dictyNews Electronic Edition Volume 30, number 7 February 22, 2008 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= Actin binding domains direct actin-binding proteins to different cytoskeletal locations Raymond W. Washington and David A. Knecht Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA BMC Cell Biol. 2008 Feb 13;9(1):10 Epub ahead of print Filamin (FLN) and non-muscle alpha-actinin are members of a family of F-actin cross-linking proteins that utilize Calponin Homology domains (CH-domain) for actin binding. Although these two proteins have been extensively characterized, little is known about what regulates their binding to F-actin filaments in the cell. We have constructed fusion proteins consisting of green fluorescent protein (GFP) with either the entire cross-linking protein or its actin-binding domain (ABD) and examined the localization of these fluorescent proteins in living cells under a variety of conditions. The full-length fusion proteins, but not the ABD’s complemented the defects of cells lacking both endogenous proteins indicating that they are functional. The localization patterns of filamin (GFP-FLN) and alpha-actinin (GFP-alphaA) were overlapping but distinct. GFP-FLN localized to the peripheral cell cortex as well as to new pseudopods of unpolarized cells, but was observed to localize to the rear of polarized cells during cAMP and folate chemotaxis. GFP-alphaA was enriched in new pseudopods and at the front of polarized cells, but in all cases was absent from the peripheral cortex. Although both proteins appear to be involved in macropinocytosis, the association time of the GFP-probes with the internalized macropinosome differed. Surprisingly, the localization of the GFP-actin-binding domain fusion proteins precisely reflected that of their respective full length constructs, indicating that the localization of the protein was determined by the actin-binding domain alone. When expressed in a cell line lacking both filamin and alpha-actinin, the probes maintain their distinct localization patterns suggesting that they are not functionally redundant. These observations strongly suggest that the regulation of the binding of these proteins to actin filaments is built into the actin-binding domains. We suggest that different actin binding domains have different affinities for F-actin filaments in functionally distinct regions of the cytoskeleton. Submitted by: David Knecht [david.knecht@uconn.edu] -------------------------------------------------------------------------------- DIF-1 induces the basal disc of the Dictyostelium fruiting body Tamao Saito1, Atsushi Kato1 and Robert R. Kay2 1 Department of Biological Sciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan 2 MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK Correspondence to TS (tasaito@sci.hokudai.ac.jp) and RRK (rrk@mrc-lmb.cam.ac.uk) Dev. Biol., in press The polyketide DIF-1 induces Dictyostelium amoebae to form stalk cells in culture. To better define its role in normal development, we examined the phenotype of a mutant blocking the first step of DIF-1 synthesis, which lacks both DIF-1 and its biosynthetic intermediate, dM-DIF-1 (des-methyl-DIF-1). Slugs of this polyketide synthase mutant (stlB-) are long and thin and rapidly break up, leaving an immotile prespore mass. They have ~30% fewer prestalk cells than their wild-type parent and lack a subset of anterior-like cells, which later form the outer basal disc. This structure is missing from the fruiting body, which perhaps in consequence initiates culmination along the substratum. The lower cup is rudimentary at best and the spore mass, lacking support, slips down the stalk. The dmtA- methyltransferase mutant, blocked in the last step of DIF-1 synthesis, resembles the stlB- mutant but has delayed tip formation and fewer prestalk-O cells. This difference may be due to accumulation of dM-DIF-1 in the dmtA- mutant, since dM-DIF-1 inhibits prestalk-O differentiation. Thus DIF-1 is required for slug migration and specifies the anterior-like cells forming the basal disc and much of the lower cup; significantly the DIF-1 biosynthetic pathway may supply a second signal – dM-DIF-1. Submitted by: Tamao Saito [tasaito@sci.hokudai.ac.jp] -------------------------------------------------------------------------------- Activation of Bacterial Ribonuclease P by Macrolides Chrisavgi Toumpeki, Anastassios Vourekas, Dimitra Kalavrizioti, Vassiliki Stamatopoulou and Denis Drainas Biochemistry, in press The effect of macrolide antibiotic spiramycin on RNase P holoenzyme and M1 RNA from Escherichia coli was investigated. Ribonuclease P (RNase P) is a ribozyme which is responsible for the maturation of 5´ termini of tRNA molecules. Spiramycin revealed a dose-dependent activation on pre-tRNA cleavage by E. coli RNase P holoenzyme and M1 RNA. The Ks and Vmax, as well as the Ks(app) and Vmax(app) values of RNase P holoenzyme and M1 RNA in the presence or absence of spiramycin, were calculated from primary and secondary kinetic plots. It was found that the activity status of RNase P holoenzyme and M1 RNA is improved by the presence of spiramycin 18 and 12 fold respectively. Primer extension analysis revealed that spiramycin induces a conformational change of the P10/11 structural element of M1 RNA which is involved in substrate recognition. Submitted by: Denis Drainas [Drainas@med.upatras.gr] ============================================================== [End dictyNews, volume 30, number 7]