Cytoskeletons from a mutant of Dictyostelium discoideum with flattened cells.

Development of a mutant of Dictyostelium discoideum, HG403, is described whose cells spread strongly on a substratum. Although the mutant cells were less clearly polarized into the front and rear ends, and usually less extensively elongated than wild-type cells, their aggregation pattern was only slightly less regular. Cells of the mutant responded well to cyclic AMP by chemotaxis, although their capability of stabilizing cell shape and maintaining dominance of a single moving front appeared to be reduced. Mutant HG403 proved to be ideal for the preparation of cytoskeletons in which the organization of the microtubular system, the network of filaments between them, the dense texture of the microfilament network at the periphery of the cells, as well as the bundling of microfilaments in spike-like extensions, could be observed.

Fractionation and characterization of two molecular variants of myosin from adult human atrium.

Monoclonal antibodies (MAb) to myosin heavy chains were prepared from one adult human ventricular myocardium. Several of these MAb reacted by indirect immunofluorescence in a heterogenous way on cryostat transverse sections of fibers from human atrial myocardium, suggesting the presence of different forms of myosin within the human atrium and prompting the further use of the MAb to attempt to fractionate preparations of native atrial myosins. Two molecular variants of human atrial myosins or myosin fragments were thus separated by immunoaffinity chromatography performed with one antiventricular myosin MAb. Seven MAb located at different positions along the myosin heavy chains, as deduced from blotting and immuno-electron microscopy experiments, were used to characterize the structural relationships between the separated human atrial isomyosins and between each of them and the main human ventricular myosin. As deduced from competitive radioimmunoassay measurements, the primary structures of the two atrial myosins differ in at least five antigenic determinants and share at least two of them; similarly located structural differences were observed between one of the atrial myosins and the ventricular myosin. Conversely, the primary structures of the other atrial myosin and of the ventricular myosin differ in at least two antigenic determinants and share at least five of them. Differences in the primary structures of the human cardiac myosins were confirmed by analysis of the peptides produced by limited enzymatic digestion of the heavy chains; a few peptide differences were consistently found. To summarize the two separated forms of atrial myosin have different heavy chains, but they have similar if not identical in vitro ATPase activities and the same light chains. One of the atrial myosins is immunologically close to the ventricular myosin, but they each differ with respect to their heavy chains, light chains, and enzymatic activities.

Incomplete contact site A glycoprotein in HL220, a modB mutant of Dictyostelium discoideum.

HL220, a modB mutant that lacks a modification of certain membrane proteins of Dictyostelium discoideum, has been shown to aggregate and to form EDTA-stable intercellular contacts typical of aggregating wild-type cells. A developmentally regulated glycoprotein of 80 X 10(3) apparent molecular weight has been identified as a target site of adhesion-blocking Fab and thought to be involved in EDTA-stable cell contact formation (Müller & Gerisch, 1978). In the HL220 mutant this glycoprotein is no longer recognized by a modB-specific antibody. Therefore, it has been suggested that the 80 X 10(3) Mr glycoprotein, or a modification on it, is not required for the EDTA-stable cell contact of aggregating cells. We show that HL220 synthesizes an equivalent of the 80 X 10(3) Mr glycoprotein with an apparent molecular weight of 68 X 10(3). The mutant product reacted with certain monoclonal antibodies highly specific for the 80 X 10(3) Mr glycoprotein in the wild type, and was developmentally regulated like the 80 X 10(3) Mr glycoprotein. These results indicate that the 68 X 10(3) Mr protein of the mutant lacks a modification, most likely an oligosaccharide residue, the absence of which causes the substantial shift of the apparent molecular weight from 80 X 10(3) to 68 X 10(3). Monoclonal antibodies that did not react with proteins of the mutant could be classified according to their reactions with different sub-sets of wild-type proteins. These results indicate that the proteins that reacted with either one or the other antibody were not modified by a uniform structure. The modification rather varies from one sub-set of cross-reacting proteins to another, suggesting differences between the glycosyl residues of the partially cross-reacting proteins. HL220 cells showed strongly reduced EDTA-stable contact formation under our conditions. EDTA-sensitive intercellular adhesion was undetectable in the mutant, whereas adhesion of the cells to the substratum appeared to be strengthened. The rear ends of the cells, in particular, were tightly attached to glass or Teflon surfaces. The mutant cells were capable of responding chemotactically. Propagated excitation waves like those known to be based on periodic cyclic AMP production and relay were clearly seen. Extracellular phosphodiesterase induction by cyclic AMP and phosphodiesterase inhibitor production were normal. These results indicate that the generation of chemotactic signals and the cellular responses to cyclic AMP are not severely affected by the mutation.

Flagella and motility behaviour of square bacteria.

Square bacteria are shown to have right-handed helical (RH) flagella. They swim forward by clockwise (CW), and backwards by counterclockwise (CCW) rotation of their flagella. They are propelled by several or single filaments arising at several or single points on the cell surface. When there are several filaments a stable bundle is formed that does not fly apart during the change from clockwise to counterclockwise rotation or vice versa. In addition to the flagella attached to the cells, large amounts of detached flagella aggregated into thick super-flagella, can be observed at all phases of growth.

Localization of two phosphorylation sites adjacent to a region important for polymerization on the tail of Dictyostelium myosin.

Phosphorylation of the myosin heavy chains of Dictyostelium discoideum is known to be inhibited following chemotactic stimulation of the cells. Effects of dephosphorylation on the assembly of myosin and on its actin-activated ATPase activity raised the question of where the phosphorylated sites are located with respect to sites responsible for polymerization and actin binding. Using seven monoclonal antibodies the binding sites of which were mapped in the electron microscope, two phosphorylation sites, i.e., threonine residues that were phosphorylated by a kinase from D. discoideum, were localized by immunoblotting of chymotryptic fragments. Two of the antibodies bound to the terminal one fifth of the tail and recognized a phosphorylated chymotryptic fragment of 38 kd. The non-phosphorylated form and single and double phosphorylated forms of this fragment were separated by two-dimensional electrophoresis. Antibody labeling of lower mol. wt. polypeptides indicated that both phosphorylation sites were located at least 32 kd from the end of the tail. A non-phosphorylated fragment, that was insoluble at low ionic strength due to polymerization, proved to be an internal cleavage product of the tail. A segment of this fragment necessary for polymerization was mapped adjacent to the phosphorylation sites.

Photomorphogenesis in Physarum: induction of tubulins and sporulation-specific proteins and of their mRNAs.

Sporangiophore formation in Physarum plasmodia starts about 10 hr after photoinduction. It is characterized by the induction of two tubulins and of at least 15 major sporangiophore morphogenetic proteins. In vitro translation of extracted mRNA revealed that differential gene expression is based on a highly synchronous temporal program of loss of plasmodial and induction of sporulation-specific mRNA species. Using a cloned cDNA encoding part of a sporangiophore morphogenetic protein from Physarum as a probe it was found that the induction of the complementary mRNA activity is due to the induction of the mRNA itself. The results suggest that light induces, with a lag phase of about 10 hr, the transient activation of sporulation-specific genes.

Responses of Dictyostelium discoideum amoebae to local stimulation by light.

Single amoebae of Dictyostelium discoideum were locally stimulated with microbeams of white and monochromatic light. Low illuminance stimulation favored formation of pseudopodia at the irradiated parts of the cells, high illuminance stimulation locally suppressed the extension of pseudopodia. When the high illuminance light spot was placed on any portion of the cell other than the moving front, no response could be observed. The results are compatible with the assumption that, during their phototactic response, single amoebae detect the direction of light by a shadowing effect caused by pigments like cytochromes, and/or by light scattering of particles in the cytoplasm.

Electron microscopic mapping of monoclonal antibodies on the tail region of Dictyostelium myosin.

The binding sites of five monoclonal antibodies against myosin of Dictyostelium discoideum have been mapped. These antibodies bind to the tail region of the myosin molecule. By rotary shadowing, images of myosin-antibody complexes were obtained in which the mean distance of the midpoint of an antibody molecule from the myosin heads was localized with a precision better than 2 nm (90% confidence limit). Other quantitative data extracted from electron micrographs provided information on the stoichiometry of antibody-myosin interaction. Certain antibodies interacted with myosin molecules only at a ratio of 1:1. Other antibodies formed complexes of two molecules bound to homologous sites on a double-stranded myosin tail. Affinities were estimated and the abilities of different antibodies to cross-connect two myosin molecules were evaluated.

Differential expression of Rous Sarcoma virus-specific transformation parameters in enucleated cells.

Chicken embryo fibroblasts transformed with the Ta and ts68 mutants of Rous Sarcoma virus (RSV) were enucleated and studied for their capacity to express reversibly the transformed phenotype in response to temperature changes. After shift to the permissive temperature (35 degrees C), the cytoplasts acquired a transformed morphology and displayed characteristic ruffles and microvilli at their surface. As detected by immunofluorescence, they also lost their actin filament cables and exhibited characteristic changes in the pattern of cell surface structures containing LETS protein. Expression of all these transformation parameters was reversible after shiftback to the nonpermissive temperature (41 degrees C). These results indicate that a whole set of changes characteristic for the transformed phenotype can be expressed independently of the cell nucleus. In contrast, ts mutant-infected cytoplasts were no longer able to respond to temperature shifts with changes in their hexose transport rate. Cytoplasts prepared from cells grown at 41 degrees C retained their low rate of hexose uptake after shift to 35 degrees C, whereas cytoplasts from cells grown at 35 degrees C exhibited a high rate of hexose transport even after 10 hr of shift to 41 degrees C. These results are in accordance with the hypothesis that the product of the src gene of RSV represents a multifunctional protein which acts independently on nuclear and extranuclear sites.

[Light microscopic and scanning electron microscopic observations on phagocytosis of Plasmodium berghei infected erythrocytes by mouse macrophages [author's transl)]. / Lichtmikroskopische und rasterelektronenmikroskopische Befunde zur Phagozytose Plasmodium berghei-infizierter roter tblutkörperchen durch Mäusemakrophagen.

Peritoneal macrophages from mice were cultured in Leighton tubes for 2 hours. Thereafter suspensions of washed red blood corpuscles originating from Plasmodium berghei-infected mice were offered to the cultured cells for phagocytosis. After 15 minutes of incubation 13,5% of the macrophages showed phagocytized parasites if the peritoneal cells came from malariaimmune mice. Cells from normal mice or from infected mice had lower rates (4,8/9,1%). After an incubation of 3 hours about 50% of the cells showed phagocytosis in all three groups. The system used allowed to estimate the halftimes for intracellular elimination of the parasites. The values are 27 minutes for cells from immune animals, 57 minutes for cells from normal animals, and 66 minutes for cells from infected animals. Details of the process of phatocytosis were recorded by scanning electron microscopic pictures.

In vitro transformation with avian myelocytomatosis virus strain CMII: characterization of the virus and its target cells.

Avian myelocytomatosis virus strain CMII induced an in vitro transformation in cells from various hematopoietic tissues and could be quantitated by focus and soft agar colony assay techniques. The CMII-transformed bone marrow cells had a high proliferative capacity in comparison to uninfected controls. The cells closely resembled hematopoietic cells transformed by strain MC29 myelocytomatosis virus, but differed from avian myeloblastosis virus (AMV)-transformed cells. They were phagocytic, became adherent under certain conditions of culturing, and required colony-stimulating factor for colony formation in semisolid medium. These properties are characteristic for cells of the granulocyte/macrophage lineage of differentiation. In contrast to avian erythroblastosis virus, CMII effectively transformed macrophage cultures suggesting that the target cell belongs to the corresponding differentiation lineage. That it is not identical to normal granulocyte/macrophage colony-forming cells was demonstrated by cell separation experiments. In addition to hematopoietic cells, CMII induced a morphological transformation in chicken fibroblasts. CMII was found to consist of a mixture of a transforming component and an associated nontransforming virus of subgroup B or D. The transforming component is defective for replication and could be complemented by standard helper viruses of subgroups B, C, and D.