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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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Filament bursting is distinct from cofilin-mediated severing. (A) Time-lapse images of two actin filaments disassembling in 10 μM cofilin, 5 μM of actin monomer, and 2 mM ATP. Long filaments were chosen to illustrate the occurrence of multiple severing events within a single filament. Bar, 1 μm. (B) Bar graph showing the fraction of disassembly events scored as bursting (red) or severing (green), either in the full depolymerizing system (left) or in cofilin alone (right). In the full system, bursting occurred with significantly higher frequency than severing (χ2 = 190, df = 1, P < 0.01). However, in the presence of high concentrations of cofilin, severing occurred with significantly higher frequency than bursting (χ2 = 55, df = 1, P < 0.01) and was the predominant disassembly mechanism observed under these conditions. We note that mean initial filament length did not differ significantly between different experiments and was not the cause of the differences observed.
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fig2: Filament bursting is distinct from cofilin-mediated severing. (A) Time-lapse images of two actin filaments disassembling in 10 μM cofilin, 5 μM of actin monomer, and 2 mM ATP. Long filaments were chosen to illustrate the occurrence of multiple severing events within a single filament. Bar, 1 μm. (B) Bar graph showing the fraction of disassembly events scored as bursting (red) or severing (green), either in the full depolymerizing system (left) or in cofilin alone (right). In the full system, bursting occurred with significantly higher frequency than severing (χ2 = 190, df = 1, P < 0.01). However, in the presence of high concentrations of cofilin, severing occurred with significantly higher frequency than bursting (χ2 = 55, df = 1, P < 0.01) and was the predominant disassembly mechanism observed under these conditions. We note that mean initial filament length did not differ significantly between different experiments and was not the cause of the differences observed.

Mentions: In the presence of cofilin, coronin, and Aip1, filaments shortened and disappeared from the field of view within the first 100 s (Fig. 1, A and B; and Video 1). We tracked the locations of filament ends using kymographs drawn along the contours of individual actin filaments (Fig. 1, B and C, filaments f1–f4). Kymographs showed that filaments did not shrink smoothly from an end; instead, they disassembled in infrequent bursts, with filaments abruptly losing mass from an end (Fig. 1 B, triangles). Most disassembly events occurred at filament ends, but events were occasionally initiated in the middle of the filament; these were scored as severing events (Fig. 1 B, blue square; see Fig. 2 for a detailed comparison of this reaction to a cofilin-mediated severing reaction). In an attempt to resolve molecular events within an endwise burst, the acquisition time per image was reduced to 16 ms using a high-speed camera (Fig. 1 B, filament f4). At this frame rate, kymographs were noisy, but disassembly still appeared primarily as bursts at ends. The abruptness of the burst argues against mechanisms involving successive dissociation of individual subunits from an end; the instantaneous off rate for such a mechanism would have to be ∼104 subunits/s.


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)

Filament bursting is distinct from cofilin-mediated severing. (A) Time-lapse images of two actin filaments disassembling in 10 μM cofilin, 5 μM of actin monomer, and 2 mM ATP. Long filaments were chosen to illustrate the occurrence of multiple severing events within a single filament. Bar, 1 μm. (B) Bar graph showing the fraction of disassembly events scored as bursting (red) or severing (green), either in the full depolymerizing system (left) or in cofilin alone (right). In the full system, bursting occurred with significantly higher frequency than severing (χ2 = 190, df = 1, P < 0.01). However, in the presence of high concentrations of cofilin, severing occurred with significantly higher frequency than bursting (χ2 = 55, df = 1, P < 0.01) and was the predominant disassembly mechanism observed under these conditions. We note that mean initial filament length did not differ significantly between different experiments and was not the cause of the differences observed.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2483518&req=5

fig2: Filament bursting is distinct from cofilin-mediated severing. (A) Time-lapse images of two actin filaments disassembling in 10 μM cofilin, 5 μM of actin monomer, and 2 mM ATP. Long filaments were chosen to illustrate the occurrence of multiple severing events within a single filament. Bar, 1 μm. (B) Bar graph showing the fraction of disassembly events scored as bursting (red) or severing (green), either in the full depolymerizing system (left) or in cofilin alone (right). In the full system, bursting occurred with significantly higher frequency than severing (χ2 = 190, df = 1, P < 0.01). However, in the presence of high concentrations of cofilin, severing occurred with significantly higher frequency than bursting (χ2 = 55, df = 1, P < 0.01) and was the predominant disassembly mechanism observed under these conditions. We note that mean initial filament length did not differ significantly between different experiments and was not the cause of the differences observed.
Mentions: In the presence of cofilin, coronin, and Aip1, filaments shortened and disappeared from the field of view within the first 100 s (Fig. 1, A and B; and Video 1). We tracked the locations of filament ends using kymographs drawn along the contours of individual actin filaments (Fig. 1, B and C, filaments f1–f4). Kymographs showed that filaments did not shrink smoothly from an end; instead, they disassembled in infrequent bursts, with filaments abruptly losing mass from an end (Fig. 1 B, triangles). Most disassembly events occurred at filament ends, but events were occasionally initiated in the middle of the filament; these were scored as severing events (Fig. 1 B, blue square; see Fig. 2 for a detailed comparison of this reaction to a cofilin-mediated severing reaction). In an attempt to resolve molecular events within an endwise burst, the acquisition time per image was reduced to 16 ms using a high-speed camera (Fig. 1 B, filament f4). At this frame rate, kymographs were noisy, but disassembly still appeared primarily as bursts at ends. The abruptness of the burst argues against mechanisms involving successive dissociation of individual subunits from an end; the instantaneous off rate for such a mechanism would have to be ∼104 subunits/s.

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