CSM News Electronic Edition Volume 4, number 4 January 28, 1995 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 ============ Cell-Substrate Interactions and Locomotion of Dictyostelium Wild-Type and Mutants Defective in Three Cytoskeletal Proteins: A Study Using Quantitative Reflection Interference Contrast Microscopy. Mathias Schindl1, Eva Wallraff2, Barbara Deubzer1, Walter Witke2, Guenther Gerisch2, and Erich Sackmann1 1 Physics Department, Biophysics Group E22, Technische Universitat Munchen, D-85747 Garching 2 Max-Planck-Institut fuer Biochemie, D-82152 Martinsried Biophysical Journal, in press Abstract Reflection interference contrast microscopy combined with digital image processing was applied to study the motion of Dictyostelium discoideum cells in their pre-aggregative state on substrata of different adhesiveness (glass, albumin-covered glass, and freshly cleaved mica). The temporal variations of the size and shape of the cell/substratum contact area and the time course of advancement of pseudopods protruding in contact with the substratum were analyzed. The major goal was to study differences between the locomotion of wild-type cells and strains of triple mutants deficient in two F-actin cross-linking proteins (alpha-actinin and the 120-kDa gelation factor) and one F-actin fragmenting protein (severin). The size of contact area, Ac, of both wild-type and mutant cells fluctuates between minimum and maximum values on the order of minutes, pointing toward an intrinsic switching mechanism associated with the mechanochemical control system. The fluctuation amplitudes are much larger on freshly cleaved mica than on glass. Wild-type and mutant cells exhibit remarkable differences on mica but not on glass. These differences comprise the population median of Ac and alterations in pseudopod protrusion. Ac is smaller by a factor of two or more for all mutants. Pseudopods protrude slower and shorter in the mutants. It is concluded that cell shape and pseudopods are destabilized by defects in the# actin-skeleton, which can be overcompensated by strongly adhesive substrata. Several features of amoeboid cell locomotion on substrata can be understood on the basis of the minimum bending energy concept of soft adhering shells and by assuming that adhesion induces local alterations of the composite membrane consisting of the protein/lipid bilayer on the cell surface and the underlying actin-cortex. ------------------------------------------------------------- Motility and substrate adhesion of Dictyostelium wild-type and cytoskeletal mutant cells: a study by RICM/bright-field double-view image analysis. Igor Weber, Eva Wallraff, Richard Albrecht and Guenther Gerisch Journal of Cell Science, in press Abstract To investigate the dynamics of cell-substrate adhesion during locomotion, a double-view optical technique and computer-assisted image analysis has been developed which combines reflection interference contrast microscopy (RICM) with bright-field imaging. The simultaneous recording of cell-substrate contact and cell body contour has been applied to aggregation-competent cells of Dictyostelium discoideum. These cells are distinguished from earlier stages of development by small areas of contact to a substratum. Three questions have been addressed in analysing the locomotion of aggregation-competent cells: (1) What is the relationship between changes in the shape of cells and their contact to a substratum during a chemotactic response? (2) What is the relationship between protrusion and retraction of the cell body, and between local attachment and detachment? (3) Are there differences between wild-type and mutant cells that lack certain cytoskeletal proteins? During a chemotactic response the front region of the amoeba can bend towards the gradient of attractant without being supported by its contact with a surface, which excludes gradients of adhesion to be necessary for the response. The finding that in locomoting cells protrusion of the leading edge often precedes retraction establishes a pioneer role of the front region. The finding that gain of contact area precedes loss provides evidence for the coordination of interactions between the cell surface and a substratum. For comparison with wild-type, aggregation-competent triple mutant cells have been used that lack two F-actin cross-linking proteins, alpha-actinin and 120 kDa gelation factor, and an actin filament fragmenting protein, severin. Disturbances in the spatial and temporal control of cytoskeletal activities have been unravelled in the mutant by RICM and quantified by cross-correlation analysis of attachment and detachment vectors. In order to detect these disturbances, it was essential to analyse cell locomotion on the weakly adhesive surface of freshly cleaved mica. ------------------------------------------------------------------ Expression of the Rotavirus SA11 Protein VP7 in the Simple Eukaryote Dictyostelium discoideum Kerry R. Emslie, Janine M. Miller, Martin B. Slade, Philip R. Dormitzer, Harry B. Greenberg, and Keith L. Williams Journal of Virology, in press. Abstract The outer capsid protein of rotavirus, VP7, is a major neutralization antigen and is considered a necessary component of any subunit vaccine developed against rotavirus infection. For this reason, significant effort has been directed towards producing recombinant VP7 that maintains the antigenic characteristics of the native molecule. Using a relately new expression system, the simple eukaryote Dictyostelium discoideum, we have cloned the portion of the simian rotavirus SA11 genome segment 9, encoding the mature VP7 protein downstream of a native D.discoideum secretion signal sequence in a high-copy-number extrachromosomal vector. The majority of the recombinant VP7 expressed by transformants was intracellular and was detected by Western (immunoblot) following gel electrophoresis as two or three bands with an apparent molecular mass of 35.5 to 37.5 kDa. A small amount of VP7 having an apparent molecular mass of 37.5 kDa wa secreted. Both the intracellular and secreted VP7 were N-glycosylated and sensitive to endoglycosidase H digestion. Under nonreducing electrophoresis conditions, over half the intracellular VP7 migrated as a monomer while the remainder migrated with an apparent molecular mass approximately twice that of the momomeric form. In an enzyme-linked immunosorbent assay, intracellular VP7 reacted with both nonneutralizing and neutralizing antibodies. The monoclonal antibody recognition pattern paralleled that found with VP7 expressed in either vaccinia virus or herpes simplex virus type 1 and confirms that D. discoideum-expressed VP7 is able to form the major neutralization domains present on viral VP7. Because D.discoideum cells are easy and cheap to grow, this expression system provides a valuable alternative for the large-scale production of recombinant VP7 protein. ------------------------------------------------------------------------ [End CSM-News, volume 4, number 3]