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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
    Correlated distribution of actin, myosin, and microtubules at the leading edge of migrating swiss 3T3 fibroblasts (1989)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 14 (1989), S. 527-543 
    Details
    ISSN: 0886-1544
    Keywords: immunofluorescence ; video-enhanced contrast microscopy ; protrusions ; lamellipodia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The formation of lamellipodia in migrating cells involves dynamic processes that occur in a cyclic manner as the leading edge of a cell slowly advances. We used video-enhanced contrast microscopy (VEC) to monitor the motile behavior of cells to classify protrusions into the temporal stages of initial and established protrusions (Fisher et al.: Cell Motility and the Cytoskeleton 11:235-247, 1988), and to monitor the fixation of cells. Multiple parameter fluorescence imaging methods (DeBiasio et al.: Journal of Cell Biology 105:1613-1622, 1987; Waggoner et al.: Methods in Cell Biology, Vol. 30, Part B, pp. 449-478, 1989) were then used to determine and to map accurately the distributions of actin, myosin and microtubules in specific types of protrusions. Initial protrustions exhibited no substructure as evidenced by VEC and actin was diffusely arranged, while myosin and microtubules were absent. Newly established protrusions contained diffuse actin as well as actin in microspikes. There was a delay in the appearance of myosin into established protrusions relative to the presence of actin. Microtubules were found in established protrusions after myosin was detected, and they were oriented parallel to the direction of migration. Actin and myosin were also localized in fibers transverse to the direction of migration at the base of initial and established protrusions. Image analysis was used to quantify the orientation of actin fibers relative to the leading edge of motile cells. The combined use of VEC, multiple parameter immunofluorescence, and image analysis should have a major impact on defining complex relationships within cells.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970140410
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  • 3
    Electronic Resource
    Electronic Resource
    Preface (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 1-1 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100102
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  • 4
    Electronic Resource
    Electronic Resource
    Subcellular compartmentalization by local differentiation of cytoplasmic structure (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 28-37 
    Details
    ISSN: 0886-1544
    Keywords: cytomatrix ; cytoplasmic ground substance ; ratio imaging ; fluorescence photobleaching recovery ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The compartmentalization of eukaryotic cells by internal membranes and the subcellular localization of endogenous macromolecules by specific binding mechanisms are familiar concepts. In this report we present evidence that the cytoplasmic ground substance, which surrounds and contains the membranebound compartments, may also be compartmentalized by local differentiations of its submicroscopic structure that sort subcellular particles on the basis of size. The subcellular distribution of size-fractionated, fluorescent tracer particles was studied in living cells by ratio imaging and fluorescence recovery after photobleaching (FRAP). Large and small particles showed different distributions within the cytoplasmic volume, suggesting that the large particles were relatively excluded from some domains. While the structural basis for this phenomenon is not yet understood in detail, ratio imaging of large and small particles can be used as an empirical tool to identify cytoplasmic compartments for further study. The cytoplasmic diffusion coefficient (Dcyto) and % mobile fraction of the large particles showed considerable spatial variation over the projected area of the cell, while Dcyto and % mobile fraction of the small particles did not. A model is presented to account for this difference. Based on this model, a method is proposed by which FRAP can be used to detect sol-gel transitions in the cytoplasmic ground substance of living cells.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100107
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  • 5
    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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  • 6
    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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  • 7
    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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  • 8
    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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  • 9
    Electronic Resource
    Electronic Resource
    Centripetal transport of cytoplasm, actin, and the cell surface in lamellipodia of fibroblasts (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 235-247 
    Details
    ISSN: 0886-1544
    Keywords: video-enhanced contrast microscopy ; transverse fibers ; transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Wound healing in Swiss 3T3 cultures was investigated with video-enhanced contrast (VEC) microscopy. The formation of protrusions at the leading edge of cells along wounds was investigated in detail during the spreading stage, which usually lasted from 1 to 4 hr postwounding. Lamellipodia exhibited a continuous rearward, or centripetal, transport of a variety of cellular constituents at rates of ∼0.26 μm/sec from the leading edge. The lamellipodia were also the sites of lateral migration as well as extension and retraction of actin microspikes. Actin fibers oriented transversely to the direction of movement were also observed to transport centripetally at similar rates. These fibers may in part give rise to large actin fibers forming at the interface between the base of the lamellipodia and the lamellae. Beads 0.5 μm in diameter attached to the dorsal surfaces of lamellipodia also transported centripetally at rates of ∼0.21 μm/sec. Thus there is an apparent correlation between transport of a variety of structures within lamellipodia and with surface movements of lamellipodia.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110403
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  • 10
    Electronic Resource
    Electronic Resource
    Actin-binding proteins regulate the work performed by myosin II motors on single actin filaments (1992)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 274-280 
    Details
    ISSN: 0886-1544
    Keywords: motility assay ; gelation ; solation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Regulation of actin/myosin II force generation by calcium [Kamm and Stull, Annu. Rev. Physiol. 51:299-313, 1989] and phosphorylation of myosin II light chains [Sellers and Adelstein, “The Enzymes,” Vol. 18, Orlando, FL: Academic Press, 1987, pp. 381-418] is well established. However, additional regulation of actin/myosin II force generation/contraction may result from actin-binding proteins [Stossel et al., Ann. Rev. Cell Biol. 1:353-402, 1985; Pollard and Cooper, Ann. Rev. Biochem. 55:987-1035, 1986] as they affect the gel state of the actin cytomatrix [reviewed in Taylor and Condeelis, Int. Rev. Cytol., 56:57-143, 1979]. Regulation of the gel state of actin may determine whether an isotonic or isometric contraction results from the interaction between myosin and actin. We have extended the single actin filament motility assay of Kron and Spudich [Proc. Natl. Acad. Sci. U.S.A. 83:6272-6276, 1986] by including filamin or α-actinin on the substrate with myosin II to examine how actin-crosslinking proteins regulate the movements of single actin filaments. Increasing amounts of actin-crosslinking proteins inhibit filament velocity and decrease the number of filaments moving. Reversal of crosslinking yields increased velocities and numbers of moving filaments. These results support the solation-contraction coupling hypothesis [see Taylor and Fechheimer, Phil. Trans. Soc. London B 299:185-197, 1982] which proposes that increased crosslinking of actin inhibits myosin-based contraction. This study also illustrates the potentially varied roles of different actin-crosslinking proteins and offers a novel method to examine actin-binding protein activity and their regulation of motility at the single molecule level. © 1992 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970220407
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