Limits...
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.

Show MeSH

Related in: MedlinePlus

Actin polymerization monitored by 1,4–PBM cross-linking. Ca– G-actin (a and a′), EGTA–G-actin (b  and b′), and Mg–G-actin (c and c′) at a  protein concentration of 1 mg/ml each  were assayed. Polymerization was initiated by addition of 100 mM KCl in the  absence (a–c) and presence (a′–c′) of a  2:1 molar excess of phalloidin over actin. Aliquots were removed at the  times indicated and cross-linked with  1,4-PBM at a cross-linker/protein molar ratio of 0.5:1 (see Materials and  Methods). Cross-linked aliquots were  analyzed by SDS-PAGE (10.5% gels).  Gel standards: myosin heavy chain, 200  kD (1); β-galactosidase, 116.25 kD (2);  phosphorylase B, 92.5 kD (3); BSA,  66.2 kD (4); and ovalbumin, 45 kD (5).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2141646&req=5

Figure 2: Actin polymerization monitored by 1,4–PBM cross-linking. Ca– G-actin (a and a′), EGTA–G-actin (b and b′), and Mg–G-actin (c and c′) at a protein concentration of 1 mg/ml each were assayed. Polymerization was initiated by addition of 100 mM KCl in the absence (a–c) and presence (a′–c′) of a 2:1 molar excess of phalloidin over actin. Aliquots were removed at the times indicated and cross-linked with 1,4-PBM at a cross-linker/protein molar ratio of 0.5:1 (see Materials and Methods). Cross-linked aliquots were analyzed by SDS-PAGE (10.5% gels). Gel standards: myosin heavy chain, 200 kD (1); β-galactosidase, 116.25 kD (2); phosphorylase B, 92.5 kD (3); BSA, 66.2 kD (4); and ovalbumin, 45 kD (5).

Mentions: To complement the pyrene fluorescence measurements, we also used intermolecular cross-linking with 1,4-PBM (a bifunctional sulfhydryl cross-linking reagent) to monitor actin polymerization (Millonig et al., 1988). More specifically, this cross-linking assay probes the oligomeric state of actin during the early stages of its polymerization into filaments. As illustrated with Ca–G-actin in Fig. 2 a, immediately after adding 100 mM KCl to initiate polymerization (t = 0 min), a single major “lower dimer” band (LD; see Mockrin and Korn, 1981, 1983; Millonig et al., 1988) with an apparent molecular mass of 86 kD was observed by SDS-PAGE in addition to the monomer band (i.e., 42 kD). During the course of the polymerization reaction, LD gradually disappeared concomitant with the appearance of increasing amounts of a second major “upper dimer” band (UD; Knight and Offer, 1978; Mockrin and Korn, 1981, 1983; Elzinga and Phelan, 1984; Millonig et al., 1988) with an apparent molecular mass of ∼130 kD (Fig. 2 a, t = 5–30 min). Since the time required to shift from LD to UD is related to the course of polymerization, it was used to qualitatively compare different polymerization conditions with each other (Millonig et al., 1988). At steady state (Fig. 2 a, t > 20 min), additional higher molecular mass bands that probably represent F-actin trimers and higher oligomers were observed beside the UD band (Knight and Offer, 1978; Elzinga and Phelan, 1984; Millonig et al., 1988).


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

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

Actin polymerization monitored by 1,4–PBM cross-linking. Ca– G-actin (a and a′), EGTA–G-actin (b  and b′), and Mg–G-actin (c and c′) at a  protein concentration of 1 mg/ml each  were assayed. Polymerization was initiated by addition of 100 mM KCl in the  absence (a–c) and presence (a′–c′) of a  2:1 molar excess of phalloidin over actin. Aliquots were removed at the  times indicated and cross-linked with  1,4-PBM at a cross-linker/protein molar ratio of 0.5:1 (see Materials and  Methods). Cross-linked aliquots were  analyzed by SDS-PAGE (10.5% gels).  Gel standards: myosin heavy chain, 200  kD (1); β-galactosidase, 116.25 kD (2);  phosphorylase B, 92.5 kD (3); BSA,  66.2 kD (4); and ovalbumin, 45 kD (5).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Actin polymerization monitored by 1,4–PBM cross-linking. Ca– G-actin (a and a′), EGTA–G-actin (b and b′), and Mg–G-actin (c and c′) at a protein concentration of 1 mg/ml each were assayed. Polymerization was initiated by addition of 100 mM KCl in the absence (a–c) and presence (a′–c′) of a 2:1 molar excess of phalloidin over actin. Aliquots were removed at the times indicated and cross-linked with 1,4-PBM at a cross-linker/protein molar ratio of 0.5:1 (see Materials and Methods). Cross-linked aliquots were analyzed by SDS-PAGE (10.5% gels). Gel standards: myosin heavy chain, 200 kD (1); β-galactosidase, 116.25 kD (2); phosphorylase B, 92.5 kD (3); BSA, 66.2 kD (4); and ovalbumin, 45 kD (5).
Mentions: To complement the pyrene fluorescence measurements, we also used intermolecular cross-linking with 1,4-PBM (a bifunctional sulfhydryl cross-linking reagent) to monitor actin polymerization (Millonig et al., 1988). More specifically, this cross-linking assay probes the oligomeric state of actin during the early stages of its polymerization into filaments. As illustrated with Ca–G-actin in Fig. 2 a, immediately after adding 100 mM KCl to initiate polymerization (t = 0 min), a single major “lower dimer” band (LD; see Mockrin and Korn, 1981, 1983; Millonig et al., 1988) with an apparent molecular mass of 86 kD was observed by SDS-PAGE in addition to the monomer band (i.e., 42 kD). During the course of the polymerization reaction, LD gradually disappeared concomitant with the appearance of increasing amounts of a second major “upper dimer” band (UD; Knight and Offer, 1978; Mockrin and Korn, 1981, 1983; Elzinga and Phelan, 1984; Millonig et al., 1988) with an apparent molecular mass of ∼130 kD (Fig. 2 a, t = 5–30 min). Since the time required to shift from LD to UD is related to the course of polymerization, it was used to qualitatively compare different polymerization conditions with each other (Millonig et al., 1988). At steady state (Fig. 2 a, t > 20 min), additional higher molecular mass bands that probably represent F-actin trimers and higher oligomers were observed beside the UD band (Knight and Offer, 1978; Elzinga and Phelan, 1984; Millonig et al., 1988).

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