A myb-related protein required for culmination in Dictyostelium.
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The avian retroviral v-myb gene and its cellular homologues throughout the animal and plant kingdoms contain a conserved DNA binding domain. We have isolated an insertional mutant of Dictyostelium unable to switch from slug migration to fruiting body formation i.e. unable to culminate. The gene that is disrupted, mybC, codes for a protein with a myb-like domain that is recognized by an antibody against the v-myb repeat domain. During development of myb+ cells, mybC is expressed only in prestalk cells. When developed together with wild-type cells mybC- cells are able to form both spores and stalk cells very efficiently. Their developmental defect is also bypassed by overexpressing cAMP-dependent protein kinase. However even when their defect is bypassed, mybC null slugs and culminates produce little if any of the intercellular signalling peptides SDF-1 and SDF-2 that are believed to be released by prestalk cells at culmination. We propose that the mybC gene product is required for an intercellular signaling process controlling maturation of stalk cells and spores and that SDF-1 and/or SDF-2 may be implicated in this process. (+info)
Genetic analysis of radiation-sensitive mutations in the slime mould Dictyostelium discoideum.
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The linkage of two mutations leading to increased sensitivity to ultraviolet light and 60Co gamma rays was determined in the slime mould Dictyostelium discoideum using a genetic analysis based on the parasexual cycle. Diploids were selected from a mixture of radiation-sensitive, temperature-resistant and radiation-resistant, temperature-sensitive haploids on the basis of simultaneous radiation and temperature resistance. Analysis of drug-resistant haploid segregants of the heterozygous diploids indicated that one of the radiation-sensitive mutations, radA20, was linked to linkage group I whereas the other, radB13, was linked to the recently defined linkage group VI. (+info)
Interaction mapping of a dynein heavy chain. Identification of dimerization and intermediate-chain binding domains.
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Cytoplasmic dynein is a multisubunit microtubule-based motor protein that is involved in several eukaryotic cell motilities. Two dynein heavy chains each form a motor domain that connects to a common cargo-binding tail. Although this tail domain is composed of multiple polypeptides, subunit organization within this region is poorly understood. Here we present an in vitro dissection of the tail-forming region of the dynein heavy chain from Dictyostelium. Our work identifies a sequence important for dimerization and for binding the dynein intermediate chain. The core of this motif localizes within an approximately 150-amino acid region that is strongly conserved among other cytoplasmic dyneins. This level of conservation does not extend to the axonemal dynein heavy chains, suggesting functional differences between the two. Dimerization appears to occur through a different mechanism than the heavy chain-intermediate chain interaction. We corroborate the in vitro interactions with in vivo expression of heavy chain fragments in Dictyostelium. Fragments lacking the interaction domain express well, without an obvious phenotype. On the other hand, the region crucial for both interactions appears to be lethal when overexpressed. (+info)
The structural gene for alpha-mannosidase-1 in Dictyostellium discoideum.
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We have isolated 4 independent mutations affecting alpha-mannosidase-1, a developmentally regulated activity in Dictyrostelium discoideum. Three of these result in a thermolabile alpha-mannosidase-1 activity. One mutation also affects the substrate affinity (Km) of the activity. In diploids these mutations show a gene dosage effect and are all alleles. The structural gene for alpha-mannosidase-1, as defined by these mutations, defines a new linkage group, linkage group VI. alpha-mammosidase 1 is probably a homopolymer with subunits of 54,000 daltons. We have also mapped two temperature-sensitive-for-growth mutations onto two previously defined linkage groups. (+info)
Cell patterning in migrating slugs of Dictyostelium discoideum.
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When front quarters of migrating slugs of Dictyostelium discoideum are isolated by surgery and induced to friut immediately they produce fruiting bodies with disproportionately large stalks (Raper, 1940). The data in this communication show that the 'stalky' character of fruits derived from front quarters persists even if the cells of the front quarter are disaggregated and hence to reaggregate before fruiting. The data also demonstrate that friuts derived from rear quarters of slugs have disproportionately large spore heads, and that this effect becomes more pronounced with increasing age of the slugs. These observations support the view that the cells of the front and rear of migrating slugs are to some extent committed to different fates. (+info)
Functional significance of the conserved residues in the flexible hinge region of the myosin motor domain.
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Analysis of the three-dimensional crystal structure of the Dictyostelium myosin motor domain revealed that the myosin head is required to bend at residues Ile-455 and Gly-457 to produce the conformation changes observed in the ternary complexes that resemble the pre- and post-hydrolysis states (Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). Asp-454, Ile-455, and Gly-457 of smooth muscle myosin were substituted by Ala, Met, and Ala, respectively, and the mechano-enzymatic activities were determined to study the role of these residues in myosin motor function. Whereas the basal steady-state Mg2+-ATPase activity of D454A was higher than that of the wild type, the rate of the hydrolytic step is reduced approximately 2,000-fold and becomes rate-limiting. M-ATP rather than M-ADP-P is the predominant steady-state intermediate, and the initial Pi burst and the ATP-induced enhancement of intrinsic tryptophan fluorescence are absent in D454A. D454A binds actin in the absence of ATP but is not dissociated from actin by ATP. Moreover, actin inhibits rather than activates the ATPase activity; consequently, D454A does not support actin translocating activity. I455M has normal actin-activated ATPase activity, Pi burst, and ATP-induced enhancement of intrinsic tryptophan fluorescence, suggesting that the enzymatic properties are normal. However, the actin translocating activity was completely inhibited. This suggests that the side chain at Ile-455 is critical for myosin motor activity but not for relatively normal enzymatic function, which indicates an apparent uncoupling between enzymatic activity and motile function. Although G457A has normal ATP-dependent actin dissociation, ATP hydrolytic step is reduced by approximately 10(5)-fold in the presence or absence of actin; consequently, G457A does not have actin translocating activity. These results indicate the importance of these conserved residues at the hinge region for normal myosin motor function. (+info)
Signal emission and signal propagation during early aggregation in Dictyostelium discoideum.
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Waves of chemotactic movement during the early phase of aggregation in Dictyostelium discoideum are of 2 kinds, concentric waves produced by cells that emit cyclic AMP signals spontaneously, and spirals generated by excitations relayed continuously around loops of excitable cells. The period of a spiral wave is the time taken for the excitation to make one complete circuit of the pacemaker loop. We have compared signal emission from the 2 types of source in time-lapse films made at a variety of temperatures. Our results show that spiral waves have a characteristic period length throughout most if not all of the early phase of aggregation, and that the period of concentric waves is generally longer and more variable. Temperature has a pronounced effect on period length and a lesser effect on propagation velocity. We find that each individual wave is propagated at constant velocity over distances of 1-2 cm but that the velocity of successive waves declines. This decline probably reflects some cumulative effect of the chemotactic excitations on the excitable properties of the aggregating cells. (+info)
Role of myosin II tail sequences in its function and localization at the cleavage furrow in Dictyostelium.
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Cytoplasmic myosin II accumulates in the cleavage furrow and provides the force for cytokinesis in animal and amoeboid cells. One model proposes that a specific domain in the myosin II tail is responsible for its localization, possibly by interacting with a factor concentrated in the equatorial region. To test this possibility, we have expressed myosins carrying mutations in the tail domain in a strain of Dictyostelium cells from which the endogenous myosin heavy chain gene has been deleted. The mutations used in this study include four internal tail deletions: Mydelta824-941, Mydelta943-1464, Mydelta943-1194 and Mydelta1156-1464. Contrary to the prediction of the hypothesis, immunofluorescence staining demonstrated that all mutant myosins were able to move toward the furrow region. Chimeric myosins, which consisted of a Dictyostelium myosin head and chicken skeletal myosin tail, also efficiently localized to the cleavage furrow. All these deletion and chimeric mutant myosins, except for Mydelta943-1464, the largest deletion mutant, were able to support cytokinesis in suspension. Our data suggest that there is no single specific domain in the tail of Dictyostelium myosin II that is required for its functioning at and localization to the cleavage furrow. (+info)