CSM News Electronic Edition Volume 8, number 8 April 5, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== A Green fluorescent protein-actin fusion protein dominantly inhibits cytokinesis, cell spreading, and locomotion in Dictyostelium Hiroyuki Aizawa, Masazumi Sameshima, and Ichiro Yahara. Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113, Japan. Cell Structure and Function (Japan), in press. ABSTRACT We transformed Dictyostelium discoideum cells by a vector for expression of a chimerical fusion protein consisting of Aequorea Victoria green fluorescent protein (GFP) and D. discoideum actin at its amino- and carboxy-terminal, respectively. The amount of expressed GFP-actin was about 3% of total actin molecules in the transformed cells. The expression of GFP-actin in D. discoideum completely inhibited cytokinesis in suspension culture. The expression decreased the rate of random cell locomotion to about a half of that of control cells. The expression also caused the cells round up. These phenotypic observation suggested that GFP-actin acts as a dominant negative form of actin in the cells. The rounding up by expression of GFP-actin was suppressed by genetical elimination of myosin II heavy chain. This result suggested myosin II is necessary for the rounding up of GFP-actin expressing cells. GFP-actin constructed cortical actin filament architectures together with intrinsic actin in the cells. Purified GFP-actin polymerized and de-polymerized repetitively according to the solution conditions in vitro. The critical concentration of GFP-actin for polymerization is the same as that of actin. The GFP-actin filaments was able to bind to coverglass surfaces coated with myosin head fragments. However, the GFP-actin filaments did not slide at all on the coverglass by addition of ATP. This indicates that the GFP-actin filaments form rigor complex with myosin II in vitro even in the presence of ATP. The formation of rigor complex may cause the cells round up. -------------------------------------------------------------------- Mitochondrial mutations impair signal transduction in Dictyostelium discoideum. Z. Wilczynska, C. Barth and P.R. Fisher. School of Microbiology, La Trobe University, Bundoora 3083, VIC, Australia. Biochem. Biophys. Res. Comm. In Press. Subpopulations of mutant mitochondria contribute to degenerative processes associated with ageing and are characteristic of many mitochondrial diseases. We have created mutants carrying plasmid insertions in the Dictyostelium mitochondrial genome. Phototaxis and thermotaxis in these mutants are more sensitive than growth and division to the presence of a subpopulation of defective mitochondria. This could result from direct impairment of a mitochondrial role in signal transduction, or indirectly from the effects of energy depletion. An obvious inference is that, directly or indirectly, signal transduction may be the first cellular activity to be compromised by the accumulation of defective mitochondria in age-related tissue dysfunction and in mitochondrial disease. --------------------------------------------------------------------- [End CSM News, volume 8, number 8] From r-chisholm@nwu.edu Thu Apr 10 18:14 CDT 1997 Return-Path: Received: from casbah.acns.nwu.edu by worms.cmb.nwu.edu (5.x/SMI-SVR4) id AA09628; Thu, 10 Apr 1997 18:14:42 -0500 Received: from dicty.cmb.nwu.edu by casbah.acns.nwu.edu with SMTP (1.40.112.8/20.4) id AA220694642; Thu, 10 Apr 1997 18:24:02 -0500 Received: by dicty.cmb.nwu.edu with Microsoft Mail id <01BC45DB.F754DEA0@dicty.cmb.nwu.edu>; Thu, 10 Apr 1997 18:21:10 -0500 Message-Id: <01BC45DB.F754DEA0@dicty.cmb.nwu.edu> From: "Rex L. Chisholm" To: "'csm-news@worms.cmb.nwu.edu'" Subject: FW: abstract for newsletter Date: Thu, 10 Apr 1997 17:53:24 -0500 Mime-Version: 1.0 Content-Transfer-Encoding: quoted-printable Content-Type: text/plain; charset="iso-8859-1" Content-Length: 2758 Status: RO ________________________________________ Rex L. Chisholm, Ph.D. (r-chisholm@nwu.edu) Cell and Molecular Biology, W129 Northwestern University Medical School 303 E. Chicago Ave. Chicago, IL 60611 Phone: 312-503-4151 Fax: 312-503-5994 -----Original Message----- From: wloomis@ucsd.edu [SMTP:wloomis@ucsd.edu] Sent: Thursday, April 10, 1997 3:18 PM To: R-CHISHOLM@nwu.edu Subject: abstract for newsletter Dear Rex, Here is a paper that has been accepted in PNAS fore your e-mail. Bill ________________________________________________________________________ Expression of an Arabidopsis Plasma Membrane Aquaporin in Dictyostelium Results in Hypo-osmotic Sensitivity and Developmental Abnormalities Fran=E7ois Chaumont, William F. Loomis and Maarten J. Chrispeels Department of Biology, University of California, San Diego, La Jolla, CA (Proc. Natl. Acad. Sci. in press) The rd28 gene of Arabidopsis thaliana encodes a water channel protein or aquaporin of the plasma membrane. We generated a construct in which transcription of the rd28 cDNA is controlled by the Dictyostelium actin = 15 regulatory region and transformed it into cells of Dictyostelium discoideum. Transformants grew well in rich medium and contained RD28 protein in their plasma membrane. However, when shifted to low ionic strength buffer, cells expressing rd28 rapidly swelled and burst = indicating that the plant aquaporin allowed water to enter rapidly in these = amoebae. Since the rate of osmotic lysis was a function of the osmotic pressure = of the buffer, this property could be used to assay suspected aquaporins. = We also selected transformants carrying a construct in which the regulatory region of the prespore gene cotB was ligated to the rd28 cDNA. These transformants accumulated rd28 mRNA uniquely in prespore cells. When developed on low ionic buffer, they aggregated and formed normally proportioned slugs but failed to form normal fruiting bodies. The = number of refractile spores was reduced 20-fold and the stalks were short after development on filters. The consequences of expressing RD28 in prespore cells could be partially overcome by adding 100 mM sorbitol to the = buffer to increase the osmolarity. Under these conditions, cotB::rd28 transformants formed more normal fruiting bodies and the number of = viable spores was slightly increased. Since prespore cells have to shrink and dehydrate to form spores, it was not unexpected that expression of an aquaporin would disrupt this process, but it was surprising to find that stalk differentiation was also affected by expression of rd28 in = prespore cells. It appears that osmotic stress on prespore cells alters their ability to signal terminal differentiation in prestalk cells. From fucini@biochem.mpg.de Tue Apr 8 12:18 CDT 1997 Return-Path: Received: from libelle.biochem.mpg.de (libelle2.biochem.mpg.de) by worms.cmb.nwu.edu (5.x/SMI-SVR4) id AA05248; Tue, 8 Apr 1997 12:18:55 -0500 Received: from pcge11.biochem.mpg.de by libelle.biochem.mpg.de via SMTP (940816.SGI.8.6.9/940406.SGI.AUTO) for id TAA09140; Tue, 8 Apr 1997 19:28:09 +0200 Message-Id: <1.5.4.32.19970408172810.88168cd0@vms.biochem.mpg.de> X-Sender: fucini@vms.biochem.mpg.de X-Mailer: Windows Eudora Light Version 1.5.4 (32) Mime-Version: 1.0 Date: Tue, 08 Apr 1997 19:28:10 +0200 To: CSM-News@worms.cmb.nwu.edu From: Paola Fucini Subject: gelation factor Content-Type: text/plain; charset="us-ascii" Content-Length: 1805 Status: RO Dear Dr. Chrisholm, attached we are sendin the abstract of our recent paper appeared in Nature Structural Biology on march 1997, volume 4 number 3, pp. 223-230: The repeating segements of the F-actin cross-linking gelation factor (ABP-120) have an immunoglobulin-like fold. Paola Fucini, Christian Renner, Christoph Herberhold, Angelika A. Noegel and Tad A. Holak. Max Planck Institute for Biochemistry, D-82152 Martinsried, F.R.G. Abstract: The 120,000 Mr gelation factor and a-actinin are the most abundant F-actin cross-linking proteins in Dictyostelium discoideum. Both molecules are rod-shaped homodimers. Each monomer chain is comprised of an actin-binding domain and a rod domain. The rod domain of the gelation factor consists of six 100-residue repetitive segments with high internal homology. We have now determined the three-dimensional structure of segment 4 of the rod domain of the gelation factor from D. discoideum using NMR spectroscopy. The segment consists of seven -sheets arranged in an immunoglobulin-like (Ig) fold. This is completely different from the a-actinin rod domain which consists of four spectrin-like -helical segments. The gelation factor is the first example of an Ig-fold found in an actin-binding protein. Two highly homologous actin-binding proteins from human with similar sequences to the gelation factor, filamin and a 280,000 Mr actin-binding protein (ABP-280), share conserved residues that form the core of the gelation factor repetitive segment structure. Thus, the segment 4 structure should be common to this subfamily of the spectrin superfamily. The structure of segment 4 together with previously published electron microscopy data, provide an explanation for the dimerization of the whole gelation factor molecule. Thank you, Fucini and co.