dictyNews Electronic Edition Volume 28, number 8 April 6, 2007 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 ========= The role of SP65 in assembly of the Dictyostelium spore coat Talibah Metcalf1, Hanke van der Wel1, Ricardo Escalante2, Leandro Sastre2 and Christopher M. West1 1Dept. of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 USA; 2Instituto de Investigaciones Biomedicas Alberto Sols. C.S.I.C./U.A.M., Arturo Duperier 4, 28029 Madrid, Spain Eukaryotic Cell, in press Like the cyst walls of other protists, the spore coat of Dictyostelium is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with GFP, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially, and that the complex contributes an essential function to outer layer architecture and function. Submitted by: Chris West [Cwest2@ouhsc.edu] -------------------------------------------------------------------------------- Chemotaxis in the absence of PIP3 gradients Oliver Hoeller and Robert R. Kay MRC Laboratory of Molecular Biology, Hills Rd, Cambridge, CB2 2QH, UK. Current Biology (in press) Chemotaxing neutrophils and Dictyostelium amoebae produce gradients of the signalling lipid PI(3,4,5)P3 (PIP3) in their plasma membranes [1-3], which are orientated with the external chemotactic gradient and have been proposed to act as an internal compass, guiding movement of the cell [4, 5]. Evidence for and against this idea exists, but in all cases it depends on the use of inhibitors or gene knockouts, which may only incompletely abolish the PIP3 gradient. We have created a multiple gene knockout strain in Dictyostelium lacking all five type-1 phosphoinositide 3-kinases encoded in the genome and the PTEN phosphatase and have thus removed all known ways for chemoattractant to produce PIP3 gradients in the plasma membrane. The resulting sextuple mutant is able to chemotax to cyclic-AMP with near wild-type efficiency and to trigger actin polymerization without apparent defect. There is however, a consistent defect in movement speed in chemotaxis and especially random movement. This work shows that polarization of membrane PIP3 is not necessary for accurate chemotaxis, but can affect cell speed. A signalling pathway from receptor to cytoskeleton must exist which is able to guide cells independently of polarized PIP3 and type-1 phosphoinositide 3-kinases. Submitted by: Rob Kay [rrk@mrc-lmb.cam.ac.uk] -------------------------------------------------------------------------------- Development of soil amoeba Dictyostelium discoideum as an expression system for recombinant human erythropoietin Bhavesh Vats, Harish Padh* *Corresponding author B. V. Patel Pharmaceutical Education and Research Development Centre, Thaltej- Gandhinagar Highway, Thaltej, Ahmedabad 380054, India. Telephone No.: +91-79-2743 9375 Fax No.: +91-79- 2745 0449 World Journal of Microbiology and Biotechnology, in press EPO is the block buster biopharmaceutical product presently being produced by recombinant DNA technology from mammalian cell lines. Other available expression systems have not been useful in producing this protein due to the requirement of N- glycosylation for in-vivo activity. In order to develop an alternative expression system, the human epo gene was expressed in the cellular slime mold Dictyostelium discoideum. The 2.43 kbp epo gene from the mammalian expression vector was cloned in the Dictyostelium expression cassette and used to transform cells. Positive clones were selected on the basis of antibiotic resistance. The clones were screened for the presence of the transgene. The copies of the gene inserted in the genome were identified and the transcript too was ascertained. The protein was identified by immuno-blotting and appears to be glycosylated though differently from that of humans or CHO cell lines. Submitted by: Harish Padh [hpadh@yahoo.com] -------------------------------------------------------------------------------- Evidence that non-coding RNA dutA is a multicopy suppressor of Dictyostelium STAT protein, Dd-STATa Nao Shimada and Takefumi Kawata* Department of Biology, Faculty of Science, Toho University, Funabashi, Japan *Corresponding author. Mailing address: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. Phone: 81-47-472-5156 FAX: 81-47-472-5156 E-mail: tkawata@bio.sci.toho-u.ac.jp Eukaryotic Cell, in press. Dd-STATa, a Dictyostelium homologue of metazoan STAT transcription factors, is necessary for culmination. We created a mutant strain with partial Dd-STATa activity, and used it to screen for unlinked suppressor genes. We screened approximately 450,000 clones from a slug-stage cDNA library for their ability to rescue the culmination defect when overexpressed. There were 12 multicopy suppressors of Dd-STATa, of which 4 encode segments of a known non-coding RNA, dutA. Expression of dutA is specific to the pstA zone, the region where Dd-STATais activated. In suppressed strains the expression patterns of several putative Dd-STATa target genes become similar to the wild-type strain. In addition, the amount of the tyrosine phosphorylated form of Dd-STATa is significantly increased in the suppressed strain. These results indicate that partial copies of dutA may act upstream of Dd-STATa to regulate tyrosine phosphorylation by an unknownmechanism. Submitted by: Takefumi Kawata [tkawata@bio.sci.toho-u.ac.jp] ============================================================ [End dictyNews, volume 28, number 8]