Proximity relationships and structural dynamics of the phalloidin binding site of actin filaments in solution and on single actin filaments on heavy meromyosin

Heidecker, Manfred; Yan-Marriott, Yuling; Marriott, Gerard

doi:10.1021/bi00035a007

Cited by 33 publications

(48 citation statements)

References 19 publications

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“…The binding site for phalloidin on actin has been previously identified and is at the interface of three actin subunits (19). The finding that jasplakinolide stabilizes an actin trimer sufficiently to ensure that it will elongate rather than dissociate (Fig.…”

Section: The Effects Of Jasplakinolide On the Time Course Of Actin Pomentioning

confidence: 92%

Effects of Jasplakinolide on the Kinetics of Actin Polymerization

Bubb

Spector

Beyer

et al. 2000

Journal of Biological Chemistry

487

399

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Jasplakinolide paradoxically stabilizes actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses. A detailed analysis of the effects of jasplakinolide on the kinetics of actin polymerization suggests a resolution to this paradox. Jasplakinolide markedly enhances the rate of actin filament nucleation. This increase corresponds to a change in the size of actin oligomer capable of nucleating filament growth from four to approximately three subunits, which is mechanistically consistent with the localization of the jasplakinolide-binding site at an interface of three actin subunits. Because jasplakinolide both decreases the amount of sequestered actin (by lowering the critical concentration of actin) and augments nucleation, the enhancement of polymerization by jasplakinolide is amplified in the presence of actin-monomer sequestering proteins such as thymosin ␤ 4 . Overall, the kinetic parameters in vitro define the mechanism by which jasplakinolide induces polymerization of monomeric actin in vivo. Expected consequences of jasplakinolide function are consistent with the experimental observations and include de novo nucleation resulting in disordered polymeric actin and in insufficient monomeric actin to allow for remodeling of stress fibers.Jasplakinolide is a cyclic peptide isolated from the marine sponge, Jaspis johnstoni, that we have previously shown to bind to and stabilize filamentous actin in vitro (1). In vivo data suggests that jasplakinolide-treated prostate cancer cells have both decreased labeling of F-actin and decreased amounts of rhodamine-phalloidin bound to cell extracts (2), results that could be explained by the observation that jasplakinolide and phalloidin bind competitively to actin (1). In addition, however, in vivo data also convincingly show that jasplakinolide disrupts actin filaments with alterations in cellular architecture (2, 3), an effect that cannot be explained simply by competitive binding. We now present kinetic data characterizing the steady state and time-dependent in vitro interactions between jasplakinolide and actin that provide a plausible explanation for the effects of jasplakinolide on actin distribution in cultured cells. EXPERIMENTAL PROCEDURESMaterials-Rabbit skeletal muscle actin was prepared from frozen muscle (Pel-Freez, Rogers, AR) in buffer G (5.0 mM Tris, 0.2 mM ATP, 0.2 mM dithiothreitol, 0.1 mM CaCl 2 , and 0.01% sodium azide, pH 7.8) (4). Non-muscle actin from bovine brain was prepared by the method of Ruscha and Himes (5). Muscle and non-muscle pyrenyl-labeled actins 1 were prepared with 0.67-0.95 mol of label/mol of protein using the method of Kouyama and Mihashi (6). Labeled and unlabeled actins were further purified by gel filtration on Superose 12 (Amersham Pharmacia Biotech). Thymosin ␤ 4 cDNA was a gift from Dr. Vivian Nachmias and was inserted in a pET-11a vector, expressed in BL21(DE3) Escherichia coli, and purified as described previously (7). Jasplakinolide was a gift from ...

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Section: The Effects Of Jasplakinolide On the Time Course Of Actin Pomentioning

confidence: 92%

Effects of Jasplakinolide on the Kinetics of Actin Polymerization

Bubb

Spector

Beyer

et al. 2000

Journal of Biological Chemistry

487

399

View full text Add to dashboard Cite

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“…Values of R 0 using Oregon Green were calculated using the quantum yield disodium fluorescein in 0.01 M NaOH as a reference (0.91, see Ref. 39) Microtubule Preparation and ATPase Assays-Microtubules were labeled with mant-GDP in their exchangeable nucleotide-binding site by polymerizing tubulin with 2 mM mant-GTP. The microtubule-activated ATPase rate was measured by mixing 20 -30 nM Eg5 with a greater than 20-fold molar excess of taxol-stabilized microtubules in 100 mM KCl, 25 mM HEPES, 2 mM MgCl 2 , 1 mM dithiothreitol, 0.1% bovine serum albumin, 2 mM ATP, pH 7.20.…”

Section: Methodsmentioning

confidence: 99%

Docking and Rolling, a Model of How the Mitotic Motor Eg5 Works

Rosenfeld

Xing²,

Jefferson

et al. 2005

Journal of Biological Chemistry

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Whereas kinesin I is designed to transport cargoes long distances in isolation, a closely related kinesin motor, Eg5, is designed to generate a sustained opposing force necessary for proper mitotic spindle formation. Do the very different roles for these evolutionarily related motors translate into differences in how they generate movement? We have addressed this question by examining when in the ATPase cycle the Eg5 motor domain and neck linker move through the use of a series of novel spectroscopic probes utilizing fluorescence resonance energy transfer, and we have compared our results to kinesin I. Our results are consistent with a model in which movement in Eg5 occurs in two sequential steps, an ATP-dependent docking of the neck linker, followed by a rotation or "rolling" of the entire motor domain on the microtubule surface that occurs with ATP hydrolysis. These two forms of movement are consistent with the functions of a motor designed to generate sustained opposing force, and hence, our findings support the argument that the mechanochemical features of a molecular motor are shaped more by the demands placed on it than by its particular family of origin.The last decade has witnessed a marked advance in our understanding of how molecular motors generate force and movement (1-6). Studies of both myosins and kinesins have revealed a variety of conserved structural elements that play key roles in mechanochemical transduction. These include switch I, switch II, and the P loop located within the catalytic site (3,5,6). Movements of these elements during ATP binding, hydrolysis, and product release lead to a series of conformational changes that are transmitted ultimately to the "business end" of the motor, the mechanical element that produces force and movement. In kinesin I, this mechanical element consists of an extended peptide sequence with variable conformation and flexibility, called the "neck linker" (2, 3). Spectroscopic and kinetic studies have led to a convincing model in which the neck linker assumes a random coil in the absence of nucleotide. ATP binding to the active site causes the neck linker to dock along a hydrophobic surface in the motor (7-10). This process, which is very rapid (Ͼ800 s Ϫ1 ) at room temperature, immobilizes the neck linker and effectively "throws" the tethered head of a kinesin dimer forward, toward the next tubulin-docking site (11,12,25). Variable flexibility and ATP-induced docking are features of the kinesin I neck linker that are well suited to the physiologic role of this motor as a transport engine. Variable flexibility in the neck linker of the tethered head allows it to undergo a diffusive search for the next microtubule-docking site, whereas ATP-induced docking of the neck linker of the attached head helps position the tethered head in a forward position and reduces the probability of backward stepping (10,(13)(14)(15)(16). Thus, the physiologic requirements placed on a transport motor like kinesin I translate into structural features of its mechanical element that s...

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“…A significant increase in the fluorescence of the labeled TCR was observed due to FRET between the labeled, bleached FP (acceptor) and a TCR-specific FITC-conjugated antibody (donor). The average R 0 (the characteristic Forester distance at which FRET efficiency is 0.5) is under 50 Å for the FITC/Rho pair (19); therefore, the detection of FRET ( Figure 2D) indicates that the donor and acceptor molecules must be fewer than 50 Å apart in the membrane. These results suggest that FP binds to the T cell membrane and preferentially colocalizes with the CD4 and TCR molecules.…”

Section: Fp Colocalizes With Cd4 and Tcrmentioning

confidence: 99%

HIV-1 fusion peptide targets the TCR and inhibits antigen-specific T cell activation

Quintana¹,

Gerber²,

Kent³

et al. 2005

J. Clin. Invest.

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The fusion peptide (FP) in the N terminus of the HIV envelope glycoprotein, gp41, functions together with other gp41 domains to fuse the virion with the host cell membrane. We now report that FP colocalizes with CD4 and TCR molecules, coprecipitates with the TCR, and inhibits antigen-specific T cell proliferation and proinflammatory cytokine secretion in vitro. These effects are specific: T cell activation by PMA/ionomycin or mitogenic antibodies is not affected by FPs, and FPs do not interfere with antigen-presenting cell function. In vivo, FPs inhibit the activation of arthritogenic T cells in the autoimmune disease model of adjuvant arthritis and reduce the disease-associated IFN-γ response. Hence, FPs might play 2 roles in HIV infection: mediating membrane fusion while downregulating T cell responses to itself that could block infection. Disassociated from HIV, however, the FP molecule provides a novel reagent for downregulating undesirable immune responses, exemplified here by adjuvant arthritis.

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Proximity relationships and structural dynamics of the phalloidin binding site of actin filaments in solution and on single actin filaments on heavy meromyosin

Cited by 33 publications

References 19 publications

Effects of Jasplakinolide on the Kinetics of Actin Polymerization

Effects of Jasplakinolide on the Kinetics of Actin Polymerization

Docking and Rolling, a Model of How the Mitotic Motor Eg5 Works

HIV-1 fusion peptide targets the TCR and inhibits antigen-specific T cell activation

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