Dicty News Electronic Edition Volume 13, number 8 25 September, 1999 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 ============= [Editors note: The following abstract appeared in the last issue. Due to problems with translation of embedded symbols, the units associated with the forces appeared incorrectly. A corrected version of the abstract is reprinted in its entirety below.] Migration Forces in Dictyostelium Measured by Centrifuge DIC Microscopy Yoshio Fukui 1, Taro Q. P. Uyeda2, Chikako Kitayama 2, and Shinya Inoué3 1 Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611-3008. 2 Biomolecular Research Group, National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki 305-8562, Japan. 3 Marine Biological Laboratory, Woods Hole, MA 02543-1015. Biological Bulletin, in press Abstract Amoeboid locomotion represents an important biological activity involved in cell growth and development . Forces that underlie movement of the giant amoeba, Chaos chaos, have been estimated to be 1.5 x 10e2 pN/micrometer2 as measured by Kamiya’s double chamber method. For a slime mold, Dictyostelium discoideum, the forces of cell locomotion have been unknown, but the cortex resists poking with a microneedle (cortical tension) at 4.1 x 10e3 pN/micrometer2 (Pasternak et al., 1989) . By micropipette aspiration, the cortical tension of D. discoideum has been measured as 1.55 x 10e3 pN/micrometer2 (Gerald et al., 1998). In the present study, we have determined the migration stalling forces of D. discoideum by using a Centrifuge Polarizing Microscope (CPM) equipped with DIC optics (Goda et al., 1998). The results demonstrated that individual wild type (NC4) amoebae can crawl centripetally on a glass surface in resistance to gravitational forces larger than 11,465 x g. NC4 amoebae can also undergo normal cytokinesis at forces of at least 8,376 x g. (Addendum) Our results also showed that the gravitational forces equivalent to the migration stall forces for NC4, AX3, HS1 (mhcA-: Ruppel et al., 1994), and A5 (myoA-/myoB-/mhcA-: Kitayama et al., 1998) are, respectively, >2.77 x 10e3 pN, 1.08 x 10e3 pN, 0.28 x 10e3 pN, and 0.30 x 103 pN. A preprint will become available at http://pubweb.acns.nwu.edu/ ~yoshifk/fukui.html. ---------------------------------------------------------------------------- Molecular mechanisms of membrane trafficking. What do we learn from Dictyostelium discoideum ? Eva M. Neuhaus and Thierry Soldati# Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany Protist, in press Summary Thanks to increasing interest, understanding of the molecular mechanisms underlying intracellular membrane transport is truly emerging. Even more rapid advance is expected since the recognition that efforts to unravel both membrane traffic and cytoskeleton need to be concerted. The two main membrane traffic pathways are the biosynthetic/secretory route, where transport occurs from the ER via the Golgi apparatus to the plasma membrane and the endocytic route involving the flow from the plasma membrane to the degradative compartment. These two pathways intersect at many compartments, both comprise multiple transport steps and both involve efficient sorting of membrane and soluble proteins. Here, we review recent studies on the dissection of endocytic trafficking in Dictyostelium discoideum and integrate the information in the light of knowledge acquired from other systems. ---------------------------------------------------------------------------- PAKa, a Putative PAK Family Member, is Required for Cytokinesis and the Regulation of the Cytoskeleton in Dictyostelium Cells during Chemotaxis Chang Y. Chung and Richard A. Firtel Department of Biology Center for Molecular Genetics University of California, San Diego 9500 Gilman Drive La Jolla, CA 92093-0634 J. of Cell Biology, in press. Abstract We have identified a Dictyostelium gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long N-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain. PAKa co-localizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its N-terminal domain. paka null cells are defective in completing cytokinesis in suspension. PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells. paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly. PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling. However, PAKa does not phosphorylate myosin II. We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase. ---------------------------------------------------------------------------- Promoter analysis of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum Naohisa Takaoka 1, Masashi Fukuzawa 2, Tamao Saito 1, Takehisa Sakaitani 1 and Hiroshi Ochiai 1 1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810 Japan, 2 Wellcome Trust Building, Department of Anatomy and Physiology, University of Dundee, Dow Street, Dundee, DD1 4HN, UK Biochem. Biophys. Acta, in press Abstract We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position - 47 to - 37 and to an initiator (Inr, PyPyCAPyPyPyPy) at position - 3 to + 5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the ß-galactosidase reporter gene in transformed cells; also was the difference of the activity between the growth conditions compared. The results indicated that there are two positive vegetative regulatory elements extending between -187 and -62 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium. ---------------------------------------------------------------------------- Identification of ?5-fatty acid desaturase from the cellular slime mold Dictyostelium discoideum Tamao SAITO and Hiroshi OCHIAI Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810 Japan Eur. J. Biochem. in press Abstract cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the first found ?5-desaturase of Mortierella alpina. We have confirmed its function as ?5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of ?5-desaturated products. This is the first report about the fatty acid desaturase in the cellular slime molds. ---------------------------------------------------------------------------- A putative receptor mediating cell-density sensing in Dictyostelium William J. Deery and Richard H. Gomer Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street Houston, TX 77005-1892 J. Biol. Chem., in press Abstract When Dictyostelium cells starve, they begin secreting a glycoprotein called conditioned medium factor (CMF). When there is a high density of starved cells, as indicated by a high concentration of CMF, the cells begin expressing some genes and aggregate using pulses of cAMP as a chemoatractant. CMF regulates gene expression via a G protein-independent pathway, while CMF regulates cAMP signal transduction via a G protein-dependent pathway. To elucidate receptors mediating cell density sensing, we used CMF-sepharose to isolate membrane proteins that bind CMF. We identified a 50 kD protein, CMFR1, that is sensitive to trypsin treatment of whole cells. We obtained partial amino acid sequence of CMFR1 and isolated the cDNA encoding it. The derived amino acid sequence has no significant similarity to known proteins, and has two or three predicted transmembrane domains. Expression of CMFR1 in insect cells caused an increase in CMF binding. Repression of CMFR1 in Dictyostelium by gene disruption resulted in a ~50% decrease of the CMF binding, and a loss of CMF-induced G protein-independent gene expression. The G protein dependent CMF signal transduction pathways appear to be functional in cmfr1 cells, suggesting that cells sense the density-sensing factor CMF using two or more different receptors. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 8]