Dicty News Electronic Edition Volume 24, number 10 April 15, 2005 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 Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Subsecond reorganization of the actin network in cell motility and chemotaxis Stefan Diez 1, Guenther Gerisch 2, Kurt Anderson 1, Annette Mueller-Taubenberger 2 and Till Bretschneider 2 1 Max-Planck-Institut fuer molekulare Zellbiologie und Genetik, D-01307 Dresden, Germany 2 Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany Proc. Natl. Acad. Sci. USA, in press Actin networks are continuously reorganized in cells that rapidly change their shape. Applying total internal reflection fluorescence (TIRF) microscopy at acquisition rates of 10 to 20 Hz, we measured an average growth rate of 3 mm x sec-1 for filamentous actin structures throughout the entire substrate-attached cortex of Dictyostelium cells. New filaments often proceed along pre-existing ones, resulting in bundle formation concurrent with filament growth. In cells that orientate in a gradient of chemoattractant, prominent assemblies of actin enriched in the Arp2/3 complex are inserted into the network, primarily at the base of filopods that point into the direction of the gradient. We propose that high turnover rates of actin filaments confer the plasticity to the cell cortex that is required for rapid accommodation to external stimuli. Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de] ----------------------------------------------------------------------------- A cysteine-rich extracellular protein containing a PA14 domain mediates quorum sensing in Dictyostelium Alexandra Kolbinger1, Tong Gao2, Debbie Brock2, Robin Ammann2 Axel Kisters1, Joseph Kellermann3, Diane Hatton2, Richard H. Gomer2*, and Birgit Wetterauer1, 1 Zoologisches Institut der Ludwig-Maximilians-UniversitŠt, Munich, Germany 2 Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, Rice University, Houston, Texas, USA 3 Max-Planck-Institut fŸr Biochemie, Martinsried, Germany Eukaryotic Cell, in press Much remains to be understood about quorum sensing factors that allow cells to sense their local density. Dictyostelium discoideum is a simple eukaryote that grows as single-celled amoebae and switches to multicellular development when food becomes limited. As the growing cells reach a high density, they begin expressing discoidin genes. The cells secrete an unknown factor, and at high cell densities the concomitant high levels of the factor induce discoidin expression. We report here the enrichment of Discoidin Inducing Complex (DIC), an ~400 kDa protein complex that induces discoidin expression during growth and development. Two proteins in the DIC preparation, DicA1 and DicB, were identified by sequencing of proteolytic digests. DicA1 and DicB were expressed in E. coli and tested for their ability to induce discoidin during growth and development. Recombinant DicB was unable to induce discoidin expression while recombinant DicA1 was able to induce discoidin expression. This suggests that DicA1 is an active component of DIC and indicates that posttranslational modification is dispensable for activity. DicA1 mRNA is expressed in vegetative and developing cells. The mature secreted form of DicA1 is 80 kDa, and has a 24 amino acid cysteine rich repeat that is similar to repeats in Dictyostelium proteins such as the extracellular matrix protein ecmB/PstA, the prespore cell inducing factor PSI, and the cAMP phosphodiesterase inhibitor PDI. Together, the data suggest that DicA1 is a component of a secreted quorum sensing signal regulating discoidin gene expression during Dictyostelium growth and development. Submitted by: Richard Gomer [richard@rice.edu] ----------------------------------------------------------------------------- Isolation and characterization of Dictyostelium thymidine kinase 1 as a calmodulin binding protein Danton H. OâDay, Munmun Chatterjee-Chakraborty, Stephanie Wagler and Michael A. Myre Department of Biology, University of Toronto at Mississauga, Mississauga, ON. Canada Biochemical Biophysical Research Communications, in press Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe (35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e-56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus two constructs (DdTKdeltaC36, DdTKdeltaC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTKdeltaN72) lacking the region failed to bind to CaM. Thus DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113 ± 0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2 ± 0.8%) and W13 (96.6 ± 0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues for research on this medically relevant protein. Submitted by: Danton H. O'Day [doday@utm.utoronto.ca] ----------------------------------------------------------------------------- An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium Michael A. Myre and Danton H. OâDay Department of Biology, University of Toronto at Mississauga, Mississauga, ON. Canada Biochemical Biophysical Research Communications, in press Nucleomorphin is a novel nuclear calmodulin (CaM) binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD (171EDVSRFIKGKLLQKQQKIYKDLERF195) containing both calcium-dependent binding motifs and an IQ-like motif for calciumöindependent binding. GFP constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues 48KKSYQDPEIIAHSRPRK64 that includes both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to 48EF49 abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the 48EF49 construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium. Submitted by: Danton H. O'Day [doday@utm.utoronto.ca] ----------------------------------------------------------------------------- Calmodulin-Mediated Signaling in Dictyostelium discoideum: CaMBOT Isolation and Characterization of the Novel Poly-Domain Protein Nucleomorphin and Other Calmodulin Binding Proteins Danton H. OâDay Department of Biology, University of Toronto at Mississauga, Mississauga, Ontario L5L 1C6, CANADA In: Progress in Cellular Signalling Research, Nova Science Publishers, Inc. New York, in press Calmodulin (CaM) is an essential protein in the model eukaryote Dictyostelium discoideum where it mediates numerous events including chemotaxis, gametogenesis, fertilization, and spore germination. Profiling using the CaM-binding overlay technique (CaMBOT) has revealed that well over four-dozen calcium-dependent and -independent CaM-binding proteins (CaMBPs) are present in Dictyostelium, some of which are linked to these CaM-mediated processes. Individual CaMBPs have been localized to specific sub-cellular locales and to show varying patterns of developmental expression. CaMBOT also is used to isolate cDNAs encoding CaMBPs from an expression library from mid-to-late multicellular development of Dictyostelium. Previously studied CaMBPs such as calcineurin A (CNA) and regulatory myosin light chain (RMLC) were identified in this way. Novel proteins were also identified. Nucleomorphin is a novel nuclear CaMBP, expressed as two main isoforms NumA1 and 2, which localizes to the nucleoplasmic periphery. NumA has at least one bipartite NLS (48KKSYQDPEIIAHSRPRK66) and a single CaM-binding domain (171EDVSRFIKGKLLQKQQKIYKDLERF195) that interacts with CaM in both a Ca2+-dependent and Ca2+-independent manner. NumA1 contains an extensive glu/asp or DEED repeat that regulates nuclear number. Yeast two hybrid and co-immunoprecipitation studies have identified calcium binding protein CBP4a as an interacting protein that binds to the DEED repeat. NumA2 also has a breast cancer C-terminus (BRCT) domain, two poly-N tracts plus a second, putative CaMBD. Proteins not previously known to be CaMBPs were also identified by CaMBOT probing of the Dictyostelium developmental expression library, including phosphoglycerate kinase, thymidine kinase and histone H1. Dictyostelium phosphoglycerate kinase (DdPGK) binds CaM in a Ca2+-dependent manner with CaM negatively regulating its activity in vitro. The identified CaMBD shows 80% identity with PGKs from diverse organisms and is localized adjacent to mutation sites underlying certain human diseases. Thymidine kinase (DdTK1) is a Ca2+-dependent CaMBP and its in vitro activity is slightly enhanced by CaM and inhibited by CaM antagonists. Histone H1 (DdH1) was isolated as a CaMBP and binds CaM in vitro but its mode of CaM-binding remains elusive. Each of the CaMBPs we have identified is discussed in terms of its individual roles as well as its potential interaction with other CaMBPs and other proteins in regulating specific CaM-dependent cellular processes including the cell cycle. Submitted by: Danton H. O'Day [doday@utm.utoronto.ca] ============================================================================== [End Dicty News, volume 24, number 10]