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  • 1
    Electronic Resource
    Electronic Resource
    A comparison of methods used to characterize gelation of actin in vitro (1984)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 4 (1984), S. 197-213 
    Details
    ISSN: 0886-1544
    Keywords: gelation ; actin ; filamin ; cytoplasm ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have compared the meniscus depletion assay and falling ball viscometry, two means of assessing the extent of gelation in actin-based systems using mixtures of actin and the actin-binding protein filamin. We examined the effect of varying the concentrations of actin and filamin in both assays. The interaction of actin and filamin was detected only above a threshold concentration of filamin. This threshold concentration was lower for falling ball viscometry than for the meniscus depletion assay at equal actin concentrations. At constant concentrations of filamin, an increase in actin concentration caused an increase in apparent viscosity measured by the falling ball assay, but a decrease in sedimentability detected by the meniscus depletion assay. The rate of sedimentation of actin was dependent on the molar ratio of actin to filamin. At each molar ratio, the sedimentation of actin was not dependent on the specific concentrations of actin and filamin used. The apparent viscosity was dependent on both the molar ratio and the specific concentrations of actin and filamin. To relate the present results to earlier studies, we examined mixtures of actin and filamin using a macroscopic assay of gelation (tube tipping assay), and polarized light microscopy. The effect of increasing filamin concentration in the four assays was compared at three actin concentrations. Mixtures of actin and filamin whose apparent viscosities were low enough to be estimated by falling ball viscometry were optically isotropic fluids that flowed out of inverted test tubes. Mixtures of actin and filamin in the range of sensitivity of the meniscus depletion assay were either viscous fluids or gels, and were either optically isotropic or anisotropic. Thus, the four assays provide different estimates of gelation. Both the meniscus depletion assay and falling ball viscometry can be used to determine relative gelation activity, but neither can be used as a quantitative assay of gelation.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970040305
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  • 2
    Electronic Resource
    Electronic Resource
    Effects of villin on the polymerization and subunit exchange of actin (1983)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 3 (1983), S. 151-165 
    Details
    ISSN: 0886-1544
    Keywords: actin ; villin ; fluorescence ; energy transfer ; polymerization ; microfilament ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have investigated the Ca2+-dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy tranfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+(∼20 μM), villin affects both the nucleation and the elongation phases of actin polymerization. Consistent with previous reports, villin stimulates the nucleation process and will form stable nuclei under depolymerization conditions. Compared to the control, the net rate of polymerization is slightly inhibited at low con-centrations of villin (villin/actin ∼ 1:400) but is stimulated at higher concentrations (villin/actin 〉 1:100). Villin also significantly increases the critical concentration of actin polymerization. Addition of either villin or villin-actin complexes induces depolymerization of preassembled actin filaments. This villin-induced depolymerization is reversible upon removal of free Ca2+ or upon the addition of phalloidin. The exchange of actin subunits at steady state is inhibited at low concentrations of villin (villin/actin ∼ 1:200) but is stimulated at higher concentrations (villin/actin ∼ 1:50). None of the above effects is observed at 〈 10-8 M free [Ca2+].
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970030205
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  • 3
    Electronic Resource
    Electronic Resource
    Selective immunocytochemical detection of fluorescent analogs with antibodies specific for the fluorophore (1984)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 4 (1984), S. 137-149 
    Details
    ISSN: 0886-1544
    Keywords: anti-fluorescein ; fluorescent analog cytochemistry ; molecular cytochemistry ; microinjection ; actin ; acetamidofluorescein-actin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Fluorescent analogs of cellular components are finding increasing use in the field of cell biology. The power of this technique can be augmented by the use of antibodies specific for the fluorophore to visualize selectively the fluorescent analog at the electron microscope level. Rabbit antibodies specific for fluorescein were elicited and purified according to published methods (Lopatin and Voss [1971]: Biochemistry 10:208). Immune sera and IgG formed precipitin lines with fluorescein-labeled proteins in Ouchterlony immunodiffusion assays, and significantly quenched the fluorescence of fluorescein-labeled proteins. Immune IgG and Fab fragments decorated fluorescein-labeled actin, but not unlabeled actin, in negative-stained preparations. Anti-fluorescein IgG was used for immunofluorescent localization of fluorescein-labeled actin following microinjection of the fluorescent analog into living cells. This approach was extended to the immunoelectron microscopic localization of the injected analog at the subcellular level by the use of an electron-dense marker coupled to goat anti-rabbit IgG. Many other fluorescent probes also can be used as haptens for production of antibodies. Therefore, a general method for localizing fluorescently labeled molecules at the electron microscopic level is now available. Several other applications of anti-fluorescein antibody in studies involving fluorescent analogs are also suggested.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970040207
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  • 4
    Electronic Resource
    Electronic Resource
    The molecular mobility of α-actinin and actin in a reconstituted model of gelation (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 64-82 
    Details
    ISSN: 0886-1544
    Keywords: D.d. α-actinin ; actin ; gelation ; fluorescence photobleaching recovery ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Dictyostelium discoideum α-actinin (D.d.α-actinin) is a calcium and pH-regulated actin-binding protein that can cross-link F-actin into a gel at a submicromolar free calcium concentration and a pH 〈7 [Fechheimer, et al., 1982]. We examined mixtures of actin and D.d. α-actinin at four pH and calcium concentrations that exhibited various degrees of gelation or solation. The macroscopic viscosities of these mixtures were measured by falling ball viscometry (FBV) and compared to the translational diffusion coefficients measured by gaussian spot and periodic-pattern fluorescence photobleaching recovery (FPR) of both the actin filaments and D.d. α-actinin. A homogeneous, macroscopic gel was not composed of a static actin network. Instead, the filament diffusion coefficient decreased to ∼65% of the control value. If the D.d. α-actinin concentration was increased, the solution became inhomogeneous, consisting of domains of higher actin concentration. These domains were often composed of a static actin network. The mobility of D.d. α-actinin consisted of a major fraction that freely diffused and a minor fraction that appeared immobile under the conditions employed. This suggested that D.d. α-actinin binding to the actin filaments was static over the time course of measurement (∼5 sec). Under solation conditions, there was no apparent interaction of actin with D.d. α-actinin. These results demonstrate that (1) actin filaments need not be cross-linked into an immobile, static array in order to have macroscopic properties of a gel, (2) interpretation of the rheological properties of actin:α-actinin gels are complicated by spatial heterogeneity of the filament concentration and mobility; and (3) a fraction of D.d. α-actinin binds statically to actin in undisturbed gels. The implications of these results are discussed in relation to cytoplasmic structure and contractility.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110107
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