Limits...
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.

Show MeSH

Related in: MedlinePlus

Actin filaments disassemble in bursts in cofilin, coronin, and Aip1. (A) Time-lapse wide-field epifluorescence images of fluorescently labeled actin filaments in the presence of 2 μM cofilin, 1 μM coronin, 200 nM Aip1, 5 μM of actin monomer, and 2 mM ATP. Filaments shorten and disappear from the field of view. Bar, 3 μm. (B) Successive time-lapse images showing a single actin filament (f1) over time, along with kymographs drawn along the contours of representative filaments (f1–f4). The red lines on the image of f1 at t = 0 denote the contour on which the kymograph was drawn. Time is given on the x axis of the kymograph, whereas the position along the filament contour is given on the y axis. Mean integration time for a single image was 400 ms for f1–f3 and 16 ms for f4. Triangles denote endwise bursting (f1–f3); yellow triangles denote initial burst (f1–f3), red triangles denote successive proximal bursts (f1 and f2), and green triangle denotes a successive distal burst (f3). Same-side bursts occurred more frequently (78%) than opposite-side bursts (22%; P < 0.001, one-tailed z test). The square denotes internal disassembly event counted as a severing event (f3). Bar, 1 μm. (C) Histogram of filament burst size. The mean burst size was 260 subunits. (D) Histogram of waiting times between successive bursts (red), fit to a single exponential (black). Single exponential fit gave characteristic decay time of τ = 14 s.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2483518&req=5

fig1: Actin filaments disassemble in bursts in cofilin, coronin, and Aip1. (A) Time-lapse wide-field epifluorescence images of fluorescently labeled actin filaments in the presence of 2 μM cofilin, 1 μM coronin, 200 nM Aip1, 5 μM of actin monomer, and 2 mM ATP. Filaments shorten and disappear from the field of view. Bar, 3 μm. (B) Successive time-lapse images showing a single actin filament (f1) over time, along with kymographs drawn along the contours of representative filaments (f1–f4). The red lines on the image of f1 at t = 0 denote the contour on which the kymograph was drawn. Time is given on the x axis of the kymograph, whereas the position along the filament contour is given on the y axis. Mean integration time for a single image was 400 ms for f1–f3 and 16 ms for f4. Triangles denote endwise bursting (f1–f3); yellow triangles denote initial burst (f1–f3), red triangles denote successive proximal bursts (f1 and f2), and green triangle denotes a successive distal burst (f3). Same-side bursts occurred more frequently (78%) than opposite-side bursts (22%; P < 0.001, one-tailed z test). The square denotes internal disassembly event counted as a severing event (f3). Bar, 1 μm. (C) Histogram of filament burst size. The mean burst size was 260 subunits. (D) Histogram of waiting times between successive bursts (red), fit to a single exponential (black). Single exponential fit gave characteristic decay time of τ = 14 s.

Mentions: We imaged the disassembly of single, fluorescently labeled actin filaments catalyzed by the three-component purified protein system consisting of cofilin, coronin, and Aip1. Concentrations of individual components were chosen to be those sufficient for full rate enhancement in L. monocytogenes comet tail disassembly (Brieher et al., 2006). All reactions also contained 5 μM of unlabeled actin monomer and 2 mM ATP, which reflects the physiological requirement that disassembly must occur in substantial concentrations of polymerizable monomer. We polymerized fluorescent-labeled actin filaments in a perfusion chamber using the actin cross-linker filamin to immobilize filaments to the coverslip surface. In all assays, filaments were polymerized for <1 min before initiation of disassembly. Filament aging leads to kinetic changes we will detail elsewhere (unpublished data). Newly assembled filaments were then disassembled by perfusion of the three-component system. Immediately after perfusion, a streaming video of actin filaments (acquisition time per image = 400 ms) was acquired by wide-field fluorescence microscopy (Fig. 1 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1).


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 in bursts in cofilin, coronin, and Aip1. (A) Time-lapse wide-field epifluorescence images of fluorescently labeled actin filaments in the presence of 2 μM cofilin, 1 μM coronin, 200 nM Aip1, 5 μM of actin monomer, and 2 mM ATP. Filaments shorten and disappear from the field of view. Bar, 3 μm. (B) Successive time-lapse images showing a single actin filament (f1) over time, along with kymographs drawn along the contours of representative filaments (f1–f4). The red lines on the image of f1 at t = 0 denote the contour on which the kymograph was drawn. Time is given on the x axis of the kymograph, whereas the position along the filament contour is given on the y axis. Mean integration time for a single image was 400 ms for f1–f3 and 16 ms for f4. Triangles denote endwise bursting (f1–f3); yellow triangles denote initial burst (f1–f3), red triangles denote successive proximal bursts (f1 and f2), and green triangle denotes a successive distal burst (f3). Same-side bursts occurred more frequently (78%) than opposite-side bursts (22%; P < 0.001, one-tailed z test). The square denotes internal disassembly event counted as a severing event (f3). Bar, 1 μm. (C) Histogram of filament burst size. The mean burst size was 260 subunits. (D) Histogram of waiting times between successive bursts (red), fit to a single exponential (black). Single exponential fit gave characteristic decay time of τ = 14 s.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483518&req=5

fig1: Actin filaments disassemble in bursts in cofilin, coronin, and Aip1. (A) Time-lapse wide-field epifluorescence images of fluorescently labeled actin filaments in the presence of 2 μM cofilin, 1 μM coronin, 200 nM Aip1, 5 μM of actin monomer, and 2 mM ATP. Filaments shorten and disappear from the field of view. Bar, 3 μm. (B) Successive time-lapse images showing a single actin filament (f1) over time, along with kymographs drawn along the contours of representative filaments (f1–f4). The red lines on the image of f1 at t = 0 denote the contour on which the kymograph was drawn. Time is given on the x axis of the kymograph, whereas the position along the filament contour is given on the y axis. Mean integration time for a single image was 400 ms for f1–f3 and 16 ms for f4. Triangles denote endwise bursting (f1–f3); yellow triangles denote initial burst (f1–f3), red triangles denote successive proximal bursts (f1 and f2), and green triangle denotes a successive distal burst (f3). Same-side bursts occurred more frequently (78%) than opposite-side bursts (22%; P < 0.001, one-tailed z test). The square denotes internal disassembly event counted as a severing event (f3). Bar, 1 μm. (C) Histogram of filament burst size. The mean burst size was 260 subunits. (D) Histogram of waiting times between successive bursts (red), fit to a single exponential (black). Single exponential fit gave characteristic decay time of τ = 14 s.
Mentions: We imaged the disassembly of single, fluorescently labeled actin filaments catalyzed by the three-component purified protein system consisting of cofilin, coronin, and Aip1. Concentrations of individual components were chosen to be those sufficient for full rate enhancement in L. monocytogenes comet tail disassembly (Brieher et al., 2006). All reactions also contained 5 μM of unlabeled actin monomer and 2 mM ATP, which reflects the physiological requirement that disassembly must occur in substantial concentrations of polymerizable monomer. We polymerized fluorescent-labeled actin filaments in a perfusion chamber using the actin cross-linker filamin to immobilize filaments to the coverslip surface. In all assays, filaments were polymerized for <1 min before initiation of disassembly. Filament aging leads to kinetic changes we will detail elsewhere (unpublished data). Newly assembled filaments were then disassembled by perfusion of the three-component system. Immediately after perfusion, a streaming video of actin filaments (acquisition time per image = 400 ms) was acquired by wide-field fluorescence microscopy (Fig. 1 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200801027/DC1).

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