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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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Barbed end–capping factors inhibit actin disassembly by cofilin, coronin, and Aip1. (A) Mean filament length over time under the following conditions: 2 μM cofilin, 1 μM coronin, 5 μM of actin monomer, and 2 mM ATP, as well as 200 nM Aip1 (red), no Aip1 (green), 5 μM CapZ (yellow), 200 nM Aip1 + 1 μM CytoD (black), or 200 nM Aip1 + 5 μM CapZ (blue). Fast monomer-insensitive disassembly required Aip1 (red vs. green, yellow). CytoD and CapZ inhibited disassembly in the full system (black, blue vs. red). (B) Bar graph showing the percentage of filaments that underwent endwise bursting or severing. The number of filaments analyzed is given below the bars. The percentages of filaments that either underwent endwise bursting or severing increased significantly in the presence of Aip1 (red vs. green, yellow) and decreased significantly when the barbed-end cappers CytoD and CapZ were added to the full system (black, blue vs. red; severing frequency of −CapZ vs. +CapZ, χ2 = 4.9, df = 1, P < 0.05; all other pairwise comparisons, χ2 > 40, df = 1, P < 0.001). (C) Polymer mass decay for bundles with either exposed barbed ends or pointed ends in 3 μM CytoD (brown and purple) or 10 μM CapZ (blue and green), as well as polymer mass decay for all filament bundles in the absence of barbed-end cappers (red). Disassembly of bundles with either exposed barbed ends or pointed ends were inhibited by CytoD/CapZ with equal efficacy. (D) Fraction of polymer mass disassembled as a function of CytoD concentration (red circles). Best fit of the data to a hyperbola (red curve) shows an IC50 of 90 nM for inhibition of disassembly. Length of filament bundles polymerized for a fixed period of time in the presence of varying concentrations of CytoD is also shown (blue squares). Hyperbolic best fit (blue curve) shows an IC50 of 30 nM for inhibition of polymerization. (E) Fraction of polymer disassembled as a function of CapZ concentration (red squares). The titration curve did not reach saturation; the red curve denotes best fit of the data to a straight line. The length of filament actin bundle polymerized in the presence of varying concentrations of CapZ is also shown (blue circles). Polymerization conditions were identical to those in D. Hyperbolic best fit (blue curve) gave an IC50 of 30 nM for inhibition of polymerization. Error bars indicate SD. (F) CytoD does not inhibit cofilin-mediated severing. The bar graph shows the percentage of filaments that severed during a 400 s video either in the absence of CytoD (left) or in the presence of 1 μM CytoD (right). Conditions: 8 μM cofilin and 5 μM of actin monomer in assay buffer.
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fig5: Barbed end–capping factors inhibit actin disassembly by cofilin, coronin, and Aip1. (A) Mean filament length over time under the following conditions: 2 μM cofilin, 1 μM coronin, 5 μM of actin monomer, and 2 mM ATP, as well as 200 nM Aip1 (red), no Aip1 (green), 5 μM CapZ (yellow), 200 nM Aip1 + 1 μM CytoD (black), or 200 nM Aip1 + 5 μM CapZ (blue). Fast monomer-insensitive disassembly required Aip1 (red vs. green, yellow). CytoD and CapZ inhibited disassembly in the full system (black, blue vs. red). (B) Bar graph showing the percentage of filaments that underwent endwise bursting or severing. The number of filaments analyzed is given below the bars. The percentages of filaments that either underwent endwise bursting or severing increased significantly in the presence of Aip1 (red vs. green, yellow) and decreased significantly when the barbed-end cappers CytoD and CapZ were added to the full system (black, blue vs. red; severing frequency of −CapZ vs. +CapZ, χ2 = 4.9, df = 1, P < 0.05; all other pairwise comparisons, χ2 > 40, df = 1, P < 0.001). (C) Polymer mass decay for bundles with either exposed barbed ends or pointed ends in 3 μM CytoD (brown and purple) or 10 μM CapZ (blue and green), as well as polymer mass decay for all filament bundles in the absence of barbed-end cappers (red). Disassembly of bundles with either exposed barbed ends or pointed ends were inhibited by CytoD/CapZ with equal efficacy. (D) Fraction of polymer mass disassembled as a function of CytoD concentration (red circles). Best fit of the data to a hyperbola (red curve) shows an IC50 of 90 nM for inhibition of disassembly. Length of filament bundles polymerized for a fixed period of time in the presence of varying concentrations of CytoD is also shown (blue squares). Hyperbolic best fit (blue curve) shows an IC50 of 30 nM for inhibition of polymerization. (E) Fraction of polymer disassembled as a function of CapZ concentration (red squares). The titration curve did not reach saturation; the red curve denotes best fit of the data to a straight line. The length of filament actin bundle polymerized in the presence of varying concentrations of CapZ is also shown (blue circles). Polymerization conditions were identical to those in D. Hyperbolic best fit (blue curve) gave an IC50 of 30 nM for inhibition of polymerization. Error bars indicate SD. (F) CytoD does not inhibit cofilin-mediated severing. The bar graph shows the percentage of filaments that severed during a 400 s video either in the absence of CytoD (left) or in the presence of 1 μM CytoD (right). Conditions: 8 μM cofilin and 5 μM of actin monomer in assay buffer.

Mentions: Omission of Aip1 from the three-component system stopped disassembly (Fig. 5 A, green) and greatly reduced the frequency of both bursts and scored severing events (Fig. 5 B, green). Omission of either cofilin or coronin had the same effect (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1), which is consistent with a requirement for all three factors for rapid monomer-insensitive disassembly in L. monocytogenes comet tails (Brieher et al., 2006). CapZ could not substitute for Aip1 in the three-component reaction (Fig. 5, A and B, yellow), which suggests a role for Aip1 in disassembly that is distinct from barbed-end capping. When added to the full three-component system, both CytoD and CapZ strongly inhibited filament disassembly (Fig. 5 A, black and blue), blocking both endwise bursting and internal severing modes of filament disassembly (Fig. 5 B, black and blue). To separately test the effect of CytoD and CapZ on disassembly from filament barbed ends and pointed ends, we imaged acrosomal filament bundles in the presence of the full three-component system and either CytoD or CapZ (Fig. 5 C). Remarkably, disassembly of filaments grown off barbed ends and pointed ends were inhibited with equal efficacy, which suggests that filament segments near both ends disassemble through the same CytoD/CapZ-sensitive mechanism (Fig. 5 C). CytoD inhibited filament bundle disassembly with an IC50 of 90 nM (measured using total polymer mass from all filament bundles in a field of view), which is similar to its IC50 for inhibition of polymerization off acrosomal fragments (30 nM; Fig. 5 D). In contrast, although CapZ also inhibited polymerization off acrosomal fragments with a low IC50 (30 nM; Fig. 5 E), its inhibitory effects on disassembly did not saturate at 10 μM (Fig. 5 E). The difference in IC50s between CytoD- and CapZ-mediated inhibition of disassembly suggests differences in the detailed mechanism between these two barbed end–capping agents. For example, barbed ends generated during disassembly may have a conformation that is recognized by CytoD but not by CapZ. Regardless of the detailed mechanism, their common inhibitory effects suggest an important role for barbed end–capping agents in controlling filament stability in the three-component system, even when disassembly occurs by bursting from the pointed end. In contrast, CytoD did not inhibit filament severing in high concentrations of cofilin alone (Fig. 5 F), which is consistent with others' results (Ono et al., 2004).


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)

Barbed end–capping factors inhibit actin disassembly by cofilin, coronin, and Aip1. (A) Mean filament length over time under the following conditions: 2 μM cofilin, 1 μM coronin, 5 μM of actin monomer, and 2 mM ATP, as well as 200 nM Aip1 (red), no Aip1 (green), 5 μM CapZ (yellow), 200 nM Aip1 + 1 μM CytoD (black), or 200 nM Aip1 + 5 μM CapZ (blue). Fast monomer-insensitive disassembly required Aip1 (red vs. green, yellow). CytoD and CapZ inhibited disassembly in the full system (black, blue vs. red). (B) Bar graph showing the percentage of filaments that underwent endwise bursting or severing. The number of filaments analyzed is given below the bars. The percentages of filaments that either underwent endwise bursting or severing increased significantly in the presence of Aip1 (red vs. green, yellow) and decreased significantly when the barbed-end cappers CytoD and CapZ were added to the full system (black, blue vs. red; severing frequency of −CapZ vs. +CapZ, χ2 = 4.9, df = 1, P < 0.05; all other pairwise comparisons, χ2 > 40, df = 1, P < 0.001). (C) Polymer mass decay for bundles with either exposed barbed ends or pointed ends in 3 μM CytoD (brown and purple) or 10 μM CapZ (blue and green), as well as polymer mass decay for all filament bundles in the absence of barbed-end cappers (red). Disassembly of bundles with either exposed barbed ends or pointed ends were inhibited by CytoD/CapZ with equal efficacy. (D) Fraction of polymer mass disassembled as a function of CytoD concentration (red circles). Best fit of the data to a hyperbola (red curve) shows an IC50 of 90 nM for inhibition of disassembly. Length of filament bundles polymerized for a fixed period of time in the presence of varying concentrations of CytoD is also shown (blue squares). Hyperbolic best fit (blue curve) shows an IC50 of 30 nM for inhibition of polymerization. (E) Fraction of polymer disassembled as a function of CapZ concentration (red squares). The titration curve did not reach saturation; the red curve denotes best fit of the data to a straight line. The length of filament actin bundle polymerized in the presence of varying concentrations of CapZ is also shown (blue circles). Polymerization conditions were identical to those in D. Hyperbolic best fit (blue curve) gave an IC50 of 30 nM for inhibition of polymerization. Error bars indicate SD. (F) CytoD does not inhibit cofilin-mediated severing. The bar graph shows the percentage of filaments that severed during a 400 s video either in the absence of CytoD (left) or in the presence of 1 μM CytoD (right). Conditions: 8 μM cofilin and 5 μM of actin monomer in assay buffer.
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fig5: Barbed end–capping factors inhibit actin disassembly by cofilin, coronin, and Aip1. (A) Mean filament length over time under the following conditions: 2 μM cofilin, 1 μM coronin, 5 μM of actin monomer, and 2 mM ATP, as well as 200 nM Aip1 (red), no Aip1 (green), 5 μM CapZ (yellow), 200 nM Aip1 + 1 μM CytoD (black), or 200 nM Aip1 + 5 μM CapZ (blue). Fast monomer-insensitive disassembly required Aip1 (red vs. green, yellow). CytoD and CapZ inhibited disassembly in the full system (black, blue vs. red). (B) Bar graph showing the percentage of filaments that underwent endwise bursting or severing. The number of filaments analyzed is given below the bars. The percentages of filaments that either underwent endwise bursting or severing increased significantly in the presence of Aip1 (red vs. green, yellow) and decreased significantly when the barbed-end cappers CytoD and CapZ were added to the full system (black, blue vs. red; severing frequency of −CapZ vs. +CapZ, χ2 = 4.9, df = 1, P < 0.05; all other pairwise comparisons, χ2 > 40, df = 1, P < 0.001). (C) Polymer mass decay for bundles with either exposed barbed ends or pointed ends in 3 μM CytoD (brown and purple) or 10 μM CapZ (blue and green), as well as polymer mass decay for all filament bundles in the absence of barbed-end cappers (red). Disassembly of bundles with either exposed barbed ends or pointed ends were inhibited by CytoD/CapZ with equal efficacy. (D) Fraction of polymer mass disassembled as a function of CytoD concentration (red circles). Best fit of the data to a hyperbola (red curve) shows an IC50 of 90 nM for inhibition of disassembly. Length of filament bundles polymerized for a fixed period of time in the presence of varying concentrations of CytoD is also shown (blue squares). Hyperbolic best fit (blue curve) shows an IC50 of 30 nM for inhibition of polymerization. (E) Fraction of polymer disassembled as a function of CapZ concentration (red squares). The titration curve did not reach saturation; the red curve denotes best fit of the data to a straight line. The length of filament actin bundle polymerized in the presence of varying concentrations of CapZ is also shown (blue circles). Polymerization conditions were identical to those in D. Hyperbolic best fit (blue curve) gave an IC50 of 30 nM for inhibition of polymerization. Error bars indicate SD. (F) CytoD does not inhibit cofilin-mediated severing. The bar graph shows the percentage of filaments that severed during a 400 s video either in the absence of CytoD (left) or in the presence of 1 μM CytoD (right). Conditions: 8 μM cofilin and 5 μM of actin monomer in assay buffer.
Mentions: Omission of Aip1 from the three-component system stopped disassembly (Fig. 5 A, green) and greatly reduced the frequency of both bursts and scored severing events (Fig. 5 B, green). Omission of either cofilin or coronin had the same effect (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1), which is consistent with a requirement for all three factors for rapid monomer-insensitive disassembly in L. monocytogenes comet tails (Brieher et al., 2006). CapZ could not substitute for Aip1 in the three-component reaction (Fig. 5, A and B, yellow), which suggests a role for Aip1 in disassembly that is distinct from barbed-end capping. When added to the full three-component system, both CytoD and CapZ strongly inhibited filament disassembly (Fig. 5 A, black and blue), blocking both endwise bursting and internal severing modes of filament disassembly (Fig. 5 B, black and blue). To separately test the effect of CytoD and CapZ on disassembly from filament barbed ends and pointed ends, we imaged acrosomal filament bundles in the presence of the full three-component system and either CytoD or CapZ (Fig. 5 C). Remarkably, disassembly of filaments grown off barbed ends and pointed ends were inhibited with equal efficacy, which suggests that filament segments near both ends disassemble through the same CytoD/CapZ-sensitive mechanism (Fig. 5 C). CytoD inhibited filament bundle disassembly with an IC50 of 90 nM (measured using total polymer mass from all filament bundles in a field of view), which is similar to its IC50 for inhibition of polymerization off acrosomal fragments (30 nM; Fig. 5 D). In contrast, although CapZ also inhibited polymerization off acrosomal fragments with a low IC50 (30 nM; Fig. 5 E), its inhibitory effects on disassembly did not saturate at 10 μM (Fig. 5 E). The difference in IC50s between CytoD- and CapZ-mediated inhibition of disassembly suggests differences in the detailed mechanism between these two barbed end–capping agents. For example, barbed ends generated during disassembly may have a conformation that is recognized by CytoD but not by CapZ. Regardless of the detailed mechanism, their common inhibitory effects suggest an important role for barbed end–capping agents in controlling filament stability in the three-component system, even when disassembly occurs by bursting from the pointed end. In contrast, CytoD did not inhibit filament severing in high concentrations of cofilin alone (Fig. 5 F), which is consistent with others' results (Ono et al., 2004).

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