Journal of Cell Science, Vol 90, Issue 1 59-71, Copyright © 1988 by Company of Biologists

JOURNAL ARTICLES

A Dictyostelium mutant with severe defects in alpha-actinin: its characterization using cDNA probes and monoclonal antibodies

M Schleicher, A Noegel, T Schwarz, E Wallraff, M Brink, J Faix, G Gerisch and G Isenberg
Max-Planck-Institutes for Psychiatry, Martinsried, Federal Republic of Germany.

Cells of a Dictyostelium discoideum mutant deficient in binding a monoclonal antibody to alpha-actinin have previously been shown to grow and develop similarly to the wild type and to exert unimpaired chemotaxis as well as patching and capping of membrane proteins. Here we show that the normal 3.0 kb message for alpha-actinin is replaced in the mutant by two RNA species of approximately 3.1 and 2.8 kb. The 3.1 kb RNA was recognized by DNA fragments from all parts of the coding region, while the 2.8 kb RNA hybridized to all but a 3'-terminal fragment. Proteins synthesized in the mutant were analysed using four monoclonal antibodies that in the wild type specifically recognize the 95 x 10(3) Mr polypeptide of alpha-actinin. Cleavage mapping indicated that the binding sites of these antibodies are distributed over a region comprising more than half of the alpha-actinin polypeptide chain. In the mutant, three of the antibodies faintly labelled two polypeptides of 95 x 10(3) Mr and 88 x 10(3) Mr; the fourth antibody, which binds closest to one end of the polypeptide chain, faintly labelled the 95 x 10(3) Mr polypeptide only. The 88 x 10(3) Mr polypeptide most probably lacks the C-terminal portion of alpha-actinin. The binding of an antibody that recognized both polypeptides was quantified by a radio-immuno competition assay using wild-type alpha-actinin as a reference. In a mutant cell extract containing total soluble proteins the antibody binding activity was decreased to 1.1% when compared with wild-type extract. After their partial purification and SDS-polyacrylamide gel electrophoresis the mutant 95 x 10(3) Mr and 88 x 10(3) Mr polypeptides were barely detectable as Coomassie Blue-stained bands, indicating that in the mutant not only certain epitopes of alpha-actinin were altered but the entire molecule is almost completely lacking. When the fitness of mutant cells relative to wild type was determined during growth in nutrient medium, a slight disadvantage for the mutant was indicated, by finding selection coefficients between 0.03 and 0.05.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				J.-Q. Wu, J. Bähler, and J. R. Pringle Roles of a Fimbrin and an {alpha}-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis Mol. Biol. Cell, April 1, 2001; 12(4): 1061 - 1077. [Abstract] [Full Text]

				F Rivero, R Furukawa, M Fechheimer, and A. Noegel Three actin cross-linking proteins, the 34 kDa actin-bundling protein, alpha-actinin and gelation factor (ABP-120), have both unique and redundant roles in the growth and development of Dictyostelium J. Cell Sci., January 8, 1999; 112(16): 2737 - 2751. [Abstract] [PDF]

				F. Rivero, A. Kuspa, R. Brokamp, M. Matzner, and A. A. Noegel Interaptin, an Actin-binding Protein of the {alpha}-Actinin Superfamily in Dictyostelium discoideum, Is Developmentally and cAMP-regulated and Associates with Intracellular Membrane Compartments J. Cell Biol., August 10, 1998; 142(3): 735 - 750. [Abstract] [Full Text] [PDF]

				J Faix, C Clougherty, A Konzok, U Mintert, J Murphy, R Albrecht, B Muhlbauer, and J Kuhlmann The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility J. Cell Sci., January 10, 1998; 111(20): 3059 - 3071. [Abstract] [PDF]

				F Rivero, B Koppel, B Peracino, S Bozzaro, F Siegert, C. Weijer, M Schleicher, R Albrecht, and A. Noegel The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development J. Cell Sci., January 11, 1996; 109(11): 2679 - 2691. [Abstract] [PDF]

				M Stoeckelhuber, A. Noegel, C Eckerskorn, J Kohler, D Rieger, and M Schleicher Structure/function studies on the pH-dependent actin-binding protein hisactophilin in Dictyostelium mutants J. Cell Sci., January 7, 1996; 109(7): 1825 - 1835. [Abstract] [PDF]

				A Jungbluth, V von Arnim, E Biegelmann, B Humbel, A Schweiger, and G Gerisch Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells J. Cell Sci., January 1, 1994; 107(1): 117 - 125. [Abstract] [PDF]

				C. Cunningham, J. Gorlin, D. Kwiatkowski, J. Hartwig, P. Janmey, H. Byers, and T. Stossel Actin-binding protein requirement for cortical stability and efficient locomotion Science, January 17, 1992; 255(5042): 325 - 327. [Abstract] [PDF]