Probing the phalloidin binding site of actin

Faulstich, Heinz; Zobeley, Suse; Heintz, Daniela; Drewes, Gerard

doi:10.1016/0014-5793(93)80515-v

Cited by 40 publications

(24 citation statements)

References 22 publications

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“…To decrease the effect of actin monomers the actin concentration was adjusted to a relatively large value, 30 M. Furthermore, the FRET efficiency was also measured in the presence of phalloidin, so that the undesired effect of the contribution of actin monomers on the measured fluorescence parameters could be estimated. The stabilizing effect of phalloidin on actin filaments was described earlier by using a number of experimental methods (20,35,(52)(53)(54)(55)(56). It was shown that phalloidin decreased significantly the rate of monomer dissociation and shifted the monomer-filament equilibrium toward the filamentous form.…”

Section: Discussionmentioning

confidence: 95%

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

Nyitrai

Hild

Hartvig

et al. 2000

Journal of Biological Chemistry

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Conformational and dynamic properties of actin filaments polymerized from ATP-or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys 374 residue of actin was smaller in filaments from ADP-actin than in filaments from ATPactin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys 374 from the longitudinal filament axis. Temperaturedependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys 374 of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.Actin is one of the most abundant proteins in biological systems and responsible for a number of cell functions in vivo (1, 2). Two principal forms of actin exist in living cells, the monomeric and the filamentous. Actins biological function depends on the actual dynamic and conformational properties of the protein and on the dynamic equilibrium between the two principal forms (1, 2).The polymerization of actin monomers with bound ATP is accompanied by the parallel hydrolysis of the nucleotide. However, the biological relevance of ATP hydrolysis by actin is still not well understood. Interestingly, ADP-monomeric actin also forms filaments (3), therefore, the presence of ATP and its hydrolysis are not essential for filament assembly. Previously, the hydrolysis of actin-bound ATP was assumed to play a key role in the steady-state treadmilling of actin filaments (4). Alternatively, Carlier (5, 6) suggested that ATP hydrolysis facilitated the rapid de-polymerization of actin.Recently, Janmey and colleagues (7) provided evidence that actin filaments polymerized from ATP-actin monomers were significantly stiffer than the ones obtained from ADP-monomers. Considering that both types of filaments consist mainly of ADP-actin protomers, this observation was explained by the assumption that the ATP-actin monomers were conformationally trapped following the hydrolysis of ATP, and the energy released during the hydrolysis was stored as elastic energy (7). The authors proposed that this elastic energy could play an important role when the actin filament interacted with actinbinding proteins. Although this exciting observation was later supported by other laboratories, a number of experimental results were contradictory.The direct effect of ATP on actin filaments was confirmed and extended to phalloidin-labeled actin by fluorescence microscopy experiments (8). Three-dimensional reconstructions from electron micrographs revealed similar effects of the nucleotides on the flexibility of actin filament (9, 10). The results of phosphorescence spectroscopic experiments apparently also supported the c...

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Section: Discussionmentioning

confidence: 95%

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

Nyitrai

Hild

Hartvig

et al. 2000

Journal of Biological Chemistry

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“…4). Phalloidin was an effective indicator of F-actin, reduced the rate of actin filament depolymerization, and prevented conversion of F-actin to G-actin [46]. It is not clear how the F-actin became distributed throughout the vesicle, since non-filamentary actin is expected to be in the G form and unavailable for phalloidin binding.…”

Section: Gpmv Actin Contentsmentioning

confidence: 99%

Pulsed-laser creation and characterization of giant plasma membrane vesicles from cells

et al. 2009

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Femtosecond-pulsed laser irradiation was found to initiate giant plasma membrane vesicle (GPMV) formation on individual cells. Laser-induced GPMV formation resulted from intracellular cavitation and did not require the addition of chemical stressors to the cellular environment. The viscosity, structure, and contents of laser-induced GPMVs Electronic supplementary material The online version of this article

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“…After further washing, gels were incubated with rhodamine phalloidin (Molecular Probes, Eugene, OR; 1:50 dilution). After a final washing in phosphate buffer, gels were mounted on glass slides with glycerol and sealed with coverslips [Faulstich et al, 1993]. Confocal imaging of cytoskeletal actin was performed using the Noran-Odyssey confocal laser scanning microscope.…”

Section: Cell Morphologymentioning

confidence: 99%

3‐D in vitro model of early skeletal muscle development

Cheema

Yang

Mudera

et al. 2003

Cell Motil. Cytoskeleton

108

138

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An understanding of the mechanical and mechano-molecular responses that occur during the differentiation of mouse C2C12 [corrected] myoblasts in 3-D culture is critical for understanding growth, which is important for progress towards producing a tissue-engineered muscle construct. We have established the main differences in force generation between skeletal myoblasts, dermal fibroblasts, and smooth muscle cells in a 3-D culture model in which cells contract a collagen gel construct. This model was developed to provide a reproducible 3-D muscle organoid in which differences in force generation could be measured, as the skeletal myoblasts fused to form myotubes within a collagen gel. Maintenance of the 3-D culture under sustained uni-axial tension, was found to promote fusion of myoblasts to form aligned multi-nucleate myotubes. Gene expression of both Insulin Like Growth Factor (IGF-1 Ea) and an isoform of IGF-1 Ea, Mechano-growth factor (IGF-1 Eb, also termed MGF), was monitored in this differentiating collagen construct over the time course of fusion and maturation (0-7 days). This identified a transient surge in both IGF-1 and MGF expression on day 3 of the developing construct. This peak of IGF-1 and MGF expression, just prior to differentiation, was consistent with the idea that IGF-1 stimulates differentiation through a Myogenin pathway [Florini et al., 1991: Mol. Endocrinol. 5:718-724]. MGF gene expression was increased 77-fold on day 3, compared to a 36-fold increase with IGF-1 on day 3. This indicates an important role for MGF in either differentiation or, more likely, a response to mechanical or tensional cues.

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Probing the phalloidin binding site of actin

Cited by 40 publications

References 22 publications

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

Pulsed-laser creation and characterization of giant plasma membrane vesicles from cells

3‐D in vitro model of early skeletal muscle development

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