CSM News Electronic Edition Volume 5, number 13 November 4, 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 =========== A Model for Pattern Formation in Dictyostelium discoideum Pauline Schaap1, Yuanhua Tang2 and Hans G. Othmer3 1.Cell Biology Unit, Institute for Molecular Plant Sciences, University of Leiden, 2300 RA Leiden, The Netherlands 2.Department of Physiology and Biophysics, Cornell University Medical College, New York, New York 10021 3.Department of Mathematics, University of Utah Salt Lake City, UT, 84112, Differentiation, in press. Summary A model is developed for regulation of cell-type proportions in the mound stage of the cellular slime mold Dictyostelium discoideum. The model is based on current information on morphogenetic signaling that controls the differentiation of prestalk and prespore cells from differentiation competent cells. The morphogens that can control the cell type transitions are i. cAMP, which is required for both prestalk and prespore differentiation, ii. DIF, which induces prestalk and inhibits prespore differentiation and iii. NH3, which is an antagonist for all DIF-induced responses. cAMP is assumed to be a background species at the mound stage produced by oscillatory cAMP signaling. Besides its effects on differentiation, cAMP also induces DIF synthesis. DIF levels are controlled by DIF- dechlorinase, which is induced in response to DIF stimulation by the prestalk population. Induction of DIF-dechlorinase is inhibited by cAMP and NH3. NH3 is predominantly produced by prestalk cells, which show relatively high catabolism. As far as possible, we have incorporated quantitative information on transition rates between cell types and their dependence on morphogen concentrations. Computations using these data show correct proportions of prestalk cells, but the ratio of prespore to undifferentiated cells is too low. This can be remedied by assuming that maintenance of prespore differentiation requires 10-fold lower cAMP concentrations than induction. In the absence of NH3 and DIF-dechlorinase production, all cells rapidly differentiate into prestalk cells. DIF-dechlorinase is of major importance in setting prestalk and prespore proportions, but inclusion of ammonia in the presence of DIF-dechlorinase is not strictly necessary in the spatially-uniform system studied here. The regulatory capacity of the system is very robust and proportion regulation is not very sensitive to changes in morphogen concentrations, beyond a certain level required for differentiation induction. -------------------------------------------------------------------- Differential Distribution of cAMP receptors cAR2 and cAR3 during Dictyostelium development Yimin Yu and Charles L. Saxe III Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322-3030 Developmental Biology, in press Summary Signal transduction via a family of cAMP receptor subtypes (cARs) is critical for proper development in the cellular slime mold Dictyostelium. Genes encoding four related subtypes have been cloned and their expression, based on RNA accumulation has been previously reported. Here we report the differential spatial and temporal distribution of cAR2 and cAR3 proteins, based on indirect double immunofluorescence. Cells were transformed with a carB::lacZ construct, and an antibody against b-galactosidase used to visualize cAR2 expression. Simultaneously, a cAR3-specific antibody was used to identify cAR3 expressing cells. Results indicate that by the time of tip formation (12-14 hr) both receptors are expressed and distribute in a virtually non-overlapping pattern, with cAR2 being expressed on anterior, prestalk cells, and cAR3 present in the rest of the organism. Differential distribution of these two receptor subtypes may result in distinct cAMP signaling mechanisms in the two major regions of the organism. ----------------------------------------------------------------------- The cAMP receptor subtype cAR2 is restricted to a subset of prestalk cells during Dictyostelium development and displays unexpected DIF-1 responsiveness Charles L. Saxe III, Yimin Yu, Cheryl Jones, Andrea Bauman+, and Chris Haynes Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322-3030 +Department of Pharmacology, Vanderbilt University School of Medicine Developmental Biology, in press Summary Dictyostelium discoidium cells express a family of cell surface cAMP receptors, and these G-protein coupled receptors are each expressed with unique spatial and temporal patterns. One of these receptors, cAR2, is present during the post aggregative stages of development and our previous work suggests that it is preferentially expressed in prestalk cells. We report here the isolation of the promoter for carB, the gene which encodes cAR2. Using this fragment to generate a carB::lacZ gene fusion construct, we investigated carB expression in detail. Expression is first detected at the tight aggregate stage and subsequently in a pattern reminiscent of the prestalk-specific gene ecmA. There are subtle differences, however, with ecmA being expressed significantly in the anterior-like cells (ALC) of the migrating pseudoplasmodium and in the basal disc and lower cup supporting the sorus during terminal development. carB is not expressed in any of these places. The presence of these different prestalk-cell subtypes was confirmed by double indirect immunofluorescence using anti-cAR2 and anti-b-galactosidase antibodies. While virtually all cAR2-expressing cells also express ecmA::lacZ, a substantial fraction of ecmA::lacZ-positive cells do not express cAR2. We also found the regulation of carB gene expression to differ from that of ecmA. carB expression is induced in vitro by extracellular cAMP, but surprisingly, not by DIF-1, a soluble molecule thought to be essential for the initiation of prestalk differentiation. Thus, cAR2 appears to be a cAMP receptor present on a restricted subset of prestalk cells and whose expression does not respond typically to the prestalk inducer DIF-1. DIF-1 sensitivity may, therefore, not be characteristic of all early prestalk differentiation. --------------------------------------------------------------------- Examination of the Endosomal and Lysosomal Pathways in Dictyostelium discoideum Myosin I Mutants. Lesly Temesvari, John Bush, Michelle Peterson, Kristine Novak, Margaret Titus and James Cardelli Journal of Cell Science, in press. Summary The role of myosin Is in endosomal trafficking and the lysosomal system was investigated in a Dictyostelium discoideum myosin I double mutant myoB-/C-, that has been previously shown to exhibit defects in fluid-phase endocytosis during growth in suspension culture (Novak et al., 1995). Various properties of the endosomal pathway in the myoB-/C- double mutant as well as in the myoB- and myoC- single mutants, including intravesicular pH, and intracellular retention time and exocytosis of a fluid phase marker, were found to be indistinguishable from wild type parental cells. The intimate connection between the contractile vacuole complex and the endocytic pathway in Dictyostelium , and the localization of a myosin I to the contractile vacuole in Acanthamoeba, led us to also examine the structure and function of this organelle in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in either the myoB-, myoC- or myoB-/C- cell lines. The transport, processing, and localization of a lysosomal enzyme, a-mannosidase, were also unaltered in all three mutants. However, the myoB- and myoB-/C- cell lines, but not the myoC- cell line, were found to oversecrete the lysosomal enzymes a-mannosidase and acid phosphatase, during growth and starvation. None of the mutants oversecreted proteins following the constitutive secretory pathway. Two additional myosin I mutants, myoA- and myoA-/B-, were also found to oversecrete the lysosomally localized enzymes a-mannosidase and acid phosphatase. Taken together, these results suggest that these myosins do not play a role in the intracellular movement of vesicles, but that they may participate in controlling events that occur at the actin rich cortical region of the cell. While no direct evidence has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fidelity of the actin cortex, and changes in cortical organization could influence such lysosomal-related membrane events such as internalization or transit of vesicles to the cell surface. ---------------------------------------------------------------------- [End CSM News, volume 5, number 13]