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  • 1
    Electronic Resource
    Electronic Resource
    A calcium- and pH-regulated actin binding protein from D. discoideum (1982)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 2 (1982), S. 287-308 
    Details
    ISSN: 0886-1544
    Keywords: actin-binding protein ; Dictyostelium ; cytoskeleton ; amoeboid movement ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A protein from Dictyostelium discoideum with an apparent subunit molecular weight of 95,000 daltons (95K protein) was previously identified as an actin-binding protein ‘Hellewell and Taylor, 1979’. In this paper, we present a method for purifying the protein, and characterize some important aspects of its structure and function. Purification of the 95K protein is achieved by fractionation with ammonium sulfate followed by chromatography on DEAE-cellulose, gel filtration on 6% agarose, and final purification on hydroxyapatite. The 95K protein is a dimer, composed of apparently identical subunits. It is a rod-shaped molecule, 38 nm in length, with a Stokes radius of 74 Å. In these structural properties, the 95K protein is similar to muscle and nonmuscle α-actinins. The 95K protein and filamin are equally competent, when compared on a weight basis, to enhance the apparent viscosity of actin as determined by falling ball viscometry. The apparent viscosity of mixtures of the 95K protein and actin is dramatically reduced at pH greater than 7.0 or free ‘Ca2+’ greater than 10-7 M. We also examine the mechanism by which calcium regulates the interaction of the 95K protein and actin. A change in free ‘Ca2+’ induces no detectable change in the quaternary structure of the 95K protein. Our experiments indicate that the 95K protein does not dramatically alter the length distribution of actin filaments in the presence of micromolar free ‘Ca2+’. A large fraction of the 95K protein cosediments with actin in the presence of low free ‘Ca2+’ (ca. 3 × 10-8M), but not in the presence of high free ‘Ca2+’ (ca. 4 × 10-6M). We conclude that increased free ‘Ca2+’ inhibits gelation of actin by the 95K protein by reducing the affinity of the 95K protein for actin. We propose that 95K protein is an important component of the cytoskeletal/contractile system in D. discoideum amoebae.
    Additional Material: 14 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970020308
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  • 2
    Electronic Resource
    Electronic Resource
    Direct binding of F-actin to ponticulin, an Integral plasma membrane glycoprotein (1991)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 18 (1991), S. 164-179 
    Details
    ISSN: 0886-1544
    Keywords: actin-binding proteins ; actin-membrane interactions ; blot overlays ; cytoskeleton ; Dictyostelium discoideum ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have developed an 125I-labeled F-actin blot overlay assay for the identification of F-actin-binding proteins after transfer to nitrocellulose from SDS-polyacryl-amide gels. Two major F-actin-binding proteins from Dictyostelium discoideum, a cytoplasmic 30 kDa protein and a 17 kDa integral membrane protein, and two minor membrane polypeptides of 19 kDa and 15 kDa were detected by this method. Using F-actin affinity and immunoaffinity chromatography, the 17 kDa polypeptide was identified as ponticulin, a previously described actin-binding glycoprotein from D. discoideum plasma membranes (Wuestehube, L.J., and Luna, E.J. [1987]: J. Cell Biol. 105:1741-1751). The binding of F-actin to ponticulin on blots is specific because unlabeled F-actin competes with 125I-labeled F-actin and because G-actin does not bind. Nitrocellulose-bound ponticulin displays binding characteristics similar to those of purified plasma membranes in solution, e.g., F-actin binding is sensitive to high salt and to elevated temperatures. Under optimal conditions, 125I-labeled F-actin blot overlays are at least as sensitive as are immunoblots with an antibody specific for ponticulin. When blotted onto nitrocellulose after 2-D gel electrophoresis, all isoforms of ponticulin and of the 19 kDa and 15 kDa polypeptides appear to bind F-actin in proportion to their abundance. Thus the actin-binding activities of these proteins do not appear to be regulated by modifications that affect isoelectric point. However, the actin-binding activity of nitrocellulose-bound ponticulin is diminished when the protein is exposed to reducing agents, suggesting an involvement of disulfide bond(s) in ponticulin function. The 125I-labeled F-actin blot overlay assay also may enable us to identify F-actin-binding proteins in other cell types and should provide a convenient method for monitoring the purification of these proteins.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970180303
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  • 3
    Electronic Resource
    Electronic Resource
    Indirect immunofluorescence localization of ponticulin in motile cells (1989)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 13 (1989), S. 245-263 
    Details
    ISSN: 0886-1544
    Keywords: actin ; actin-binding protein ; plasma membranes ; cytoskeleton ; immunofluorescence microscopy ; cell motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microcopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggreating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrace and that the actin-binding activity of ponticulin may be tightly controlled.Indirect immupofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified aganst D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970130404
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  • 4
    Electronic Resource
    Electronic Resource
    Actin-Associated proteins in Dictyostelium discoideum (1990)
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 11 (1990), S. 328-332 
    Details
    ISSN: 0192-253X
    Keywords: Cellular slime molds ; cytoskeleton ; actin-binding proteins ; review ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The cellular slime mold Dictyostelium discoideum is becoming the premier system for the explication of the biochemical and cellular events that occur during motile processes. Proteins associated with the actin cytoskeleton, in particular, appear to play key roles in cellular responses to many external stimuli. This review summarizes our present understanding of the actin-associated proteins in Dictyostelium, including their in vitro activities and their structural and/or functional analogues in mammalian cells.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/dvg.1020110503
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  • 5
    Electronic Resource
    Electronic Resource
    Ponticulin, a developmentally-regulated plasma membrane glycoprotein, mediates actin binding and nucleation (1990)
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 11 (1990), S. 354-361 
    Details
    ISSN: 0192-253X
    Keywords: Amoeboid movement ; cell adhesion ; cytoskeleton ; cell motility ; Dictyostelium ; microfilaments ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Ponticulin is a 17,000-dalton transmembrane glycoprotein that is involved in the binding and nucleation of actin filaments by Dictyostelium discoideum plasma membranes. The major actin-binding protein isolated from these membranes by F-actin affinity chromatography, ponticulin also binds F-actin on blot overlays. The actin-binding activity of ponticulin in vitro is identical to that observed for purified plasma membranes: it resists extraction with 0.1 N NaOH, is sensitive to high salt concentrations, and is destroyed by heat, proteolysis, and thiol reduction and alkylation. A cytoplasmic domain of ponticulin mediates binding to actin because univalent antibody fragments directed against the cytoplasmic surface of this protein inhibit 96% of the actin-membrane binding in sedimenlation assays. Antibody specific for ponticulin emoves both ponticu-lin and the ability to reconstitute actin nucleation activity from detergent extracts of solubilized plasma membranes. Levels of plasma membrane ponticulin increase 2- to 3-fold during aggregation streaming, when cells adhere to each other and are highly motile. Although present throughout the plasma membrane, ponticulin is preferentially localized to some actin-rich membrane structures, including sites of cell-cell adhesion and arched regions of the plasma membrane reminiscent of the early stages of pseudopod formation. Ponticulin also is present but not obviously enriched at phagocytic cups of log-phase amebae. These results indicate that ponticulin may function in vivo to attach and nucleate actin filaments at the cytoplas-mic surface of the plasma membrane. A 17,000-dalton analogue of ponticulin has been identified in human polymorphonuclear leukocyte plasma membranes by immunoblotting and immunofluo-rescence microscopy. These findings suggest that the structure and function of ponticulin in motile cells has been evolutionarily conserved.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/dvg.1020110506
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