Junctional plasma membrane domains isolated from aggregating Dictyostelium discoideum amebae (original) (raw)
Related papers
2000
In novel, low-speed sedimentation assays, highly purified, sonicated Dictyostelium discoideum plasma membrane fragments bind to F-actin beads (fluoresce in-labeled F-actin on antifluorescein IgG-Sephacryl S-1000 beads) . Binding was found to be (a) specific, since beads containing bound fluorescein-labeled ovalbumin or beads without bound fluorescein-labeled protein do not bind membranes, (b) saturable at -0.6 ug of membrane protein per microgram of bead-bound F-actin, (c) rapid with a b,, of 4-20 min, and (d) apparently of reasonable affinity since the off rate is too slow to be measured by present techniques. Using low-speed sedimentation assays, we found that sonicated plasma membrane fragments, after extraction with chaotropes, still bind F-actin beads. Heat-denatured membranes, proteolyzed membranes, and D. discoideum lipid vesicles did not bind F-actin beads. These results indicate that integral membrane proteins are responsible for the binding between sonicated membrane fragments and F-actin on beads . This finding agrees with the previous observation that integral proteins mediate interactions between D. discoideum plasma membranes and F-actin in solution (Luna, E. J ., V. M. Fowler, J . Swanson, D. Branton, and D. L. Taylor, 1981, J. Cell Biol., 88 :396-409). We conclude that low-speed sedimentation assays using F-actin beads are a reliable method for monitoring the associations between F-actin and membranes . Since these assays are relatively quantitative and require only micrograms of membranes and F-actin, they are a significant improvement over other existing techniques for exploring the biochemical details of F-actin-membrane interactions .
Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum
Journal of Cell Biology, 1986
The binding of native, 125I-Bolton-Hunterlabeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. In the presence of gelsolin, the amount of actin bound at saturation to three different membrane preparations was 80, 120, and 200 ~g/mg of membrane protein. The respective concentrations of actin at half-saturation were 8, 12, and 18 ~g/ml. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape.
The EMBO journal, 1985
Expression of developmentally regulated membrane proteins of aggregating cells of Dictyostelium discoideum is subject to several control mechanisms. One of them involves periodic cyclic-AMP pulses as signals for gene expression. To increase the probability of selecting mutants specifically defective in the contact site A (csA) glycoprotein, one of the characteristic proteins of aggregating cells, we have bypassed the requirement for both cyclic-AMP pulses and another control element by two runs of mutagenesis. A ;double bypass' mutant, HG592, was obtained which aggregated in nutrient medium where wild-type did not develop. Mutants defective in expression of the csA-glycoprotein were selected from HG592 by fluorescence-activated cell sorting and colony immunoblotting using a monoclonal antibody specific for that protein. One among 51 csA-negative mutants, HG693, specifically lacked the capability of forming EDTA-stable intercellular contacts. It acquired chemotactic responsivenes...
The Journal of Cell Biology, 1987
During the early phase of Dictyostelium discoideum development, cells undergo chemotactic migration to form tight aggregates. A developmentally regulated surface glycoprotein of Mr 80,000 (gp80) has been implicated in mediating the EDTA-resistant type of cell cohesion at this stage. We have used a monoclonal antibody directed against gp80 to study the topographical distribution of gp80 on the cell surface. Indirect immunofluorescence studies showed that gp80 was primarily localized on the cell surface, with a higher concentration at contact areas. Immunoelectron microscopy was carried out by indirect labeling using protein A-gold, and a nonrandom distribution of gp80 was revealed. In addition to contact regions, gold particles were found preferentially localized on filopodia. Quantitative analysis using transmission electron microscopy (TEM) showed that approximately 60% more gold particles were localized in contact regions in comparison with the noncontact regions, and the filopodi...
Molecular and cellular biology, 1983
WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized...
How actin binds and assembles onto plasma membranes from Dictyostelium discoideum
Journal of Cell Biology, 1988
We have shown previously . J. Cell Biol. 102: 2067-2075.) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross-linking experiments show that membrane-bound actin is multimeric. Thus, Samples with or without 350 ktg/ml plasma membranes and with or without 5 ~tM phalloidin were prepared in 20 I.tl of polymerization buffer with 0.02 % Tween 20. Then, either ~25I-labeled EF actin or ~25I-labeled untreated actin
2000
Dictyosteliumdiscoideum plasma membranes isolatedby each of three procedures bind F-actin.The interactions between these membranes and actin are examined by a novel application of fallingball viscometry .Treating the membranes as multivalent actin-binding particlesanalogous to divalent actin-gelation factors,we observe large increases in viscosity (actincross-linking)when membranes depleted of actinand myosin are incubated with rabbit skeletalmuscle F-actin.Pre-extractionof peripheral membrane proteins with chaotropes or the
Journal of Cell Biology, 1987
F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gpl7) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gpl7 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gpl7 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gpl7 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gpl7 9 The Rockefeller University Press, 0021-9525/
Cell Research, 2009
To investigate the roles of substrate adhesion in cytokinesis, we established cell lines lacking paxillin (PAXB) or vinculin (VINA), and those expressing the respective GFP fusion proteins in Dictyostelium discoideum. As in mammalian cells, GFP-PAXB and GFP-VINA formed focal adhesion-like complexes on the cell bottom. paxB − cells in suspension grew normally, but on substrates, often failed to divide after regression of the furrow. The efficient cytokinesis of paxB − cells in suspension is not because of shear forces to assist abscission, as they divided normally in static suspension culture as well. Double knockout strains lacking mhcA, which codes for myosin II, and paxB or vinA displayed more severe cytokinetic defects than each single knockout strain. In mitotic wild-type cells, GFP-PAXB was diffusely distributed on the basal membrane, but was strikingly condensed along the polar edges in mitotic mhcA − cells. These results are consistent with our idea that Dictyostelium displays two forms of cytokinesis, one that is contractile ringdependent and adhesion-independent, and the other that is contractile ring-independent and adhesion-dependent, and that the latter requires PAXB and VINA. Furthermore, that paxB − cells fail to divide normally in the presence of substrate adhesion suggests that this adhesion molecule may play additional signaling roles.