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A correlative analysis of actin filament assembly, structure, and dynamics.

Steinmetz MO, Goldie KN, Aebi U - J. Cell Biol. (1997)

Bottom Line: Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found.However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes.Hence, we conclude that the structure and dynamics of the Mg-F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca2+ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.

View Article: PubMed Central - PubMed

Affiliation: M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
The effect of the type of metal ion (i.e., Ca2+, Mg2+, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on filament assembly, structure, and dynamics was investigated in the absence and presence of the mushroom toxin phalloidin. In agreement with earlier reports, we found the polymerization reaction of G-actin into F-actin filaments to be tightly controlled by the type of divalent cation residing in its HAS. Moreover, novel polymerization data are presented indicating that LD, a dimer unproductive by itself, does incorporate into growing F-actin filaments. This observation suggests that during actin filament formation, in addition to the obligatory nucleation- condensation pathway involving UD, a productive filament dimer, a facultative, LD-based pathway is implicated whose abundance strongly depends on the exact polymerization conditions chosen. The "ragged" and "branched" filaments observed during the early stages of assembly represent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in highly motile cells. Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found. However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes. Together, our data indicate that whereas the G-actin conformation is tightly controlled by the divalent cation in its HAS, the F-actin conformation appears more robust than this variation. Hence, we conclude that the structure and dynamics of the Mg-F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca2+ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.

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Statistical analysis of  crossover spacing (a) and maximum crossover width (b) of negatively stained Ca–F-actin filaments imaged by STEM ADF  (see Fig. 5 a). The axial spacing  and maximum width of 240 filament crossovers were evaluated  and the corresponding values  were histogrammed in a and b.  Gaussian fits have been superimposed onto the histograms,  and the corresponding means  and standard deviations are displayed. In c, the 240 crossover  spacings/crossover widths are  plotted against each other and  displayed as a gray level representation with eight discrete  contouring levels (i.e., corresponding to two to nine occurrences, respectively).
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Figure 6: Statistical analysis of crossover spacing (a) and maximum crossover width (b) of negatively stained Ca–F-actin filaments imaged by STEM ADF (see Fig. 5 a). The axial spacing and maximum width of 240 filament crossovers were evaluated and the corresponding values were histogrammed in a and b. Gaussian fits have been superimposed onto the histograms, and the corresponding means and standard deviations are displayed. In c, the 240 crossover spacings/crossover widths are plotted against each other and displayed as a gray level representation with eight discrete contouring levels (i.e., corresponding to two to nine occurrences, respectively).

Mentions: The crossover spacing, i.e., the axial spacing between two subsequent crossovers of the two long-pitch helical strands as revealed from projection images of negatively stained F-actin filaments, has been determined (Bremer et al., 1991) from high magnification STEM ADF micrographs (see Fig. 5). For each one-crossover-long filament segment evaluated, its maximum width was determined from its radial mass density profile (Steven et al., 1988). Fig. 6, a and b, yields the crossover spacing and the corresponding maximum crossover width frequency distributions for Ca–F-actin polymerized with 100 mM KCl. In Fig. 6 c, we also plotted the crossover spacings against the corresponding maximum crossover widths for the data set displayed in Fig. 6, a and b. As documented in Table I, all native filament types (i.e., those polymerized in the absence of phalloidin) yielded virtually the same crossover spacing (35.2 ± 2.1 nm for the overall mean) and maximum crossover width (9.5 ± 0.5 nm for the overall mean) distributions. The calculated screw angle ψ is −166.19 ± 0.83° (for details see Materials and Methods). These mean values (i.e., the crossover spacing and screw angle) correspond to a mean integer helical selection rule l = −6n + 13m and are in good agreement with previously published data (Aebi et al., 1986; Bremer et al., 1991, 1994; Orlova and Egelman, 1992).


A correlative analysis of actin filament assembly, structure, and dynamics.

Steinmetz MO, Goldie KN, Aebi U - J. Cell Biol. (1997)

Statistical analysis of  crossover spacing (a) and maximum crossover width (b) of negatively stained Ca–F-actin filaments imaged by STEM ADF  (see Fig. 5 a). The axial spacing  and maximum width of 240 filament crossovers were evaluated  and the corresponding values  were histogrammed in a and b.  Gaussian fits have been superimposed onto the histograms,  and the corresponding means  and standard deviations are displayed. In c, the 240 crossover  spacings/crossover widths are  plotted against each other and  displayed as a gray level representation with eight discrete  contouring levels (i.e., corresponding to two to nine occurrences, respectively).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141646&req=5

Figure 6: Statistical analysis of crossover spacing (a) and maximum crossover width (b) of negatively stained Ca–F-actin filaments imaged by STEM ADF (see Fig. 5 a). The axial spacing and maximum width of 240 filament crossovers were evaluated and the corresponding values were histogrammed in a and b. Gaussian fits have been superimposed onto the histograms, and the corresponding means and standard deviations are displayed. In c, the 240 crossover spacings/crossover widths are plotted against each other and displayed as a gray level representation with eight discrete contouring levels (i.e., corresponding to two to nine occurrences, respectively).
Mentions: The crossover spacing, i.e., the axial spacing between two subsequent crossovers of the two long-pitch helical strands as revealed from projection images of negatively stained F-actin filaments, has been determined (Bremer et al., 1991) from high magnification STEM ADF micrographs (see Fig. 5). For each one-crossover-long filament segment evaluated, its maximum width was determined from its radial mass density profile (Steven et al., 1988). Fig. 6, a and b, yields the crossover spacing and the corresponding maximum crossover width frequency distributions for Ca–F-actin polymerized with 100 mM KCl. In Fig. 6 c, we also plotted the crossover spacings against the corresponding maximum crossover widths for the data set displayed in Fig. 6, a and b. As documented in Table I, all native filament types (i.e., those polymerized in the absence of phalloidin) yielded virtually the same crossover spacing (35.2 ± 2.1 nm for the overall mean) and maximum crossover width (9.5 ± 0.5 nm for the overall mean) distributions. The calculated screw angle ψ is −166.19 ± 0.83° (for details see Materials and Methods). These mean values (i.e., the crossover spacing and screw angle) correspond to a mean integer helical selection rule l = −6n + 13m and are in good agreement with previously published data (Aebi et al., 1986; Bremer et al., 1991, 1994; Orlova and Egelman, 1992).

Bottom Line: Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found.However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes.Hence, we conclude that the structure and dynamics of the Mg-F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca2+ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.

View Article: PubMed Central - PubMed

Affiliation: M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
The effect of the type of metal ion (i.e., Ca2+, Mg2+, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on filament assembly, structure, and dynamics was investigated in the absence and presence of the mushroom toxin phalloidin. In agreement with earlier reports, we found the polymerization reaction of G-actin into F-actin filaments to be tightly controlled by the type of divalent cation residing in its HAS. Moreover, novel polymerization data are presented indicating that LD, a dimer unproductive by itself, does incorporate into growing F-actin filaments. This observation suggests that during actin filament formation, in addition to the obligatory nucleation- condensation pathway involving UD, a productive filament dimer, a facultative, LD-based pathway is implicated whose abundance strongly depends on the exact polymerization conditions chosen. The "ragged" and "branched" filaments observed during the early stages of assembly represent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in highly motile cells. Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found. However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes. Together, our data indicate that whereas the G-actin conformation is tightly controlled by the divalent cation in its HAS, the F-actin conformation appears more robust than this variation. Hence, we conclude that the structure and dynamics of the Mg-F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca2+ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.

Show MeSH
Related in: MedlinePlus