Dicty News Electronic Edition Volume 13, number 11 November 13, 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" ================ Announcement ================ An All Dicty Blast site is available at [http://www.sdsc.edu/mpr/dicty/] and can be accessed from DictyDB [http://glamdring.ucsd.edu/others/dsmith/dictydb.html] or from the Genomics section of the Dicty Web site http://dicty.cmb.nwu.edu/dicty/dicty.html. This site gives the same graphical display of regions of overlaps that the Jena site does but gives instant accesses to all the Baylor, Jena and Sanger updated raw reads as well as all Genbank/EMBL Dicty sequences. The other Dicty sites give direct access only to the local reads. Various BLAST programs are available and run relatively quickly on the San Diego Supercomputer. We hope this is a service to the Dicty community. Michael Gribskov Negin Iranfar Bill Loomis Doug Smith ============= Abstracts ============= Dynein Intermediate Chain Mediated Dynein-Dynactin Interaction is Required for Interphase Microtubule Organization and Centrosome Replication and Separation in Dictyostelium Shuo Ma, Leda Triviños-Lagos, Ralph Gräf# and Rex L. Chisholm* Dept of Cell and Molecular Biology and Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, 303 E. Chicago Ave. Chicago, IL 60611 # Adolf-Butenandt-Institut/Zellbiologie, Universitaet Muenchen, Schillerstr. 42, D-80336 Muenchen/Germany J. Cell Biology, in press Abstract Cytoplasmic dynein intermediate chain (IC) mediates dynein-dynactin interaction in vitro (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995). To investigate the physiological role of IC and dynein-dynactin interaction, we expressed IC truncations in wild type Dictyostelium cells. ICDC associated with dynactin but not with dynein heavy chain, while ICDN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal Golgi localization, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase microtubule network as well as centrosome replication and separation in Dictyostelium. ---------------------------------------------------------------------------- Evidence of intramolecular regulation of the Dictyostelium discoideum 34,000 dalton F-actin bundling protein. Rita W. L. Lim, Ruth Furukawa, and Marcus Fechheimer Department of Cellular Biology, University of Georgia, Athens, GA 30602 Biochemistry, in press. ABSTRACT: Intramolecular interaction within the Ca2+-regulated 34 kDa actin bundling protein from Dictyostelium discoideum was found to contribute to the regulation of its actin binding activity. Recombinant N-terminally truncated proteins aa77-295, 124-295, and 139-295 bound actin at > 2:1 stoichiometry which is 5-fold greater than the intact protein aa1-295 as assessed by co-sedimentation with F-actin. These proteins also have enhanced cross-linking activity as assessed by viscometry and electron microscopy. All truncated 34 kDa proteins failed to bind 45Ca2+ on blots and displayed Ca2+-insensitive binding with actin, although most proteins possessed intact putative EF-hand Ca2+ binding motifs. An intramolecular interaction within the 34 kDa protein was inferred from direct demonstrations of domain-domain interaction among the truncated 34 kD proteins both in the presence and absence of actin. The intramolecular interaction between interaction zone 1 (aa71-123) and interaction zone 2 (aa193-254) is proposed to maintain the N-terminal inhibitory region (aa1-76) in close proximity with the strong actin binding site (aa193-254) in order to modulate the interaction of the intact protein with actin filaments. ---------------------------------------------------------------------------- SEVERE DEVELOPMENTAL DEFECTS IN DICTYOSTELIUM NULL MUTANTS FOR ACTIN BINDING PROTEINS Eleonora Ponte, Francisco Rivero#, Marcus Fechheimer*, Angelika Noegel# and Salvatore Bozzaro Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale S.Luigi, 10043-Orbassano, Italy; #Institut für Biochemie I, Universität zu Köln, Joseph-Stelzmann-str. 52, 50931-Köln, Germany, *Department of Cellular Biology, University of Georgia, 724 Biological Sciences Building, Athens, GA 30602, USA. Mechanisms of Development, in press ABSTRACT The actin cytoskeleton is implicated in many cellular processes, such as cell adhesion, locomotion, contraction and cytokinesis, which are central to any development. The extent of polymerization, crosslinking, and bundling of actin is regulated by several actin-binding proteins. Knock-out mutations in these proteins have revealed in many cases only subtle, if any, defects in development, suggesting that the actin system is redundant, with multiple proteins sharing overlapping functions. The apparent redundancy may, however, reflect limitations of available laboratory assays in assessing the developmental role of a given protein. By using a novel assay, which reproduces conditions closer to the natural ones, we have re-examined the effects of disruption of many actin-binding proteins, and show here that deletion of alpha-actinin, interaptin, synexin, 34-kDa actin-bundling protein, and gelation factor affect to varying degrees the efficiency of Dictyostelium cells to complete development and form viable spores. No phenotypic defects were found in hisactophilin or comitin-null mutants. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 11]