Dicty News Electronic Edition Volume 18, number 2 January 26, 2002 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============= Abstracts ============= Molecular analysis of the cytosolic Dictyostelium gamma-tubulin complex Christine Daunderer and Ralph Grf Adolf-Butenandt-Institut/Zellbiologie, Universitt Mnchen, Germany Ralph.Graef@lrz.uni-muenchen.de Eur. J. Cell Biol., in press Gamma-tubulin plays an essential role in microtubule nucleation and organization and occurs, besides its centrosomal localization, in the cytosol, where it forms soluble complexes with other proteins. We investigated the size and composition of gamma-tubulin-complexes in Dictyostelium, using a mutant cell line in which the endogenous copy of the gamma-tubulin gene had been replaced by a tagged version of the gene. Dictyostelium gamma-tubulin complexes were generally much smaller than the large gamma-tubulin ring complexes found in higher organisms. The stability of the small Dictyostelium gamma-tubulin complexes depended strongly on the purification conditions, with a striking stabilization of the complexes under high salt conditions. Furthermore, we cloned the Dictyostelium homolog of Spc97 and an almost complete sequence of the Dictyostelium homolog of Spc98, which are both components of gamma-tubulin complexes in other organisms. Both proteins localize to the centrosome in Dictyostelium throughout the cell cycle and are also present in a cytosolic pool. We could show that the prevailing small complex present in Dictyostelium consists of DdSpc98 and gamma-tubulin, whereas DdSpc97 does not associate. Dictyostelium is thus the first organism investigated so far where the three proteins do not interact stably in the cytosol. ----------------------------------------------------------------------------- Visualization of Actin Dynamics During Macropinocytosis and Exocytosis Eunkyung Lee and David A. Knecht Department of Molecular and Cell Biology, University of Connecticut Storrs, CT 06269 Traffic, in press Macropinocytosis and exocytosis of post-lysosomes have been visualized using an GFP probe that binds specifically to F-actin filaments. F-actin association with macropinocytosis begins as a V-shaped infolding of the membrane. Vesicle enlargement occurs through an inward movement of the proximal point of the V as well as an outward protrusion at the tip of the V to form an elongated invagination. The protrusion eventually closes at its distal margin to become a vesicle and is moved centripetally while recovering its circular shape. The vesicle loses its actin coat within one minute after internalization. One hour later, post-lysosomal vesicles became very weakly surrounded by actin while still cytoplasmic. Some of these vesicles moved to the plasma membrane, docked, and then expelled their contents. Slightly before the vesicle content began to disappear, an increase in F-actin association with the vesicle was observed. This was followed by rapid contraction of the vesicle and then disappearance of the actin signal once the internal content was released. These results show that dynamic changes in actin filament association with the vesicle membrane accompany both endocytosis and exocytosis. ----------------------------------------------------------------------------- Dynacortin is a novel actin bundling protein that localizes to dynamic actin structures. Douglas N. Robinson*, Stephani S. Ocon, Ronald S. Rock and James A. Spudich Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307 *Contact information: Department of Cell Biology, Johns Hopkins Medical Institute, 725 N. Wolfe St., Baltimore, MD 21205, Ph: 410-502-2850 Email: Douglas.Robinson@jhu.edu J. Biol. Chem., In press Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin-bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7 nm Stoke s radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 mM Kd. Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin- bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 seconds. These results indicate that cells have evolved different actin- filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes. ----------------------------------------------------------------------------- Morphology and dynamics of the endocytic pathway in Dictyostelium discoideum Eva M. Neuhaus, Wolfhard Almers and Thierry Soldati Mol. Biol. Cell, in press Dictyostelium discoideum is a genetically and biochemically tractable social amoeba belonging to the crown group of eukaryotes. It performs some of the tasks characteristic of a leukocyte such as chemotactic motility, macropinocytosis and phagocytosis that are not performed by other model organisms or are difficult to study. D. discoideum is becoming a popular system to study molecular mechanisms of endocytosis, but the morphological characterization of the organelles along this pathway and the comparison with equivalent and/or different organelles in animal cells and yeasts were lagging. Here, we used a combination of evanescent wave microscopy and electron microscopy of rapidly frozen samples to visualize primary endocytic vesicles, vesicular-tubular structures of the early and late endo-lysosomal system, such as multi-vesicular bodies, and the specialized secretory lysosomes. In addition, we present biochemical and morphological evidence for the existence of a micropinocytic pathway, which contributes to the uptake of membrane alongside macropinocytosis, which is the major fluid phase uptake process. This complex endosomal compartment underwent continuous cycles of tubulation/vesiculation as well as homo- and heterotypic fusions, in a way very reminiscent of mechanisms and structures documented in leukocytes. Finally, egestion of fluid phase from the secretory lysosomes was directly observed. ----------------------------------------------------------------------------- The dynamin A ring complex: molecular organisation and nucleotide-dependent conformational changes Boris Klockow, Willem Tichelaar*, Dean R. Madden*, Hartmut H. Niemann, Toshihiko Akiba, Keiko Hirose$, Dietmar J. Manstein Department of Biophysics and *Ion Channel Structure Research Group, Max-Planck-Institute for Medical Research, Jahnstr. 29, D-69120 Heidelberg, Germany National Institute for Advanced Interdisciplinary Research and $ Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan The EMBO Journal, in press. The GTPase dynamin A of the lower eukaryote Dictyostelium discoideum functions in multiple membrane severing events. Here we show by electron microscopy that dynamin A self-assembles into rings and helices similar to human dynamin 1. Single particle analysis of the rings led to a three- dimensional map of the nucleotide-free dynamin A ring complex. The complex consists of two layers, each layer comprising two concentric rings with 11- fold symmetry. The inner ring forms spike-like structures towards the centre, ideally situated to penetrate into a tubulated lipid membrane. The GTP-bound state, visualised using the non-hydrolysable GTP analogue GppNHp, is associated with reorganisation of the ring complex leading to the generation of short helical assemblies. Subsequent steps in the hydrolytic cycle induce further conformational changes including an increase in helical pitch and culminate in the disassembly of the complex. The results support a model for the mechanochemical action of dynamin family proteins that involves both constriction and stretching in membrane fission. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 2]