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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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Negatively stained F-actin filaments imaged by CTEM after 0 (a), 5 (b), 10 (c), and >30 min (d) after onset of the polymerization reaction by addition of KCl to 100 mM to Ca–ATP–G-actin (1 mg/ml). At each time point, 5 μl aliquots were directly applied for 15 s  to an EM grid, washed, and negatively stained with uranyl formate (for details see Materials and Methods). Bar, 200 nm.
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Figure 3: Negatively stained F-actin filaments imaged by CTEM after 0 (a), 5 (b), 10 (c), and >30 min (d) after onset of the polymerization reaction by addition of KCl to 100 mM to Ca–ATP–G-actin (1 mg/ml). At each time point, 5 μl aliquots were directly applied for 15 s to an EM grid, washed, and negatively stained with uranyl formate (for details see Materials and Methods). Bar, 200 nm.

Mentions: To gain more insight into the mechanism of actin polymerization, we monitored F-actin filament formation and maturation by EM. Fig. 3 displays CTEM micrographs of negatively stained F-actin filaments 0 (Fig. 3 a), 5 (Fig. 3 b), 10 (Fig. 3 c), and >30 min (Fig. 3 d) after the onset of polymerization by adding 100 mM KCl to Ca–G-actin. Comparison of the corresponding time points of Fig. 2 a (i.e., t = 5 and 10 min) with the apparently ragged and branched filaments displayed in Fig. 3, b and c, indicates that LD might incorporate into growing filaments during the elongation phase. Gradually, the surplus bound material dissociates from the “partially decorated” filaments to yield smooth, bona fide mature F-actin filaments at steady state (Fig. 3 d).


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

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

Negatively stained F-actin filaments imaged by CTEM after 0 (a), 5 (b), 10 (c), and >30 min (d) after onset of the polymerization reaction by addition of KCl to 100 mM to Ca–ATP–G-actin (1 mg/ml). At each time point, 5 μl aliquots were directly applied for 15 s  to an EM grid, washed, and negatively stained with uranyl formate (for details see Materials and Methods). Bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Negatively stained F-actin filaments imaged by CTEM after 0 (a), 5 (b), 10 (c), and >30 min (d) after onset of the polymerization reaction by addition of KCl to 100 mM to Ca–ATP–G-actin (1 mg/ml). At each time point, 5 μl aliquots were directly applied for 15 s to an EM grid, washed, and negatively stained with uranyl formate (for details see Materials and Methods). Bar, 200 nm.
Mentions: To gain more insight into the mechanism of actin polymerization, we monitored F-actin filament formation and maturation by EM. Fig. 3 displays CTEM micrographs of negatively stained F-actin filaments 0 (Fig. 3 a), 5 (Fig. 3 b), 10 (Fig. 3 c), and >30 min (Fig. 3 d) after the onset of polymerization by adding 100 mM KCl to Ca–G-actin. Comparison of the corresponding time points of Fig. 2 a (i.e., t = 5 and 10 min) with the apparently ragged and branched filaments displayed in Fig. 3, b and c, indicates that LD might incorporate into growing filaments during the elongation phase. Gradually, the surplus bound material dissociates from the “partially decorated” filaments to yield smooth, bona fide mature F-actin filaments at steady state (Fig. 3 d).

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