Dicty News Electronic Edition Volume 9, number 12 Novewmber 8, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== Migration and bidirectional phototaxis in Dictyostelium discoideum slugs lacking the actin cross-linking 120 kDa gelation factor. Eva Wallraff and Hans G. Wallraff J. Exp. Biol., in press Absract. Three mutant strains of Dictyostelium discoideum, lacking different actin-binding proteins, were tested for behavioural deficits in the multicellular pseudoplasmodium (slug) stage. Two strains, defective in the production of either a actinin (an actin cross-linker) or severin (an actin capping and severing protein), did not show changes in slug behaviour. Slugs of the mutant lacking another actin cross-linker, the 120 kDa gelation factor (ABP120), however, migrated shorter distances in darkness as well as in horizontally directed light. More remarkably, they migrated at an angle of approximately 45° to the left or right of the incident light, whereas wild-type slugs migrated on fairly straight paths towards the light. We discuss the hypothesis that this bidirectional oblique-angle phototaxis is due to changes in the optical properties of the pseudoplasmodia. Normally, in wild type slugs, a lens effect causes stronger stimulation on the side distal from the light. We propose that in the mutant the lens quality is reduced, so that at small angles between the slug axis and the rays of light the proximal side is stimulated more intensely. As a result, the intended symmetrical stimulation is achieved at a certain angle to the left or right of the incident light. We assume that the absence of ABP120 alters the shape of the lens and/or enhances internal light scattering via deterioration of intercellular coherence; however, intracellular attenuation of light remains an additional or alternative possibility. --------------------------------------------------------------------- Membrane Bending Modulus and Adhesion Energy of Wild-Type and Mutant Cells of Dictyostelium Lacking Talin or Cortexillins Rudolf Simson, Eva Wallraff, Jan Faix, Jens Niewöhner, Günther Gerisch and Erich Sackmann. Biophysical Journal, in press Abstract. We have employed an interferometric technique for the local measurement of bending modulus, membrane tension, and adhesion energy of motile cells adhering to a substrate. Wild-type and mutant cells of Dictyostelium discoideum were incubated in a flow chamber. The flow-induced deformation of a cell near to its adhesion area was determined by quantitative reflection interference contrast microscopy (RICM) and analyzed in terms of the elastic boundary conditions: equilibrium of tensions and bending moments at the contact line. This technique was employed to quantify changes caused by the lack of talin, a protein that couples the actin network to the plasma membrane, or by the lack of cortexillin I or II, two isoforms of the actin-bundling protein cortexillin. Cells lacking either cortexillin I or II exhibited reduced bending moduli of 95 and 160 kBT, respectively, as compared to 390 kBT, obtained for wild-type cells. No significant difference was found for the adhesion energies of wild-type and cortexillin mutant cells. In cells lacking talin not only a strongly reduced bending modulus of 70 kBT, but also a low adhesion energy of one fourth the value in wild-type cells was measured. ------------------------------------------------------------------------- Targeted gene disruption reveals a role of vacuolin B in the late endocytic pathway and exocytosis Nicole Jenne, Robert Rauchenberger, Ulrike Hacker, Thomas Kast and Markus Maniak* Abt. Zellbiologie, Max-Planck-Institut fur Biochemie, D-82152 Martinsried, Germany J Cell Science, in press. Summary: Cells of Dictyostelium discoideum take up fluid by macropinocytosis. The contents of macropinosomes are acidified and digested by lysosomal enzymes. Thereafter, endocytic marker progresses in an F-actin dependent mechanism from the acidic lysosomal phase to a neutral post-lysosomal phase. From the post-lysosomal compartment indigestible remnants are released by exocytosis. This compartment is characterised by two isoforms of vacuolin, A and B, which are encoded by different genes. Fusions of the vacuolin isoforms to the green fluorescent protein associate with the cytoplasmic side of post-lysosomal vacuoles in vivo. Vacuolin isoforms also localise to patches at the plasma membrane. Since vacuolins have no homologies to known proteins and do not contain domains of obvious function, we investigated their role by knocking out the genes separately. Although the sequences of vacuolins A and B are about 80% identical, only deletion of the vacuolin B gene results in a defect in the endocytic pathway; the vacuolin A knock-out appeared to be phenotypically normal. In vacuolin B- mutants endocytosis is normal, but the progression of fluid-phase marker from acidic to neutral pH is impaired. Furthermore, in the mutants post-lysosomal vacuoles are dramatically increased in size and accumulate endocytic marker, suggesting a role for vacuolin B in targeting the vacuole for exocytosis. ------------------------------------------------------------------------- Tdd-3, a transfer RNA gene-associated poly(A) retrotransposon from Dictyostelium discoideum Thomas Winckler (1), Christina Tschepke (1), Eugenio L. de Hostos (2), Andreas Jendretzke (1) and Theodor Dingermann (1) 1 Institut für Pharmazeutische Biologie, Universität Frankfurt/M. (Biozentrum), Marie-Curie-Strasse 9, D-60439 Frankfurt/M, Germany 2 Department of Biochemistry and Cell Biology, MS 140, Rice University, 6100 Main St., Houston TX, 77005-1892, U.S.A. Mol. Gen. Genetics, in press Summary: The full length 5218 base pair DNA sequence of the mobile genetic element Tdd-3 from Dictyostelium discoideum is described. Tdd-3 encodes two overlapping open reading frames (ORFs) flanked by nonredundant, untranslated regions. The deduced amino acid sequence of ORF2 is homologous to reverse transcriptases (RTs) encoded by the class of poly(A) retrotransposons. ORF2 also encodes a putative protein domain related to the family of apurinic/apyrimidinic (AP) endonucleases, whose retroelement-encoded homologs have recently been proposed to represent the integrase function of poly(A) retrotransposons. Comparison of several genomic Tdd-3 copies revealed that the element inserted orientation-specifically about 100 bp downstream of transfer RNA genes in the D. discoideum genome. The above properties of Tdd-3 suggest that the element is a tRNA gene-associated poly(A) retroelement present in the D. discoideum genome. Analysis of several cloned copy DNAs of Tdd-3-specific plus strand RNAs indicate that the element is transcribed and polyadenylated during the growth of D. discoideum cells. ------------------------------------------------------------------------- Dictyostelium discoideum Nuclear Plasmid Ddp5 is a Chimera Related to the Ddp1 and Ddp2 Plasmid Families, W. K. Rieben,Jr., C. M. Gonzales, S. T. Gonzales, K. J. Pilkington, H. Kiyosawa, J. E. Hughes and D. L. Welker Molecular Biology Program, Biology Department, Utah State University, Logan, Utah 84322-5305, Email: dlw@cc.usu.edu, Phone: 435-797-3552, Fax:435-797-1575 Genetics, in press. 14955 bp Dictyostelium discoideum nuclear plasmid Ddp5 contains six transcribed ORFs. One of these is related to the rep gene of the Ddp2 plasmid and the other five are related to genes present on the Ddp1 plasmid. The absence of a homolog of the Ddp1 G1 gene, coupled with the presence of the Ddp2 rep gene homolog and of a 1.6 kb inverted repeat analogous to the inverted repeats on members of the Ddp2 plasmid family, suggests that Ddp5 uses Ddp2-like replication and copy number control mechanisms and that it should be assigned to the Ddp2 plasmid family. Ddp5 carries genes homologous to the D1/D3 and D2 genes of the Ddp1 plasmid as well as the Ddp1 G2/G3/D4, G5/D6, and G6/G4/D5 genes. The products of the Ddp5 G2-like, G5-like and G6-like genes are likely to be transcription factors regulating the expression of themselves and of the other Ddp5 genes. The D1-like and D2-like genes may confer a selective advantage to plasmid-bearing cells, since they can be deleted from plasmid-based shuttle vectors with no apparent effect on vector maintenance. Updated sequence information for the Ddp1 G5/D6, D1/D3, and D2 genes as well as the Dmp1 and Dmp2 G5-like genes is presented. The locations of introns in the G5-like and D1-like genes of Ddp5 and in the homologous genes of the Ddp1, Dmp1, and Dmp2 plasmids were identified. These introns all have GU at the 5’ intron border and AG at the 3’ intron border, are short (59 to 71 nucleotides), and are AT rich. A conserved HHCC domain was identified in the G5 proteins; this is a putative zinc binding domain and may be involved in protein-DNA interaction. ----------------------------------------------------------------------- Dictyostelium discoideum Nuclear Plasmid Ddp6 is a New Member of the Ddp2 Plasmid Family Shammat, I., C. M. Gonzales, and D. L. Welker Molecular Biology Program, Biology Department, Utah State University, Logan, Utah 84322-5305, Email: dlw@cc.usu.edu, Phone: 435-797-3552, Fax:435-797-1575 Current Genetics, in press Summary, Ddp6 is a high copy number, circular plasmid found in the nucleus of the simple eukaryote Dictyostelium discoideum wild isolate NC47.2. The complete nucleotide sequence, 5257-bp, shows that Ddp6 has a structure similar to that of other members of the Ddp2 plasmid family: a single long 2.8 kb open reading frame (rep gene) and an inverted repeat containing a pair of 654-bp elements. A single constitutively- expressed 3.3 kb transcript of the rep gene was detected in RNA prepared from vegetative and developmental cells. Maintenance assays revealed that sequences within the inverted repeat and the intact Ddp6 ORF are essential for maintenance of the plasmid. Mutation of the inverted repeat or of the rep gene lowered plasmid copy number but did not affect autonomous replication of shuttle vector constructs. Comparisons of the predicted protein products of the rep genes of five members of the Ddp2 plasmid family identified a set of ten conserved features distributed throughout the peptides. ------------------------------------------------------------------------- Coinsertional replication is responsible for tandem multimer formation during plasmid integration into the Dictyostelium genome. C. Barth, D.J. Fraser and P.R. Fisher School of Microbiology, La Trobe University, Bundoora VIC 3083, Australia. Plasmid. In Press. We investigated the establishment of integrating transformation vectors in the genome of Dictyostelium discoideum to gain insight into the formation of the plasmid insertions and to investigate the conditions that determine the number of plasmid copies present in such insertions. Transformation vectors conferring resistance to neomycin and/or blasticidin were introduced into the cell as a calcium phosphate coprecipitate or by electroporation. The integration of the plasmid DNA was based on either recombinational integration of plasmids (RIP) or restriction enzyme-mediated integration (REMI). The genomic DNA of the resulting transformants was examined by Southern blot analysis of pulsed field gels and by the recently published method of direct electroporation into E. coli (Barth et al., 1996). The number of insertion sites was found to be dependent on the transformation method used, and the minimum number of plasmid copies per insertion site required for resistance depended on the type and the concentration of the selective drug. Cotransformation studies revealed a strictly homogeneous composition of vector multimers from any given insertion site. This suggests that multimers arise by coinsertional replication of a single plasmid monomer, rather than by subsequent additional insertion events involving homologous recombination. The multimerization of the integrated vector only occurred when the insertion was established by homologous recombination. Moreover, the number of plasmid copies appeared to be random, was established at the time of the transformation and did not change with subsequent alterations to the selection regime. ------------------------------------------------------------------------- In vitro reconstituted Dictyostelium discoideum early endosome fusion is regulated by Rab7 but proceeds in the absence of ATP-Mg2+ from the bulk solution. Olivier Laurent, Franz Bruckert, Celine Adessi* and Michel Satre Laboratoire de Biochimie et Biophysique des Systemes Integres, UMR 314 CEA-CNRS, and *Laboratoire de Chimie des Proteines, CEA-Grenoble, France J. Biol. Chem., in press. Abstract: We characterised the in vitro fusion of endosomal compartments from Dictyostelium discoideum. Fusion activity was restricted to early compartments, was dependent on cytosolic proteins and was activated by GTP and GTPgammaS. This stimulation suggests the involvement of a small G-protein, that we propose to be Rab7 on the basis of the strong inhibitory effect of anti-Rab7 antibodies. Noteworthy, in the presence of GTPgammaS, the concentration of ATP-Mg2+ could be reduced to less than 1 nM without loss of fusion activity. Under these conditions, competing residual ATP with ATPgammaS-Mg2+ also failed to inhibit endosome fusion. The presence of an ATP-depleting system alone blocked fusion probably because endogenous GTP was removed by coupling through NDP kinase. Moreover, whether ATP was present or not, GTPgammaS-activated fusion was equally sensitive to anti-Rab7 antibodies or N-ethylmaleimide and was restricted to early compartments. These results show that soluble ATP-Mg2+ is not needed for endosome fusion. Since homotypic fusion of endosomes in Dictyostelium discoideum has been shown to depend on the ATPase N-ethylmaleimide Sensitive Factor (Lenhard et al. 1992, J.Biol. Chem. 267 :1896-903), the nucleotide exchange on NSF must take place before GTPgammaS activation in this system. ------------------------------------------------------------------------- Identification of the Dictyostelium discoideum homolog of the N-ethylmaleimide-sensitive fusion protein Marianne Weidenhaupt, Franz Bruckert and Michel Satre Laboratoire de Biochimie et Biophysique des Systemes Integres, UMR 314 CEA-CNRS, CEA-Grenoble, France Gene, in press. Abstract: The N-ethylmaleimide-sensitive fusion protein (NSF) is required for vesicular membrane fusion in multiple cellular functions. We have cloned a cDNA encoding the Dictyostelium discoideum homolog of the NSF protein. This cDNA hybridizes with a single fragment in Southern blots suggesting that NSF is encoded by a single gene in the amoeba. It is expressed constitutively during vegetative growth and throughout the differentiation cycle. The encoded gene product comprises 738 aa with a predicted molecular mass of 82 kDa. It shows the characteristic three domain structure of NSF proteins. A more divergent amino-terminal part is followed by two highly conserved ATP-binding domains featuring Walker A and B signature sequences. The D. discoideum protein presents an overall aa sequence identity of 44% when compared to known NSF homologs. The monoclonal antibody 2E5 directed against Cricetellus griseus NSF recognizes a protein with a molecular weigth of approximatively 80,000 in a D. discoideum crude extract and the recombinant D. discoideum His6-NSF expressed in Escherichia coli. ------------------------------------------------------------------------- [End Dicty News, volume 9, number 12]