Dicty News Electronic Edition Volume 17, number 1 June 30, 2001 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 ============== Requirements for the Adenylyl Cyclases in Development of Dictyostelium Christophe Anjard, Fredrik Sderbom, and William F. Loomis Center for Molecular Genetics, Division of Biology University of California San Diego, La Jolla, CA 92093 Development, in press ABSTRACT It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high. ----------------------------------------------------------------------------- Myosin II Dynamics and Cortical Flow during Contractile Ring Formation in Dictyostelium Cells. Shigehiko Yumura Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan. J. Cell Biol., In press. Abstract Myosin II is a major component of a contractile ring. In order to examine if myosin II turns over in contractile rings, fluorescence of GFP-myosin II expressed in Dictyostelium cells was bleached locally by laser illumination, and the recovery was monitored. The fluorescence recovered with a half time of 7.01 2.62 sec. This recovery was not caused by lateral movement of myosin II from the non-bleached area but by an exchange with endoplasmic myosin II. Similar experiments were performed in cells expressing GFP-3ALA myosin II, of which three phosphorylatable threonine residues were replaced with alanine residues. In this case, recovery was not detected within a comparable time range. These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation. Because GFP-3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time. Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings. ----------------------------------------------------------------------------- Brief Communications Genomics: Genes lost during evolution Jeroen Roelofs and Peter J.M. van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Nature 411, 1013 - 1014 (2001) One of the main conclusions presented by the International Human Genome Sequencing Consortium is that "hundreds of gene appear to have resulted from horizontal gene transfer from bacteria at some point in the vertebrate lineage". We noticed that a significant proportion of these human genes have closely related orthologues in the primitive eukaryote Dictyostelium. This observation supports independent gene loss in multiple lineages (worm, fly, yeast, plants) rather than horizontal gene transfer from bacteria. ----------------------------------------------------------------------------- The Dictyostelium homologue of mammalian soluble adenylyl cyclase encodes a guanylyl cyclase Jeroen Roelofs1, Marcel Meima2, Pauline Schaap2 and Peter J.M. Van Haastert1 1) GBB, Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands 2) Department of Biochemistry, University of Dundee, Dundee DD1 5EH, UK EMBO journal, 2001, in press Abstract A new Dictyostelium cyclase gene was identified that encodes a protein (sGC) with 35% similarity to mammalian soluble adenylyl cyclase (sAC). Gene disruption of sGC has no effect on adenylyl cyclase activity and results in more then a ten-fold reduction of guanylyl cyclase activity. The scg- null mutants show reduced chemotactic sensitivity and aggregate poorly under stringent conditions. With Mn2+/GTP as substrate most of sGC activity is soluble, but with the more physiological Mg2+/GTP the activity is detected in membranes and stimulated by GTPgS. Unexpectedly, orthologues of sGC and sAC are present in bacteria and vertebrates, but absent from Drosophila, C. elegans, Arabidopsis and S. cerevisiae. ----------------------------------------------------------------------------- A Diffusion-Translocation Model for Gradient Sensing by Chemotactic Cells Marten Postma and Peter J.M. Van Haastert GBB, Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands Biophysical Journal, 2001, in press Abstract Small chemotactic cells like Dictyostelium and neutrophils transduce shallow spatial chemoattractant gradients into strongly localized intracellular responses. We show that the capacity of a second messenger to establish and maintain localized signals, is mainly determined by its dispersion range l = (Dm/k-1)0.5, which must be small compared to the cells length. Therefore short living second messengers (high k-1) with diffusion coefficients Dm in the range of 0 - 5 mm2s-1 are most suitable. Additional to short dispersion ranges, gradient sensing may include positive feedback mechanisms that lead to local activation and global inhibition of second messenger production. To introduce the essential non-linear amplification we have investigated models in which one or more components of the signal transduction cascade translocate from the cytosol to the second messenger in the plasma membrane. A one-component model is able to amplify a 1.5-fold difference of receptor activity over the cell length into a 15-fold difference of second messenger concentration. Amplification can be improved considerably by introducing an additional activating component that translocates to the membrane. In both models, communication between the front and the back of the cell is mediated by partial depletion of cytosolic components, which leads to both, local activation and global inhibition. The results suggest that a biochemically simple and general mechanism may explain various signal localization phenomena, not only in chemotactic cells but also those occurring in morphogenesis and cell differentiation. ----------------------------------------------------------------------------- cAMP and DIF-1 repress the expression of the Dictyostelium MADS-box gene srfA at early stages of development Ricardo Escalante1and Leandro Sastre Instituto de Investigaciones Biomdicas del Consejo Superior de Investigaciones Cientficas , C/Arturo Duperier,4. 28029 Madrid. Spain. B.B.R.C. (in press) 1 To whom correspondence should be addressed. Fax: 34-1-5854587. e-mail: rescalante@iib.uam.es Abstract The MADS-box containing gene srfA from Dictyostelium discoideum codes for a putative transcription factor that plays multiple roles in the development of this social amoeba. We have investigated the regulation of srfA gene expression after disaggregation of the cells from developing structures. The steady-state level of srfA mRNA was strongly and transiently induced shortly after disaggregation. srfA is maximally expressed 20 minutes after cell disaggregation and decreases thereafter. Induction was not dependent on protein synthesis, PKA, the kinase SplA and SrfA itself. This phenomena does not occur when cells are disaggregated in a small volume of buffer, suggesting the presence of extracellular molecules that repress srfA gene expression. In order to test this hypothesis, several well-known extracellular signaling molecules were studied. We found that srfA mRNA induction can be efficiently repressed by addition of exogenous cAMP and DIF-1 to the buffer in which the cells were disaggregated. Addition of other extracellular compounds such as ammonia, adenosine, SDF-1 and SDF-2 had no effect. srfA promoter P2, specifically induced during slug migration, was responsible for this regulation by extracellular compounds. ----------------------------------------------------------------------------- [End Dicty News, volume 17, number 1]