The Influence of Divalent Cations on the Dynamic Properties of Actin Filaments: A Spectroscopic Study

Hild, Gábor; Nyitrai, Miklós; Belágyi, Joseph; Somogyi, B

doi:10.1016/s0006-3495(98)77742-2

Cited by 26 publications

(25 citation statements)

References 39 publications

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“…This observation seems to support recent results, which report nucleotide-induced flexibility differences in actin filaments (7)(8)(9)(10)(11). However, care should be taken when comparing results obtained by using experimental methods characteristic for motions on substantially different time scales (40). The methods applied in previous experiments (7)(8)(9)(10)(11)(12)(13)(14) to describe the nucleotide-dependent changes in the structure of actin filaments were probably informative on a time scale longer than the one in the present measurements.…”

Section: Discussionsupporting

confidence: 90%

“…The actin monomers usually bind Ca 2ϩ after standard preparations. However, the efficiency of the nucleotide exchange is better (34) and the polymerization process is faster (40) in the presence of Mg 2ϩ than in the presence of Ca 2ϩ .…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, the filaments from both ATP-and ADP-actin monomers were assembled following a Ca 2ϩ -Mg 2ϩ exchange procedure with bound Mg 2ϩ so that the time, which the actin monomers spend in ADP form, could be minimized and also the known effect of different cations could be excluded (10,24,40,41).…”

Section: Discussionmentioning

confidence: 99%

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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: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

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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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“…The dynamic behavior of the filaments was sensitive to whether they were polymerized in the presence of calcium (Ca-F-actin) or magnesium ions (Mg-F-actin) [12][13][14]. In previous FRET experiments the intermonomer flexibility of Ca-F-actin was greater than that of Mg-F-actin [12].…”

Section: Resultsmentioning

confidence: 97%

The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments

VISEGRADY¹

2004

FEBS Letters

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In this work the effect of phalloidin and jasplakinolide on the dynamic properties and thermal stability of actin filaments was studied. Temperature dependent fluorescence resonance energy transfer measurements showed that filaments of Ca-actin became more rigid in the presence of phalloidin or jasplakinolide. Differential scanning calorimetric data implied that the stiffer filaments also had greater thermal stability in the presence of phalloidin or jasplakinolide. The fluorescence and calorimetric measurements provided evidences that the extent of stabilization by jasplakinolide was greater than that by phalloidin.

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“…There are a number of methods used previously to describe the in vitro properties and in vivo cellular localization and interactions of actin (15)(16)(17)(18). Amongst the numerous in vitro techniques fluorescence spectroscopy proved to be a powerful tool to study the conformational and dynamic properties of proteins.…”

mentioning

confidence: 99%

The effects of detergents on the polymerization properties of actin

et al. 2010

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Effects of some detergents-most frequently used in membrane raft studies-on the polymerization properties of actin were examined under in vitro and in vivo conditions, for protein and cellular investigations, respectively. Under in vitro conditions the polymerization rates were measured with pyrene-labeled actin. We found that polymerization rate depended on the detergent concentration by following either biphasic characteristics or only decreasing tendency. The strongest effects were observed at relatively low detergent concentrations. SDS-PAGE electrophoresis and dynamic light-scattering measurements provided further evidences for the size distribution of actin filaments formed under the influence of detergents. Comparing the polymerization rates measured in the presence of different detergents to those obtained with various magnesium and KCl concentrations showed that detergents may influence the actin polymerization at three levels by modifying: (i) the monomer-monomer interaction, (ii) the local ionic strength, and (iii) the affinity of actin for various cations. In vivo studies on NIH 3T3MDR1 cells using TRITC-phalloidin detected fast depolymerization of large extent around the critical micellar concentrations of the detergents. We concluded that microdomain insolubility observed in the presence of detergents is hardly to be the result of the stabilization of the submembrane actin cytoskeleton merely; rather inter-lipid and lipid-protein interactions are also involved within the detergent-resistant membranes. '

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The Influence of Divalent Cations on the Dynamic Properties of Actin Filaments: A Spectroscopic Study

Cited by 26 publications

References 39 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

The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments

The effects of detergents on the polymerization properties of actin

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