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The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins.

Galkin VE, Orlova A, VanLoock MS, Rybakova IN, Ervasti JM, Egelman EH - J. Cell Biol. (2002)

Bottom Line: The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments.When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin.The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.

ABSTRACT
Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

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Related in: MedlinePlus

A schematic diagram illustrating how the IHRSR method was used to sort the filament images into three class: half-decorated, singly decorated, and poor or mixed decoration. An electron micrograph (top, center) shows a typical field of filaments, and from these, 16,070 segments were extracted from filaments identified as light (white arrow), and 6,240 segments were extracted from filaments identified as dark (black arrow). Five examples of such segments are shown for each set. Two initial three-dimensional reconstructions were then generated from ∼2,000 segments from each set and yielded a mean symmetry of both sets of ∼166° (in this instance, the variable twist of F-actin was averaged). A third reconstruction, of pure F-actin, was taken from previously published work (Orlova et al., 2001). These initial reconstructions were clearly distinguishable from each other and from pure F-actin, but suffered from inhomogeneity of decoration, mixture of modes, and variable twist. The next step was to generate reference projections from these three-dimensional volumes for sorting. The light segments were then cross-correlated against projections of both pure F-actin and the light reconstruction, whereas the dark segments were cross-correlated against projections of both the light and dark reconstructions. Although only two reference projections are shown for each case, in actuality, 90 reference projections were generated for each symmetry (corresponding to azimuthal rotations of 4° about the helical axis), and actin symmetries from 154° to 174° in 2° increments were used. Thus, the 16,070 light segments and the 6,240 dark segments were each cross-correlated against 1,980 reference projections. This then yielded a sorting of segments by both twist and decoration. New reconstructions were then iteratively generated using IHRSR for all classes, including poorly, half- and singly decorated subsets showing different symmetries. The largest subsets for all classes had a final symmetry of ∼166°, and reconstructions from these subsets are shown at the bottom. For the segments initially collected from light filaments, ∼38% (6,155 out of 16,070) showed stronger correlation with pure F-actin than they did to the half-decorated reconstruction. For the segments initially collected from dark filaments, ∼50% (3,079 out of 6,240) showed stronger correlation with the half decoration than they did to the singly decorated reconstruction. These percentages cannot be interpreted in terms of stoichiometries or incomplete binding, since we are sorting primarily on ordered binding rather than actual occupancy.
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fig2: A schematic diagram illustrating how the IHRSR method was used to sort the filament images into three class: half-decorated, singly decorated, and poor or mixed decoration. An electron micrograph (top, center) shows a typical field of filaments, and from these, 16,070 segments were extracted from filaments identified as light (white arrow), and 6,240 segments were extracted from filaments identified as dark (black arrow). Five examples of such segments are shown for each set. Two initial three-dimensional reconstructions were then generated from ∼2,000 segments from each set and yielded a mean symmetry of both sets of ∼166° (in this instance, the variable twist of F-actin was averaged). A third reconstruction, of pure F-actin, was taken from previously published work (Orlova et al., 2001). These initial reconstructions were clearly distinguishable from each other and from pure F-actin, but suffered from inhomogeneity of decoration, mixture of modes, and variable twist. The next step was to generate reference projections from these three-dimensional volumes for sorting. The light segments were then cross-correlated against projections of both pure F-actin and the light reconstruction, whereas the dark segments were cross-correlated against projections of both the light and dark reconstructions. Although only two reference projections are shown for each case, in actuality, 90 reference projections were generated for each symmetry (corresponding to azimuthal rotations of 4° about the helical axis), and actin symmetries from 154° to 174° in 2° increments were used. Thus, the 16,070 light segments and the 6,240 dark segments were each cross-correlated against 1,980 reference projections. This then yielded a sorting of segments by both twist and decoration. New reconstructions were then iteratively generated using IHRSR for all classes, including poorly, half- and singly decorated subsets showing different symmetries. The largest subsets for all classes had a final symmetry of ∼166°, and reconstructions from these subsets are shown at the bottom. For the segments initially collected from light filaments, ∼38% (6,155 out of 16,070) showed stronger correlation with pure F-actin than they did to the half-decorated reconstruction. For the segments initially collected from dark filaments, ∼50% (3,079 out of 6,240) showed stronger correlation with the half decoration than they did to the singly decorated reconstruction. These percentages cannot be interpreted in terms of stoichiometries or incomplete binding, since we are sorting primarily on ordered binding rather than actual occupancy.

Mentions: Analysis of a total of ∼22,500 segments, each containing ∼14 actin subunits, revealed that three different populations could be found within the decorated filaments. These are two different modes of decoration by ut261, and a third category which contained undecorated, partially decorated actin, or disordered binding. The IHRSR approach (Figs. 2, 3, and 4) was key to the separation of these modes, since conventional helical analysis (DeRosier and Klug, 1968) would tend to average these states together. Although we find that there is a cooperativity in the mode of binding, as evidenced by the fact that different filament types can be differentiated by eye in electron micrographs (Fig. 1), detailed analysis shows that this cooperativity is far from complete. Two different independent methods were employed to sort and classify the filament segments used for three-dimensional reconstruction. One approach is based upon cross-correlations with projections of reference volumes (Fig. 2), whereas the second approach is based upon using differences in the two-dimensional radial density distributions within images for sorting (Fig. 3).


The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins.

Galkin VE, Orlova A, VanLoock MS, Rybakova IN, Ervasti JM, Egelman EH - J. Cell Biol. (2002)

A schematic diagram illustrating how the IHRSR method was used to sort the filament images into three class: half-decorated, singly decorated, and poor or mixed decoration. An electron micrograph (top, center) shows a typical field of filaments, and from these, 16,070 segments were extracted from filaments identified as light (white arrow), and 6,240 segments were extracted from filaments identified as dark (black arrow). Five examples of such segments are shown for each set. Two initial three-dimensional reconstructions were then generated from ∼2,000 segments from each set and yielded a mean symmetry of both sets of ∼166° (in this instance, the variable twist of F-actin was averaged). A third reconstruction, of pure F-actin, was taken from previously published work (Orlova et al., 2001). These initial reconstructions were clearly distinguishable from each other and from pure F-actin, but suffered from inhomogeneity of decoration, mixture of modes, and variable twist. The next step was to generate reference projections from these three-dimensional volumes for sorting. The light segments were then cross-correlated against projections of both pure F-actin and the light reconstruction, whereas the dark segments were cross-correlated against projections of both the light and dark reconstructions. Although only two reference projections are shown for each case, in actuality, 90 reference projections were generated for each symmetry (corresponding to azimuthal rotations of 4° about the helical axis), and actin symmetries from 154° to 174° in 2° increments were used. Thus, the 16,070 light segments and the 6,240 dark segments were each cross-correlated against 1,980 reference projections. This then yielded a sorting of segments by both twist and decoration. New reconstructions were then iteratively generated using IHRSR for all classes, including poorly, half- and singly decorated subsets showing different symmetries. The largest subsets for all classes had a final symmetry of ∼166°, and reconstructions from these subsets are shown at the bottom. For the segments initially collected from light filaments, ∼38% (6,155 out of 16,070) showed stronger correlation with pure F-actin than they did to the half-decorated reconstruction. For the segments initially collected from dark filaments, ∼50% (3,079 out of 6,240) showed stronger correlation with the half decoration than they did to the singly decorated reconstruction. These percentages cannot be interpreted in terms of stoichiometries or incomplete binding, since we are sorting primarily on ordered binding rather than actual occupancy.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199260&req=5

fig2: A schematic diagram illustrating how the IHRSR method was used to sort the filament images into three class: half-decorated, singly decorated, and poor or mixed decoration. An electron micrograph (top, center) shows a typical field of filaments, and from these, 16,070 segments were extracted from filaments identified as light (white arrow), and 6,240 segments were extracted from filaments identified as dark (black arrow). Five examples of such segments are shown for each set. Two initial three-dimensional reconstructions were then generated from ∼2,000 segments from each set and yielded a mean symmetry of both sets of ∼166° (in this instance, the variable twist of F-actin was averaged). A third reconstruction, of pure F-actin, was taken from previously published work (Orlova et al., 2001). These initial reconstructions were clearly distinguishable from each other and from pure F-actin, but suffered from inhomogeneity of decoration, mixture of modes, and variable twist. The next step was to generate reference projections from these three-dimensional volumes for sorting. The light segments were then cross-correlated against projections of both pure F-actin and the light reconstruction, whereas the dark segments were cross-correlated against projections of both the light and dark reconstructions. Although only two reference projections are shown for each case, in actuality, 90 reference projections were generated for each symmetry (corresponding to azimuthal rotations of 4° about the helical axis), and actin symmetries from 154° to 174° in 2° increments were used. Thus, the 16,070 light segments and the 6,240 dark segments were each cross-correlated against 1,980 reference projections. This then yielded a sorting of segments by both twist and decoration. New reconstructions were then iteratively generated using IHRSR for all classes, including poorly, half- and singly decorated subsets showing different symmetries. The largest subsets for all classes had a final symmetry of ∼166°, and reconstructions from these subsets are shown at the bottom. For the segments initially collected from light filaments, ∼38% (6,155 out of 16,070) showed stronger correlation with pure F-actin than they did to the half-decorated reconstruction. For the segments initially collected from dark filaments, ∼50% (3,079 out of 6,240) showed stronger correlation with the half decoration than they did to the singly decorated reconstruction. These percentages cannot be interpreted in terms of stoichiometries or incomplete binding, since we are sorting primarily on ordered binding rather than actual occupancy.
Mentions: Analysis of a total of ∼22,500 segments, each containing ∼14 actin subunits, revealed that three different populations could be found within the decorated filaments. These are two different modes of decoration by ut261, and a third category which contained undecorated, partially decorated actin, or disordered binding. The IHRSR approach (Figs. 2, 3, and 4) was key to the separation of these modes, since conventional helical analysis (DeRosier and Klug, 1968) would tend to average these states together. Although we find that there is a cooperativity in the mode of binding, as evidenced by the fact that different filament types can be differentiated by eye in electron micrographs (Fig. 1), detailed analysis shows that this cooperativity is far from complete. Two different independent methods were employed to sort and classify the filament segments used for three-dimensional reconstruction. One approach is based upon cross-correlations with projections of reference volumes (Fig. 2), whereas the second approach is based upon using differences in the two-dimensional radial density distributions within images for sorting (Fig. 3).

Bottom Line: The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments.When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin.The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.

ABSTRACT
Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

Show MeSH
Related in: MedlinePlus