Dicty News Electronic Edition Volume 22, number 12 May 14, 2004 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 ============= Specificity of a Soluble UDP-Galactose:Fucoside a1,3Galactosyltransferase that Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium* Catherine Ketcham¤, Fei Wang¤, S. Z. Fisher¤, Altan Ercan||, Hanke van der Wel¤||, R. D. Locke¦, S. ud-Doulah.k¦, Khushi L. Matta¦ and Christopher M. West¤|| From the ¤Department of Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235 USA, the ¦Department of Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263 USA, and the ||Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 USA J. Biol. Chem., in press Skp1 is an adaptor-like protein in E3SCF-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Gala1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc-conjugates in the presence of Mn2+ and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Gala1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to a1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower Km and 35-fold higher Vmax compared to a simple a-fucoside, but this advantage was abolished by denaturation or alkylation of Cys-residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di- and tri-saccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a Vmax advantage, but still much less active than Skp1 itself owing to a Km difference. These findings indicate that aGalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Gala1,3Fuc-linkage is deficient in expressed mutant Skp1 proteins. Submitted by: Chris West [Christopher-West@ouhsc.edu] ----------------------------------------------------------------------------- Chemoattractant-Induced PI(3,4,5)P3 Accumulation is Spatially Amplified and Adapts, Independent of the Actin Cytoskeleton 1Chris Janetopoulos, 2Lan Ma, 1Peter N. Devreotes, and 2,3Pablo A. Iglesias 1Dept Cell Biology Johns Hopkins University School of Medicine 725 N. Wolfe St Baltimore, MD 21205 410-955-3225 410-614-9461 (Fax) 2Dept Electrical & Computer Engineering Johns Hopkins University 3400 N. Charles St. Baltimore, MD, 21218 410-516-6026 410-516-5566 (Fax) 3Corresponding Author Proceedings of the National Academy of Sciences USA, in press Experiments in amoebae and neutrophils have shown that local accumulations of PI(3,4,5)P3 mediate the ability of cells to migrate during gradient sensing. To define the nature of this response, we subjected D. discoideum cells to measurable temporal and spatial chemotactic inputs and analyzed the accumulation of PI(3,4,5)P3 on the membrane, as well as the recruitment of the enzymes PI3K and PTEN. In latrunculin-treated cells, spatial gradients elicited a PI(3,4,5)P3 response only on the front portion of the cell where the response increased more steeply than the gradient and did not depend on its absolute concentration. PI3K bound to the membrane only at the front, although it was less sharply localized than PI(3,4,5)P3. Membrane-bound PTEN was highest at the rear and varied inversely with receptor occupancy. The localization of PI(3,4,5)P3 was enhanced further in untreated, polarized cells, containing an intact cytoskeleton. Interestingly, the treated cells could respond to two independent gradients simultaneously, demonstrating that a response at the front does not necessarily inhibit the back. Combinations of temporal and spatial stimuli provided evidence of an inhibitory process and showed that a gradient generates a persistent steady-state response independent of previous history of exposure to chemoattractant. These results support a local excitation/global inhibition model and argue against other schemes proposed to explain directional sensing. Submitted by: Chris Janetopoulos [cjanetop@jhmi.edu] ----------------------------------------------------------------------------- A gene encoding prespore-cell-inducing factor in Dictyostelium discoideum Takefumi Kawata1,4,*, Manabu Nakagawa3, Nao Shimada1, Shigeru Fujii3 and Akiko A. Oohata2,4 1Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, JAPAN 2Biological Laboratory and 3Chemical Laboratory, Kansai Medical University, Hirakata, Osaka 573-1136, JAPAN 4Correspondence authors Dev. Growth Diff., in press Two factors that exist in conditioned medium (CM) of Dictyostelium discoideum induce amoebae to differentiate into prespore cells when they are incubated at a very low cell density in submerged monolayer culture. Previously, we purified one of them, a glycoprotein factor with an apparent molecular mass of 106 kDa, and we named it y factor (psi, prespore-inducing factor). Based on partial amino acid sequence of the purified y factor, we have isolated the corresponding cDNA clone, which is expressed maximally at the loose mound stage. The cDNA encodes a novel protein and the predicted molecular mass of the mature secreted protein is 60 kDa. Knockout mutant strains of the y factor gene, psiA-, were created by targeted integration. Although these mutant strains appear to develop normally, CM from these mutants showed reduced prespore-cell-inducing activity. Rescuing the mutant strains by expression of y factor under control of a constitutive promoter causes overproduction of y factor protein and CM from such cells showed a 20-fold higher level of prespore-cell-inducing activity than that from wild-type cells. Further, CM from parental cells induced prespore cell division, while that from psiA null strains showed no cell division inducing activity. Our results indicate that y factor protein is a novel type of growth factor that does not belong to any of the families of growth factor so far identified in animals. Submitted by: Takefumi Kawata [tkawata@bio.sci.toho-u.ac.jp] ----------------------------------------------------------------------------- Cell movements and mechanical force distribution during the migration of Dictyostelium slugs Jean-Paul Rieu 1,*, Catherine Barentin 1, Satoshi Sawai 2, Yasuo Maeda 3 and Yasuji Sawada 4 1 Laboratoire de Physique de la Matiere Condensee et des Nanostructures, Universite J. Biol. Phys., in press Migration of Dictyostelium discoideum slugs results from coordinated movement of its constituent cells. It is generally assumed that each cell contributes to the total motive force of the slug. However, the basic mechanisms by which mechanical forces (traction and resistive forces) are transmitted to the substrate, their magnitude and their location, are largely unknown. In this work, we performed detailed observations of cell movements by fluorescence microscopy using two-dimensional (2D) slugs. We show that 2D slugs share most of the properties of 3D ones. In particular, waves of movement propagate in long 2D slugs, and slug speed correlates with slug length as found in 3D slugs. We also present the first measurements of the distribution of forces exerted by 2D and 3D slugs using the elastic substrate method. Traction forces are mainly exerted in the central region of the slug. The large perpendicular forces around slug boundary and the existence of parallel resistive forces in the tip and/or the tail suggest an important role of the sheath in the transmission of forces to the substrate. Submitted by: Yasuo Maeda [ymaeda@mail.tains.tohoku.ac.jp] =============================================================================== [End Dicty News, volume 22, number 12]