Dicty News Electronic Edition Volume 14, number 3 January 29, 2000 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 ============= Multiple O-glycoforms on the spore coat protein SP96 in Dictyostelium discoideum Marcus Mreyen, Alan Champion, Keith L. Williams and Nicolle H. Packer J. Biol.Chem., In press Abstract A decreased level of fucosylation on certain spore coat proteins of Dictyostelium discoideum alters the permeability of the spore coat. Here the post-translational modifications of a major spore coat protein, SP96, are studied in a wild-type strain (X22) and a fucosylation defective mutant (HU2470). A novel phosphoglycan structure on SP96 of the wild type strain, consisting of Fuc-GlcNAc- alpha-1-P-Ser, was identified by electrospray ionisation mass spectrometry (ESI-MS). It was shown using monosaccharide and gas chromatography mass spectrometry (GC-MS) analysis that SP96 in the mutant HU2470 contained approximately 20% of wild type levels of fucose, as a result of a missing terminal fucose on the novel glycan structure. The results support previous predictions, based on inhibition studies on different fucose deficient strains, about the nature of monoclonal antibody (mAb) epitopes identified by mAbs MUD62 and MUD166, which are known to identify O-linked glycans (Champion et al. (1995). Microbiology 141, 785-97). Quantitative studies on wild-type SP96 indicated there were approximately 60 sites with phosphodiester linked N-acetyl- glucosamine-fucose disaccharide units and a further approximately 20 sites with fucose directly linked to the protein. Over 70% of the serine sites are modified, with less than 1% of these sites phosphorylated without further glycosylation. Threoinine and tyrosine residues were not found to be modified. ---------------------------------------------------------------------------- Dictyostelium amoebae lacking an F-box protein form spores rather than stalk in chimeras with wild-type Herbert L. Ennis*, Dee N. Dao*, Stefan U. Pukatzki, and Richard H. Kessin Department of Anatomy and Cell Biology, Columbia University, 630 West 168th St. N.Y., N.Y. 10032, USA Accepted, Proceedings of the National Academy of Sciences, USA Summary: Using a selection for Dictyostelium mutants that preferentially form spores we have recovered a mutant called CheaterA. In chimeras with isogenic wild-type cells, the CheaterA mutant preferentially forms viable spores rather than inviable stalk cells. The mutant causes wild-type cells that have begun to express spore-specific genes to migrate to the prestalk compartment of the developing organism. In the wild-type, the chtA transcript is absent in growing cells and appears early in development. No transcript was detected in mutant slugs by northern blot. The chtA gene codes for a protein with an F-box and WD40 domains. This class of protein usually forms part of a complex that targets specific protein substrates for ubiquitination and degradation. Note: Our colleagues Margaret Nelson, Jeff Williams, and Rick Firtel have also cloned and studied this gene. ---------------------------------------------------------------------------- The actin cytoskeleton of Dictyostelium: a story from mutants Angelika A. Noegel and Michael Schleicher* Institut für Biochemie I, Medizinische Fakultät, Universität zu Köln, Joseph-Stelzmann-Str. 52, 50931 Köln, FRG, *Adolf-Butenandt-Institut für Zellbiologie, Ludwig-Maximilians-Universität, Schillerstr. 42, 80336 München, FRG J. Cell Sci. SUMMARY Actin-binding proteins are effectors of cell signalling and coordinators of cellular behaviour. Emphasis in research of the Dictyostelium actin cytoskeleton focusses both on the elucidation of the function of bona fide actin-binding proteins as well as on proteins involved in signalling to the cytoskeleton. A major part of this work is concerned with the analysis of Dictyostelium mutants. The results derived from these investigations have added to our understanding of the role of the actin cytoskeleton in growth and development. Furthermore, the studies identified several cellular and developmental stages, that are particularly sensitive to an unbalanced cytoskeleton. In addition, use of GFP fusion proteins unravels the spatial and temporal patterns of their association with the cytoskeleton. ---------------------------------------------------------------------------- The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis Sharon Chien, Chang Y. Chung, Sujatha Sukumaran, Nicholas Osborne, Susan Lee, Charlene Ellsworth, James G. McNally, and Richard A. Firtel Molecular Biology of the Cell, in press. ABSTRACT We have identified limB, a gene encoding a novel LIM domain containing protein, LIM2 in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin-enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell-cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not co-localized with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways controlling cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains is presented. ---------------------------------------------------------------------------- Charge Changes in Loop 2 Affect the Thermal Unfolding of the Myosin Motor Domain Bound to F-Actin. Michael A. Ponomarev‡, Marcus Furch, Dmitrii I. Levitsky‡, and Dietmar J. Manstein* ‡ A.N.Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prosp. 33, Moscow 117071, Russia, and Max-Planck-Institut für Medizinische Forschung, Jahnstr. 29, 69120 Heidelberg, Germany Biochemistry, in press ABSTRACT The thermal unfolding of Dictyostelium discoideum myosin head fragments with alterations in the actin-binding surface loop 2 was studied by differential scanning calorimetry (DSC). Lengthening of loop 2 without concomitant charge changes led to decreases in the transition temperature of not more than 1.8oC. Insertions with multiple positive or negative charges had a stronger destabilizing effect and led to reductions in the thermal transition temperature of up to 3.7oC. In the presence of nucleotide, most mutants displayed similar or higher transition temperatures than M765. Only constructs M765(11/+6) and M765(20/+12) with long positively charged inserts showed transition temperatures that were more than 2oC below the values measured for M765 in the presence of ADP, ADP-AlF4 and ADP-BeF3. Interaction with F-actin in the presence of ADP shifted the thermal transition of M765 by 6oC, from 49.1°C to 55.1°C. The actin-induced increase in thermal stability varied between 1.2 and 9.1°C and showed a strong correlation with the mutant constructs’ affinity for actin. Our results show that length and charge changes in loop 2 do not significantly affect nucleotide-induced structural changes in the myosin motor domain, but they affect structural changes that occur when the motor domain is strongly bound to actin and affect the coupling between the actin- and nucleotide binding sites. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 3]