CSM News Electronic Edition Volume 2, number 23 June 25, 1994 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 through www.nwu.edu. ----------- Abstracts ----------- STAGE-SPECIFIC REQUIREMENT FOR MYOSIN II DURING DICTYOSTELIUM DEVELOPMENT M. L. Springer*, B. Patterson, and J. A. Spudich Departments of Biochemistry and Developmental Biology Stanford University School of Medicine Stanford, CA 94305-5307 *Present address: Department of Molecular Pharmacology Stanford University School of Medicine Stanford, CA 94305-5332 Development, in press Summary Dictyostelium cells that lack a functional myosin II heavy chain are motile and are capable of aggregation, but fail to undergo further multicellular development. We have used a Dictyostelium mutant expressing a cold-sensitive myosin heavy chain to examine the requirement for myosin throughout the course of development. The loss of myosin function upon cooling is rapid and reversible. Temperature shift experiments reveal that myosin is essential during two different stages of development. During aggregation, myosin function appears to be necessary for cells to sort correctly in a way that allows further development to occur. During the final stage of development, it is required for the formation of a complete stalk and the raising of the spore head. Development between those stages, however, proceeds normally in the absence of myosin function. Aggregates at non-permissive temperature undergo an aberrant form of development resulting in a ball of cells. Calcofluor staining and reporter gene fusions reveal that these structures contain defective spores and a miniature stalk. -------------------------------------------------------------------- EXPRESSION OF cAMP DEPENDENT PROTEIN KINASE IN PRESPORE CELLS IS SUFFICIENT TO INDUCE SPORE CELL DIFFERENTIATION IN DICTYOSTELIUM Sandra K. O. Mann1, Delwood L. Richardson2, Susan Lee1, Alan R. Kimmel2, and Richard A. Firtel1 1Department of Biology Center for Molecular Genetics University of California, San Diego La Jolla, CA 92093-0634 and 2Laboratory of Cellular and Developmental Biology NIDDK (Building 6, B122) National Institutes of Health Bethesda, MD 20892 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, IN PRESS ABSTRACT The activity of cAMP-dependent Protein Kinase (PKA) is required for proper development at several stages during the Dictyostelium life cycle. We present evidence suggesting that activation of PKA is rate-limiting for the differentiation of prespore cells to spores during normal development and that the activation of PKA may be the developmental trigger for sporulation. Strains that overexpress the catalytic subunit of PKA (PKAcat) or lack a functional regulatory subunit (rdeC strains) have been shown to have rapid, heterochronic development. In this manuscript, we show that overexpression of PKAcat in prespore cells is sufficient to directly induce the expression of the spore maturation marker spiA in these cells and their differentiation to spores. This induction occurs early in multicellular development, very shortly after induction of the prespore pathway and appears to be cell autonomous. In contrast, wild-type cells do not express this marker until culmination is almost complete. Moreover, overexpression of PKAcat in prespore cells can bypass a mutation that blocks an earlier developmental stage to induce spiA expression. This suggests that the regulatory pathway in prespore cells between the activation of PKA and spiA induction/spore maturation is quite short and that PKAcat expression in prespore cells is sufficient to induce spore maturation and may be mediating this effect at the level of transcription. The induction of sporulation requires the prior activation of the prespore cell pathway since the overexpression of PKAcat in wild-type prestalk cells does not lead to induction of spiA. In addition, using a PKAcat/lacZ reporter construct, we show that PKAcat expression is highly enriched in the anterior prestalk A region during the tipped aggregate, slug, and early culminant stages and that this pattern switches abruptly to a prespore pattern at the time of spore maturation, supporting the proposed role of PKA in this process. ---------------------------------------------------------------- A GBF-binding site and a novel AT element define the minimal sequences sufficient to direct prespore-specific expression in Dictyostelium Jo Anne Powell-Coffman, Gavin R. Schnitzler, and Richard A. Firtel Department of Biology, Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive La Jolla, CA 92093-0634 USA tel. (619) 534-2788 fax (619) 534-7073 MOLECULAR AND CELLULAR BIOLOGY, IN PRESS SUMMARY In order to better understand the molecular mechanisms of cellular differentiation in Dictyostelium, we have identified the minimum regulatory sequences of the prespore-specific gene SP60/cotC that are sufficient to confer cell-type-specific expression to a heterologous promoter. This region includes at least two essential cis-acting elements: a novel AT-rich element (or elements) and CAE3. The essential function of the AT element is confirmed through point mutations that decrease expression below the level of detection. CAE3 is one of three CA-rich elements (CAEs) required for the induction of SP60/cotC during development or in response to extracellular cAMP. The CAEs have differential affinities for a specific developmentally-induced nuclear activity (CAE1 > CAE2 >> CAE3). Here, we identify this activity as G-Box Binding Factor (GBF) and show that in vitro transcribed/translated GBF binds all three SP60/cotC CAEs in a sequence-specific manner. Previous studies have suggested that GBF mediates the induction of some prestalk genes, and these results demonstrate that it also has a specific role in prespore gene activation. ------------------------------------------------------------------- [End CSM News, volume 2, #23]