CSM News Electronic Edition Volume 3, number 9 September 17, 1994 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. =========== Abstracts =========== [[NO TITLE PROVIDED]] Eamonn K. Rooney (1), Julian D. Gross (2) and Michel Satre (1) (1) Laboratoire de Biologie Cellulaire (URA 1130 CNRS), CEA, D=E9partement d= e Biologie Mol=E9culaire et Structurale, Centre d'Etudes Nucl=E9aires de Grenoble, 38054 Grenoble Cedex 9, France (2) Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom Cell Calcium, in press. Abstract Calcium uptake by microsomal membranes form the cellular slime mould Dictyostelsium discoideum was measured using Calcium Green-2 as a fluorescent probe of external free Ca2+ concentration. High-affinity Ca2+ uptake was found to be completely inhibited by low concentrations of vanadate, but not by thapsigargin, suggesting that the activity is mediated by a Ca2+-ATPase distinct from the sarco(endo)plasmic reticumum type of higher animal cells. On sucrose gradients, Ca2+ uptake distributes with vacuolar proton pump activity and part of the observed Ca2+ uptake is dependent on the pH gradient generated by the vacuolar-type H+-ATPase, indicating that the Ca2+ pump is located on both acidic and non-acidic vesicles, possibly derived from the H+-ATPase-rich contractile vacuole complex. --------------------------------------------------------------------- A GENERAL METHOD FOR PLASMA MEMBRANE ISOLATION BY COLLOIDAL GOLD DENSITY SHIFT Theodore L. Steck and Malti Lavasa Department of Biochemistry and Molecular Biology, The University Of Chicago, Chicago, IL 60637 Analytical Biochemistry, in press. A general method for isolating plasma membranes is described. Vegetative amoebae of Dictyostelium discoideum were allowed to directly adsorb raw colloidal gold of particle diameter 10-20 nm. After quenching the gold surface, the cells were lysed and the lysates diluted in 60% sucrose and centrifuged through a 65% sucrose cushion so as to selectively pellet the gold-laden membranes. Three generally-applicable exogenous cell surface markers were used to follow the plasma membranes: intercalated [3H]cholesterol and octadecylrhodamine and the adsorbed gold colloid itself. The isolates routinely contained ~60% of these tags, enriched ~15-fold with respect to protein. The recovery and degree of enrichment of contaminating markers in the plasma membrane fraction were: lysosomes (3% and 1-fold); mitochondria (11% and 3-fold); rough endoplasmic reticulum, as refleceted by RNA (3% and 0.7-fold); and DNA (9% and 4-fold). Membrane proteins and lipids were quantitatively solubilized from the gold by detergents. We conclude that this methodology provides an approach to the isolation of plasma membranes which compares favorably to existing techniques with respect to yield, purity and ease. ---------------------------------------------------------------------- Molecular genetic truncation analysis of filament assembly and phosphorylation domains of Dictyostelium myosin heavy chain. [[Author list not provided]] Tom Egelhoff and colleagues J. Cell Sci., in press. Abstract Conventional myosin ("myosin II") is a major component of the cytoskeleton in a wide variety of eukaryotic cells, ranging from lower amoebae to mammalian fibroblasts and neutrophils. Gene targeting technologies available in the Dictyostelium discoideum system have provided the first genetic proof that this molecular motor protein is essential for normal cytokinesis, capping of cell surface receptors, normal chemotactic cell locomotion and morphogenetic shape changes during development. Although the roles of myosin in a variety of cell functions are becoming clear, the mechanisms that regulate myosin assembly into functional bipolar filaments within cells is poorly understood. Dictyostelium is currently the only system where mutant forms of myosin can be engineered in vitro, then expressed in their native context in cells which are devoid of the wild type isoform. We have utilized this technology in combination with nested truncation and deletion analysis to map domains of the myosin tail necessary for in vivo and in vitro filament assembly, and for normal myosin heavy chain (MHC) phosphorylation. This analysis defines a region of 35 amino acids within the tail which is critical for filament formation both for purified myosin molecules and for myosin within the in vivo setting. Phosphorylation analysis of these mutants in intact cytoskeletons demonstrates that the carboxy-terminal tip of the myosin heavy chain is required for complete phosphorylation of the myosin tail. ------------------------------------------------------------------- [[End CSM News, volume 3, number 9]]