Dicty News Electronic Edition Volume 11, number 8 October 24, 1998 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 ============= Dissection of the cAMP induced cytosolic calcium response in Dictyostelium discoideum: the role of cAMP receptor subtypes and G protein subunits Jürgen Sonnemann, Annette Aichem and Christina Schlatterer Fakultät für Biologie, Universität Konstanz, D-78457 Konstanz FEBS Letters, in press Abstract The cAMP signaling cascade leading to changes in [Ca2+]i in Dictyostelium discoideum was analyzed using cell lines overexpressing single cAMP receptor subtypes (cAR1-cAR3) or lacking the Galpha2 or Gbeta subunit of the G-protein. Imaging of fura2-dextran-loaded amoebae revealed cAMP-induced [Ca2+]i-changes characteristic for each receptor subtype activated. Cells expressing distinct subtypes sort to defined zones during multicellular development suggesting involvement of the specific [Ca2+]i-transients in patterning processes. Whereas generation of the [Ca2+]i-increase was Galpha2-independent, only few cells devoid of Gbeta displayed a [Ca2+]i-change after stimulation indicating its participation in the regulation of the calcium homeostasis. ---------------------------------------------------------------------------- A computational model of ameboid deformation and locomotion Dean C. Bottino *(University of Utah) and Lisa J. Fauci (Tulane University) European Biophysics Journal (1998) 27:532-539 Abstract: Traditional continuum models of ameboid deformation and locomotion are limited by the computational difficulties intrinsic in free boundary conditions. A new model using the immersed boundary method overcomes these difficulties by representing the cell as a force field immersed in a fluid domain. The forces can be derived from a direct mechanical interpretation of such cell components as the cell membrane, the actin cortex, and the transmembrane adhesions between the cytoskeleton and the substratum. The numerical cytoskeleton, modeled as a dynamic network of immersed springs, is able to qualitatively model the passive mechanical behavior of a shear-thinning viscoelastic fluid (Bottino, J. Comp. Phys., 147,1(1998)). The same network is used to generate active protrusive and contractile forces. When coordinated with the attachment-detachment cycle of the cell’s adhesions to the substratum, these forces produce directed locomotion of the model ameba. With this model it is possible to study the effects of altering the numerical parameters upon the motility of the model cell in a manner suggestive of genetic deletion experiments. In the context of this ameboid cell model and its numerical implementation, simulations involving multicellular interaction, detailed internal signaling, and complex substrate geometries are tractable. ---------------------------------------------------------------------------- Random insertion of GFP into the cAMP dependent protein kinase regulatory subunit from Dictyostelium discoideum Ricardo M. Biondi,1 Pascal J. Baehler,2 Christophe D. Reymond2,** and Michel Véron1 *Unité de Régulation Enzymatique des Activités Cellulaires, CNRS-URA 1773, Institut Pasteur, 25 rue du Dr. Roux, 75724, Paris Cedex 15, France 2Institut de Biologie Cellulaire et de Morphologie, Université de Lausanne, rue du Bugnon 9, CH-1005 Lausanne, Switzerland Footnote: The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors NUCLEIC ACIDS RESEARCH (In Press) ABSTRACT The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+ sensor. We show here that GFP fusions can also be used to obtain informations on regions essential for protein function. As FRET requires the two GFP's to be very close, N- or C- terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP dependent manner. Random insertion of GFP within the R subunit sets the path to develope two component FRET with the C subunit. ---------------------------------------------------------------------------- DISRUPTION OF A DYNAMIN HOMOLOGUE AFFECTS ENDOCYTOSIS, ORGANELLE MORPHOLOGY AND CYTOKINESIS IN DICTYOSTELIUM DISCOIDEUM Dirk C. Wienke*, Menno L.W. Knetsch*, Eva M. Neuhaus§, Mary C. Reedy, and Dietmar J. Manstein* * Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Jahnstr. 29, D-69120 Heidelberg, Germany; § Abteilung Molekulare Zellforschung, Max-Planck-Institut für Medizinische Forschung, Jahnstr. 29, D-69120 Heidelberg, Germany; Dept. of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA. Mol. Biol. Cell, in press SUMMARY The identification and functional characterization of Dictyostelium discoideum dynamin A, a protein composed of 853 amino acids, that shares up to 44% sequence identity with other dynamin-related proteins is described. Dynamin A is present during all stages of D. discoideum development and is predominantly found in the cytosolic fraction and in association with endosomal and post-lysosomal vacuoles. Overexpression of the protein has no adverse effect on the cells while depletion of dynamin A by gene targeting techniques leads to multiple and complex phenotypic changes. Cells lacking a functional copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucleated due to a failure to complete normal cytokinesis. These pleiotropic effects of dynamin A-depletion can be rescued by complementation with the cloned gene. Morphological studies using cells producing GFP-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double-labeling with vacuolin, a marker of a post-lysosomal compartment in D. discoideum, showed an almost complete colocalization of vacuolin and dynamin A. Our results suggest that that dynamin A is likely to function in membrane trafficking processes along the endo-lysosomal pathway of D. discoideum but not at the plasma membrane. ---------------------------------------------------------------------------- NUCLEAR PLASMIDS OF DICTYOSTELIUM Joanne E. Hughes and Dennis L. Welker Department of Biology, Utah State University, Logan, Utah 84322-5305 Genetic Engineering, Volume 21, in press (1999) This is a review of the information to date on the nuclear plasmids from cellular slime molds. It covers basic plasmid characterization, plasmid families, plasmid maintenance functions, other potential plasmid functions, and extrachromosomal vectors. ---------------------------------------------------------------------------- [End Dicty News, volume 11, number 8]