Dicty News Electronic Edition Volume 10, number 4 February 7, 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 =========== DdLIM is a Cytoskeleton-associated Protein Involved in the Protrusion of Lamellipodia in Dictyostelium Josef Prassler, Alexander Murr, Susanne Stocker, Jan Faix, John Murphy, and Gerard Marriott Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany Mol. Biol. Cell, in press Abstract: DdLim, a multi-domain member of the cysteine rich family of LIM domain proteins, was isolated from Dictyostelium cells where it localizes in lamellipodia and at sites of membrane ruffling. The transcription and expression of DdLim is developmentally regulated and the timing of its increased association with the actin cytoskeleton coincides with the acquisition in starved cells of a motile, chemotactic behavior. Vegetative cells that overexpress DdLim contain large lamella and exhibit ruffling at the cortex. The high frequency of large, multi-nucleated mutant cells found in suspension culture suggests that excess DdLim interferes with cytokinesis. DdLim was also identified as a protein in a Dictyostelium cell lysate that associated indirectly, but in a GTP-dependent manner, with a GST-rac1 fusion protein. The data presented suggest that DdLim acts as an adapter protein at the cytoskeleton-membrane interface where it is involved in a receptor-mediated rac1 signaling pathway that leads to actin polymerization in lamellipodia and ultimately cell motility. ------------------------------------------------------------------------ Ethane-freezing/Methanol-fixation of Cell Monolayers. A Procedure for Improved Preservation of Structure and Antigenicity for Light and Electron Microscopies Eva M. Neuhaus, Heinz Horstmann, Wolfhard Almers, Markus Maniak*, and Thierry Soldati Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany * Department of Cell Biology, Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany Journal of Structural Biology, in press Abstract: In order to dissect at the ultrastructural level the morphology of highly dynamic processes such as cell motility, membrane trafficking events and organelle movements, it is necessary to fix/stop time- dependent events in the millisecond range. Ideally, immunoelectron microscopical labelling experiments requires the availability of high- affinity antibodies and accessibility to all compartments of the cell. The biggest challenge is to define an optimum between significant preservation of the antigenicity in the fixed material without compromising the intactness of fine structures. Here, we present a procedure which offers an opportunity to unify preparation of cell monolayers for immunocytochemistry in fluorescence and electron microscopy. This novel strategy combines a rapid ethane-freezing technique with a low temperature methanol-fixation treatment (EFMF), and completely avoids chemical fixatives. It preserves the position and delicate shape of cells and organelles and leads to improved accessibility of the intracellular antigens and to high antigenicity preservation. We illustrate the establishment of this procedure using Dictyostelium discoideum, a powerful model organism to study molecular mechanisms of membrane trafficking and cytoskeleton. ------------------------------------------------------------------------- The cAMP-dependent protein kinase of Dictyostelium : role of the amino-terminal domain in catalytic activity and development. DAMMANN, H. TTRAINCARD, F. ANJARD, C. Van BEMMELEN, M. REYMOND, C. and VERON, M. Mechanism of Development, in press ABSTRACT The catalytic subunit of the cAMP dependent protein kinase (PKA) from Dictyostelium discoideum contains several domains including an unusually long N-terminal extension preceding a highly conserved catalytic core. We transformed the aggregationless PkaC-null strain with several deletion constructs of both domains. Strains transformed with genes expressing catalytically inactive polypeptides could not rescue development. Cotransformation with constructs encoding the N-terminal extension and the catalytic core, both unable to rescue development by themselves, yielded transformants able to proceed to late development. A 27 amino acids long hydrophobic region, immediately upstream the catalytic core, was found indispensable for PKA function. A putative role of this sequence in the acquisition of the active conformation of the protein is discussed. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 4]