Dicty News Electronic Edition Volume 10, number 1 January 3, 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" A Happy and Productive New Year to everyone! =========== Abstracts =========== The Signals for Starvation Response are Transduced through Elevated [Ca2+]i in Dictyostelium cells YUKIKO TANAKA*, RYU ITAKURA, AIKO AMAGAI and YASUO MAEDA Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-77, Japan Exp. Cell. Res., in press. Abstract: The mechanism by which cells recognize starvation to allow subsequent cellular development was analyzed using Dictyostelium discoideum, with special emphasis on Ca2+ as a crucial signal transducer in intra- and inter-cellular communications. As was expected, the cytosolic Ca2+- concentration ([Ca2+]i) in aequorin-expressing cells (RHI76 derived from D. discoideum Ax-3) was temporarily increased, when 3-5 uM thapsigargin (Tg), a specific inhibitor of the Ca2+-ATPase was added into the cells incubated in semi-starvation medium (SS-medium: 1 volume of growth medium plus 8 volumes either of 20 mM Na2/K-phosphate buffer (pH 6.2) or of Bonner's salt solution (BSS)). Essentially the same result was obtained by the application of 5 uM nigericin (Ng), an acid ionophore to cells under the semi-starved condition. Here it is of interest to note that in the SS-medium Tg and Ng are capable of enhancing cell differentiation as exemplified well by the earlier acquisition of chemotactic response to cAMP, possibly inducing the starvation response through the [Ca2+]i increase. From Western blot analysis of phosphotyrosine (pTyr)-containing proteins using anti-pTyr antibody, it was found that the pTyr-phosphorylation levels of 97 kDa, 80 kDa, and 45 kDa proteins increase specifically in response to starvation. Interestingly, Tg and Ng induced such a change of the 80 kDa protein in the cells incubated in the SS-medium. Taken together these results strongly suggest that the temporal increase of [Ca2+]i may be a matter of importance for signal transduction coupled with starvation response. ------------------------------------------------------------------------- Co-expression of a constitutively active plasma membrane calcium pump with GFP identifies roles for intracellular calcium in controlling cell sorting during morphogenesis in Dictyostelium Andrew B. Cubitt1, Indira Reddy2, Susan Lee1, James G. McNally2, and Richard A. Firtel1 1Department of Biology, Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634 and 2Department of Biology, Washington University, St. Louis, MO 63130 Develop. Biol. in press ABSTRACT To examine the potential role of calcium in regulating Dictyostelium development, we reduced free cytosolic and total cell Ca2+ in Dictyostelium cells by expressing a constitutively active form of a human erythrocyte plasma membrane calcium pump. The pump-expressing cells lacked a thapsigargin-mediated increase in cytoplasmic calcium, consistent with a reduced level of total cellular Ca2+. During aggregation, the cells initially form large number of aggregation centers, many of which coalesced to form mounds that were smaller than those of wild-type cells, and the cells did not exhibit the normal formation of elongated aggregation streams. The majority of the mounds either arrested at this stage with the formation of small protrusions or formed very aberrant finger-like structures, indicating an essential role for cellular calcium in morphogenesis. We used pump and wild-type cells differentially-labelled by expressing different wavelength (green and blue) forms of green fluorescent protein and three-dimensional (3-D) reconstruction of serial fluorescent imaging to visualize the movement of pump and wild-type cells within the aggregate. The results showed that the pump cells exhibited very aberrant cell movement and sorting within the forming mound, suggesting that the reduced cytosolic calcium is affecting movement required for tip formation. When allowed to form chimeric organisms with wild-type cells, pump cells preferentially localized to two bands, one of which was at the prestalk/prespore boundary and the other in the very posterior of the organism, also suggesting that pump cells are unable to properly sort. Expression of the calcium pump had little effect on the induction of prestalk- or prespore-specific genes, whereas extended treatment with EGTA blocked induction of both classes of cell-type- specific genes. Our results suggest a role for intracellular Ca2+ in controlling cell sorting and morphogenesis in Dictyostelium. ------------------------------------------------------------------------- Simultaneous detection of two GFP spectral mutants during in-vivo confocal microscopy of migrating Dictyostelium cells Timo Zimmermann & Florian Siegert * Zoologisches Institut, Universität München, Luisenstr. 14, 80333 München, Germany BioTechniques, in press Abstract A method is described that allows simultaneous measurement of two spectrally distinguishable green fluorescent proteins (GFP) mutants with a confocal microscope. In contrast to previously described methods, neither UV excitation nor repetition of scans are required. Thus the method is well suited to the long-time observation of living cells in 3D microscopy and time series recording, as demonstrated with GFP expressing Dictyostelium discoideum cells. ------------------------------------------------------------------------- Dictyostelium discoideum fatty acid amidase II (FAA II) has de-acylase activity on Rhizobium nodulation factors. Peggy J. Sutherland1#, Andrea E. Tobin1#, Charles L. Rutherford2 and Neil P.J. Price1* 1Department of Chemistry at SUNY College of Environmental Science and Forestry, Syracuse, NY 13210. 2Department of Biology Virginia Tech, Blacksburg Virginia 24061 Abstract Dictyostelium discoideum (Amoebidae) secretes cell lysing enzymes; esterases, amidases and glycosylases, many of which degrade soil bacteria to provide a source of nutrients. Two of these enzymes, fatty acyl amidase I (FAA I) and fatty acyl amidase II (FAA II) act sequentially on the N-linked long chain acyl groups of lipid A, the lipid anchor of gram negative bacterial lipopolysaccharide (LPS). FAA I selectively hydrolyzes the 3-hydroxymyristroyl group N-linked to the proximal glucosamine residue of de-O-acylated lipid A. Substrate specificity for FAA II is less selective, but does require prior de-N-acylation of the proximal sugar, i.e. the bis-N-acylated lipid A is not a substrate. We have synthesized a 14C-labeled substrate analog for FAA II, and used this in a novel assay to monitor its purification. Inhibitory studies indicate that FAA II is not a serine protease but may have a catalytic mechanism similar to metalloprotein de-N-acetylases such as LpxC. Interestingly, Rhizobial Nod factor signal oligosaccharides that induce root nodules on leguminous plants have many of the structural requirements for substrate recognition by FAA II. In vitro evidence indicate that R. fredii Nod factors are selectively de-N-acylated by purified FAA II suggesting that the enzyme may reduce the N2-fixing efficiency of Rhizobium-legume symbioses. In contrast, N-methylated Nod factors from transgenic R. fredii carrying the rhizobial nodS gene were resistant to FAA II, suggesting a mechanism by which Nod factors may be protected from enzymatic de-N-acylation. Since FAA II and Nod factors are both secreted, and Nod factors that lack the N-acyl group are unable to induce nodules, dictyostelial FAA II may decrease the efficiency of symbiotic nitrogen fixation in the environment by reducing the available biologically active nodule inducer signal. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 1]