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Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers.

Kueh HY, Charras GT, Mitchison TJ, Brieher WM - J. Cell Biol. (2008)

Bottom Line: Mitchison. 2006.CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not.The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315-324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.

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Actin filaments disassemble with similar kinetics from both ends. (A) Time-lapse images of actin filament bundles grown off fragments of L. polyphemus acrosomal processes. Filaments in the long bundle have exposed barbed ends (b), whereas filaments in the short bundle have exposed pointed ends (p). The two bundles shown were elongated from opposite ends of the same L. polyphemus acrosomal fragment. The brightness of the shorter bundle was increased relative to the longer one for ease of visualization. At 0 s, filament bundles were perfused with 2 μM cofilin, 1 μM coronin, 300 nM Aip1, 5 μM of actin monomer, and 2 mM ATP. Bar, 1 μm. (B) Total actin polymer mass in the filament bundles as a function of time measured for all bundles (red), bundles with exposed barbed ends (green), and bundles with exposed pointed ends (blue). To compare the rates of rapid disassembly, the slowly varying component of each decay curve was removed (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1; see Materials and methods). (C) Bar graph comparing decay rates for bundles with exposed barbed ends (b) with those with exposed pointed ends (p). Data represent mean and standard deviation of three independent experiments.
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fig3: Actin filaments disassemble with similar kinetics from both ends. (A) Time-lapse images of actin filament bundles grown off fragments of L. polyphemus acrosomal processes. Filaments in the long bundle have exposed barbed ends (b), whereas filaments in the short bundle have exposed pointed ends (p). The two bundles shown were elongated from opposite ends of the same L. polyphemus acrosomal fragment. The brightness of the shorter bundle was increased relative to the longer one for ease of visualization. At 0 s, filament bundles were perfused with 2 μM cofilin, 1 μM coronin, 300 nM Aip1, 5 μM of actin monomer, and 2 mM ATP. Bar, 1 μm. (B) Total actin polymer mass in the filament bundles as a function of time measured for all bundles (red), bundles with exposed barbed ends (green), and bundles with exposed pointed ends (blue). To compare the rates of rapid disassembly, the slowly varying component of each decay curve was removed (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1; see Materials and methods). (C) Bar graph comparing decay rates for bundles with exposed barbed ends (b) with those with exposed pointed ends (p). Data represent mean and standard deviation of three independent experiments.

Mentions: Actin filaments are structurally polarized, so we next tested whether the bursts of disassembly occur preferentially from a particular end of the filament. To separately probe disassembly near barbed ends and pointed ends, we imaged disassembly of filaments elongated from fragments of Limulus polyphemus,acrosomal processes, which are highly bundled arrays of filaments all oriented in the same direction (Bonder and Mooseker, 1983). Filaments grown off the barbed end of acrosomal bundles have free barbed ends but pointed ends that are connected to the acrosome. The reverse applies at the pointed end. We polymerized bundles of filaments off the barbed ends and pointed ends of L. polyphemus acrosomes using 5 μM of fluorescently labeled actin monomer and 2 mM ATP and then perfused them with cofilin, coronin, and Aip1, as well as unlabeled actin monomer and ATP. To minimize filament aging effects, filaments were perfused within 2 min after initiation of polymerization. Interestingly, filament bundles grown from either barbed ends (Fig. 3 A, b) or pointed ends (Fig. 3 A, p) of acrosomal processes disassembled at comparable rates (Fig. 3 A). Most of the polymer mass in the filament bundles disappeared within the first 2 min; the remainder, which manifest as a short stub of filaments at the acrosomal barbed end, disassembled with considerably slower kinetics (Fig. S1 and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1). To quantify the fast component of disassembly, we first subtracted out the slowly varying component of the decay curve (see Materials and methods). Bundles with either exposed barbed ends or exposed pointed ends exhibited similar polymer mass decay curves and inferred decay rate constants (Fig. 3, B and C). These results suggest that bursting does not occur preferentially from a particular end of the filament but can proceed vectorially from either end. We also note that in our single-filament assay, internal filament disassembly events catalyzed by cofilin, coronin, and Aip1 frequently gave rise to two new filament ends that both underwent bursting disassembly (unpublished data), which is consistent with both ends undergoing bursting disassembly at similar rates.


Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers.

Kueh HY, Charras GT, Mitchison TJ, Brieher WM - J. Cell Biol. (2008)

Actin filaments disassemble with similar kinetics from both ends. (A) Time-lapse images of actin filament bundles grown off fragments of L. polyphemus acrosomal processes. Filaments in the long bundle have exposed barbed ends (b), whereas filaments in the short bundle have exposed pointed ends (p). The two bundles shown were elongated from opposite ends of the same L. polyphemus acrosomal fragment. The brightness of the shorter bundle was increased relative to the longer one for ease of visualization. At 0 s, filament bundles were perfused with 2 μM cofilin, 1 μM coronin, 300 nM Aip1, 5 μM of actin monomer, and 2 mM ATP. Bar, 1 μm. (B) Total actin polymer mass in the filament bundles as a function of time measured for all bundles (red), bundles with exposed barbed ends (green), and bundles with exposed pointed ends (blue). To compare the rates of rapid disassembly, the slowly varying component of each decay curve was removed (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1; see Materials and methods). (C) Bar graph comparing decay rates for bundles with exposed barbed ends (b) with those with exposed pointed ends (p). Data represent mean and standard deviation of three independent experiments.
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Related In: Results  -  Collection

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fig3: Actin filaments disassemble with similar kinetics from both ends. (A) Time-lapse images of actin filament bundles grown off fragments of L. polyphemus acrosomal processes. Filaments in the long bundle have exposed barbed ends (b), whereas filaments in the short bundle have exposed pointed ends (p). The two bundles shown were elongated from opposite ends of the same L. polyphemus acrosomal fragment. The brightness of the shorter bundle was increased relative to the longer one for ease of visualization. At 0 s, filament bundles were perfused with 2 μM cofilin, 1 μM coronin, 300 nM Aip1, 5 μM of actin monomer, and 2 mM ATP. Bar, 1 μm. (B) Total actin polymer mass in the filament bundles as a function of time measured for all bundles (red), bundles with exposed barbed ends (green), and bundles with exposed pointed ends (blue). To compare the rates of rapid disassembly, the slowly varying component of each decay curve was removed (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1; see Materials and methods). (C) Bar graph comparing decay rates for bundles with exposed barbed ends (b) with those with exposed pointed ends (p). Data represent mean and standard deviation of three independent experiments.
Mentions: Actin filaments are structurally polarized, so we next tested whether the bursts of disassembly occur preferentially from a particular end of the filament. To separately probe disassembly near barbed ends and pointed ends, we imaged disassembly of filaments elongated from fragments of Limulus polyphemus,acrosomal processes, which are highly bundled arrays of filaments all oriented in the same direction (Bonder and Mooseker, 1983). Filaments grown off the barbed end of acrosomal bundles have free barbed ends but pointed ends that are connected to the acrosome. The reverse applies at the pointed end. We polymerized bundles of filaments off the barbed ends and pointed ends of L. polyphemus acrosomes using 5 μM of fluorescently labeled actin monomer and 2 mM ATP and then perfused them with cofilin, coronin, and Aip1, as well as unlabeled actin monomer and ATP. To minimize filament aging effects, filaments were perfused within 2 min after initiation of polymerization. Interestingly, filament bundles grown from either barbed ends (Fig. 3 A, b) or pointed ends (Fig. 3 A, p) of acrosomal processes disassembled at comparable rates (Fig. 3 A). Most of the polymer mass in the filament bundles disappeared within the first 2 min; the remainder, which manifest as a short stub of filaments at the acrosomal barbed end, disassembled with considerably slower kinetics (Fig. S1 and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1). To quantify the fast component of disassembly, we first subtracted out the slowly varying component of the decay curve (see Materials and methods). Bundles with either exposed barbed ends or exposed pointed ends exhibited similar polymer mass decay curves and inferred decay rate constants (Fig. 3, B and C). These results suggest that bursting does not occur preferentially from a particular end of the filament but can proceed vectorially from either end. We also note that in our single-filament assay, internal filament disassembly events catalyzed by cofilin, coronin, and Aip1 frequently gave rise to two new filament ends that both underwent bursting disassembly (unpublished data), which is consistent with both ends undergoing bursting disassembly at similar rates.

Bottom Line: Mitchison. 2006.CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not.The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315-324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.

Show MeSH
Related in: MedlinePlus