Dicty News Electronic Edition Volume 18, number 5 March 30, 2002 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============= Abstracts ============= A putative serpentine receptor gene tasA required for normal morphogenesis of primary stalk and branch structure in Polysphondylium pallidum. Yoshinori Kawabe, Hidekazu Kuwayama, Takahiro Morio, Hideko Urushihara, and Yoshimasa Tanaka Gene 285, 291-299 (2002) Abstract The fruiting body of Polysphondylium pallidum is composed of whorls of branches along the axis of a primary stalk. In the course of fruiting body formation, the interval between neighboring whorls and the number and the spacing of branches in a whorl are highly regulated. In this study, using REMI (restriction enzyme mediated integration) mutagenesis, we have obtained a mutant (strain M6226) with thicker and aberrant primary stalk. The gene responsible for the mutant phenotype, confirmed by homologous recombination, encodes an ORF with 383 aa residues (46.3 kDa) and was named tasA (thick and aberrant stalk A). TasA is highly homologous to Dictyostelium discoideum cAMP receptors. A tasA transcript is expressed strictly at the late aggregation stage. Cells expressing a tasA::gfp fusion DNA are localized at the posterior region of the primary sorogen where secondary sorogens and branches originate. This result indicates the existence of "prebranch" and "pretrunk" regions in P. pallidum instead of the prespore and prestalk regions in D. discoideum. The analyses of the gene disruptant and chimeric fruiting bodies also suggests that TasA affects the normal morphogenesis of the primary stalk and the process of cell differentiation into prebranch cells, but not into spore or stalk cells directly. ----------------------------------------------------------------------------- Expression and One Step Purification of a Developmentally Regulated Protein from Dictyostelium discoideum Muatasem Ubeidat and Charles L. Rutherford* Biology Department, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA Accepted for publication in: Protein Expression and Purification ABSTRACT To overexpress Dictyostelium 5NT, a 1506 bp fragment of the cDNA encoding the gene was cloned into a pET32c+ vector and expressed in the E. coli expression host BL21 CodonPlus"!(DE3)-RIL by Isopropyl-b-D-thiogalactoside (IPTG) induction. Maximum induction of insoluble recombinant protein was reached after incubation of the culture for 3 h with 1.0 mM IPTG. High level of 5NT expression was confirmed by SDS-PAGE and immunoblotting analysis. The recombinant 5NT was purified to homogeneity by a one step purification using continuous-elution electrophoresis. Ten mg of recombinant 5NT was purified per liter of growth medium. To achieve one of the goals of this study, polyclonal antibody against the recombinant 5NT was produced in a rabbit. We have shown previously by Northern analysis and reporter gene analysis that 5nt is developmentally regulated. In this report we used polyclonal antibody against the recombinant protein in Western analysis of membrane protein extracts from different developmental stages of Dictyostelium. 5NT protein levels were first detected at the tight aggregation stage of development. Thus, there is no significant delay between transcription and translation of 5nt. ----------------------------------------------------------------------------- A novel disintegrin domain protein affects early cell type specification and pattern formation in Dictyostelium. Timothy R. Varney, Hoa Ho, Chere Petty, and Daphne D. Blumberg Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 Development, in press SUMMARY The cellular slime mold, Dictyostelium discoideum is a non-metazoan organism yet we demonstrate here that a disintegrin domain containing protein, the product of the ampA gene, plays a role in cell type specification. Disintegrin domain containing proteins are involved in Notch signaling in Drosophila and C. elegans by an ectodomain shedding mechanism that depends on a metalloprotease domain. The Dictyostelium protein lacks a metalloprotease domain. Nonetheless, analysis of cell type specific reporter gene expression during development of the ampA null strain identifies patterning defects that define two distinct roles for the AmpA protein in specifying cell fate. In the absence of a functional ampA gene, cells prematurely specify as prespore cells. Prestalk cell differentiation and migration are delayed. Both of these defects can be rescued by the inclusion of 10% wild-type cells in the developing null mutant aggregates, indicating that the defect is non-cell autonomous. The ampA gene is also demonstrated to be necessary in a cell autonomous manner for the correct localization of anterior-like cells to the upper cup of the fruiting body. When derived from ampA null cells, the anterior-like cells are unable to localize to positions in the interior of the developing mounds. Wild-type cells can rescue defects in morphogenesis by substituting for null cells when they differentiate as anterior-like cells but they can not rescue the ability of ampA null cells to fill this role. Thus, in spite of its simpler structure, the Dictyostelium ampA protein carries out the same diversity of functions that have been observed for the ADAM and ADAMTS families in metazoans. ----------------------------------------------------------------------------- Nucleomorphin: A novel, acidic, nuclear calmodulin-binding protein from Dictyostelium that regulates nuclear number Michael A. Myre* and Danton H. O Day* *University of Toronto at Mississauga, Department of Zoology, 3359 Mississauga Rd. Rm. 3030, Mississauga, ON. L5L 1C6 Canada J. Biol. Chem., in press Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using 35S-recombinant calmodulin as a probe has revealed approximately three-dozen Ca2+-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression and subcellular localization of a gene encoding a novel CaMBP, we have called nucleomorphin, from D. discoideum. A ZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe (35S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular weight of 38.7 kDa, and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain, and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca2+-dependent manner. Amino-terminal fusion to the green fluorescent protein showed that GFP-NumA localized to the nucleus as distinct arc like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery but the number of nuclei in these cells was increased markedly compared to control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery, but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, this data presents the first identification of a member of the nucleoplasmin family as a calmodulin- binding protein and suggests nucleomorphin has a role in nuclear structure in Dictyostelium. ----------------------------------------------------------------------------- Dictyostelium EB1 is a genuine centrosomal component required for proper spindle formation Markus Rehberg and Ralph Grf* Adolf-Butenandt-Institut / Zellbiologie, Ludwig-Maximilians-Universitt Mnchen, Schillerstrasse 42, D-80336 Mnchen, Germany. Mol. Biol. Cell, in press EB1 proteins are ubiquitous microtubule-associated proteins involved in microtubule search-and-capture, regulation of microtubule dynamics, cell polarity and chromosome stability. We have cloned a complete cDNA of Dictyostelium EB1 (DdEB1), the largest known EB1 homologue (57 kDa). Immunofluorescence analysis and expression of a GFP-DdEB1 fusion protein revealed that DdEB1 localizes along microtubules, at microtubule tips, centrosomes and protruding pseudopods. During mitosis, it was found at the spindle, spindle poles and kinetochores. DdEB1 is the first EB1-homologue which is also a genuine centrosomal component, because it was localized at isolated centrosomes that are free of microtubules. Furthermore, centrosomal DdEB1 distribution was unaffected by nocodazole treatment. DdEB1 colocalized with DdCP224, the XMAP215 homologue, at microtubule tips, the centrosome and kinetochores. Furthermore, both proteins were part of the same cytosolic protein complex suggesting that they may act together in their functions. DdEB1 deletion mutants expressed as GFP or maltose-binding fusion proteins indicated that microtubule binding requires homo-oligomerization which is mediated by a coiled-coil domain. A DdEB1 null mutant was viable but retarded in prometaphase progression due to a defect in spindle formation. As spindle elongation was normal, DdEB1 seems to be required for the initiation of the outgrowth of spindle microtubules. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 5]