics (5). The results demonstrated that individual wild type (NC4) amoebae (6) can crawl centripetally on a glass surface in resistance to gravitational forces larger than 11,465 x g. NC4 amoebae can also undergo normal cytokinesis at forces of at least 8,376 x g. ---------------------------------------------------------------------------- Activated G(alpha) subunits can inhibit multiple signal transduction pathways during Dictyostelium development Jaishree Srinivasan, Robert E. Gundersen, and Jeffrey A. Hadwiger Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078-3020; Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono Maine 04469. Developmental Biology (in press) Abstract Mutations impairing the GTPase activity of G protein G(alpha) subunits can result in activated G(alpha) subunits that affect signal transduction, cellular responses, and, in some cases, promote tumor formation. An analogous mutation in the Dictyostelium G(alpha)4 subunit gene (Q200L substitution) was constructed and found to inhibit G(alpha)4-mediated responses to folic acid, including the accumulation of cyclic nucleotides and chemotactic cell movement. The G(alpha)4-Q200L subunit also severely inhibited responses to cAMP, including cyclic nucleotide accumulation, cAMP chemotaxis, and cellular aggregation. An analogous mutation in the G(alpha)2 subunit (Q208L substitution), previously reported to inhibit cAMP responses (Okaichi et al., 1992), was also found to partially inhibit folic acid chemotaxis. Chemotactic responses to folic acid and cAMP and developmental aggregation were also inhibited by a mutant G(alpha)5 subunit with the analogous alteration (Q199L substitution). All aggregation- defective G(alpha) mutants were capable of multicellular development after a temporary incubation at 4o C and this development was found to be dependent on wild-type G(alpha)4 function. This study indicates mutant G(alpha) subunits can inhibit signal transduction pathways mediated by other G(alpha) subunits. ---------------------------------------------------------------------------- An Adenylyl Cyclase that Functions during Late Development of Dictyostelium Fredrik Söderbom, Christophe Anjard, Negin Iranfar, Danny Fuller and William F. Loomis Development (in press) SUMMARY A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA- mutant cells can be rescued by developing them in mixtures with wild type cells suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25 fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA- mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA- cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk. ---------------------------------------------------------------------------- Functional Characterization of the Secondary Actin Binding Site of Myosin II Juliette Van Dijk, Marcus Furch, Chrystel Lafont, Dietmar J. Manstein, and Patrick Chaussepied CRBM du CNRS, IFR 24, Montpellier, France and Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany Biochemistry, in press ABSTRACT The role of the interaction between actin and the "secondary actin binding site" of myosin (segment 565-579 of rabbit skeletal muscle myosin referred to as loop 3 in this work) has been studied using proteolytically generated smooth- and skeletal muscle myosin subfragment-1, and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or non-muscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e. in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and non-muscle myosin chimera showed a four to six fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k-A) with no significant change in the rate for actin binding (k+A). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actin-myosin interface nor for the modulation of actomyosin ATPase activity. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 7]