Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy

  1. Kien Xuan Ngo
  2. Noriyuki Kodera  Is a corresponding author
  3. Eisaku Katayama
  4. Toshio Ando
  5. Taro QP Uyeda  Is a corresponding author
  1. National Institute of Advanced Industrial Science and Technology, Japan
  2. Kanazawa University, Japan
  3. Japan Science and Technology Agency, Japan
  4. Osaka City University, Japan
  5. University of Tsukuba, Japan
  6. Yokohama City University, Japan
6 figures, 12 videos, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
HS-AFM observation of control and cofilin-bound actin filaments.

(A) Control actin filaments without cofilin. (B) Actin filaments fully bound with cofilin over an extended distance. (C) Paracrystals of actin filaments. Bars: 25 nm, Z-scale: 0–12 nm. (D and E) …

https://doi.org/10.7554/eLife.04806.003
Figure 1—figure supplement 1
Models of control actin filaments and cofilin-decorated filaments on a flat substrate.

(A and B) show predicted peak heights of control actin filaments (PDB ID 3G37; Murakami et al. (2010)), and (C and D) show fully cofilin-decorated filaments (PDB ID 3J0S; Galkin et al. (2011)). …

https://doi.org/10.7554/eLife.04806.004
Asymmetric structure of bare actin zones neighboring a cofilin cluster.

(A and B) HS-AFM image of a short cofilin cluster transiently associating with two S1 molecules (yellow arrowheads), which persisted for ∼1 s, enabling identification of the filament polarity (B). …

https://doi.org/10.7554/eLife.04806.005
Figure 3 with 4 supplements
Actin filaments with bound cofilin or cofilin-rod fusion protein.

(AC) are electron micrographs of negatively stained samples, and (D) is a HS-AFM image of a sample similar to that shown in (B). (A) Actin filaments bound with cofilin-rod. Arrowheads show …

https://doi.org/10.7554/eLife.04806.007
Figure 3—figure supplement 1
Co-sedimentation of cofilin (with or without His-tag) with actin filaments.

(A and B) are SDS-PAGE of supernatant and pellet fractions after ultracentrifugation of mixtures of various concentrations of cofilin with (A) or without (B) His-tag and 1 µM actin filaments, …

https://doi.org/10.7554/eLife.04806.008
Figure 3—figure supplement 2
Actin binding curves of cofilin and cofilin without His tag.

Band intensities of gels shown in Figure 3—figure supplement 1 were quantified, and fractions of actin-bound with cofilin ([cofilin]ppt/[actin]ppt) were shown as a function of free cofilin …

https://doi.org/10.7554/eLife.04806.009
Figure 3—figure supplement 3
Co-sedimentation of cofilin-rod with (+) and without (−) His-tag.

(A and B) SDS-PAGE analysis of binding of cofilin-rod with and without His-tag to actin filaments, respectively. Experiments were carried out in F buffer containing 1 mM ATP, and protein …

https://doi.org/10.7554/eLife.04806.010
Figure 3—figure supplement 4
Representative still images from Video 1, demonstrating cluster formation and severing function of cofilin-rod without His-tag.

Red arrowheads: severing points in end half helices in cofilin clusters; white arrowheads: cofilin-rod clusters. Z-scale: 0–12 nm. Scale bar: 25 nm.

https://doi.org/10.7554/eLife.04806.011
Growth of cofilin clusters along actin filaments.

Growth of cofilin clusters along actin filaments in F buffer containing 1 mM ADP and 0.1 mM ATP (A), along actin filaments carrying ADP, prepared by incubating filaments in F buffer containing 1 mM …

https://doi.org/10.7554/eLife.04806.014
Directional preference of the growth of cofilin clusters.

The growth of cofilin clusters was observed under three buffer conditions: in the presence of 1 mM ADP and 0.1 mM ATP (+ADP +ATP); 1 mM ADP (+ADP) and 1 mM ADP and 10 mM Pi (+ADP +Pi), as in Figure 4

https://doi.org/10.7554/eLife.04806.018
Figure 6 with 4 supplements
Severing of actin filaments near cofilin clusters.

(AC) Typical cases of filament severing (red arrowheads) within or near cofilin clusters (white arrowheads). The observation buffers were F-buffer containing 1 mM ADP (A and C) and 1 mM ATP (B). …

https://doi.org/10.7554/eLife.04806.019
Figure 6—figure supplement 1
Representative still images from Video 9, demonstrating severing of actin filaments by cofilin with His-tag.

Red, blue, and green arrowheads indicate severing in half helices in cofilin clusters, in a bare half helix immediately neighboring a cofilin cluster, and in bare zones more than half a helix away …

https://doi.org/10.7554/eLife.04806.020
Figure 6—figure supplement 2
Representative still images from Video 10, showing severing of actin filaments by cofilin without His-tag.

Red, blue, and green arrowheads indicate severing in half helices in cofilin clusters, in a bare half helix immediately neighboring a cofilin cluster, and in bare zones more than half a helix away …

https://doi.org/10.7554/eLife.04806.021
Figure 6—figure supplement 3
Representative still images from Video 11, showing severing of actin filaments in the presence and absence of low concentration of cofilin.

Blue arrowheads: severing in a bare half helix immediately neighboring a cofilin cluster; green arrowheads: severing in bare zones more than half a helix away from cofilin clusters; white …

https://doi.org/10.7554/eLife.04806.022
Figure 6—figure supplement 4
Representative still images from Video 12, showing severing in actin filaments decorated with high concentrations of cofilin.

Red arrowheads: severing in half helices in cofilin clusters; blue arrowheads: severing in a bare half helix immediately neighboring a cofilin cluster; white arrowheads: cofilin clusters. Z-scale: …

https://doi.org/10.7554/eLife.04806.023

Videos

Video 1
Cluster formation and severing functions of cofilin-rod.

Conditions: F buffer containing 1 mM ATP, and 300 nM cofilin-rod without His-tag, imaging rate: 2 frames/s, and playing rate: 5 frames/s. White arrowheads indicate clusters of cofilin-rod, and red …

https://doi.org/10.7554/eLife.04806.012
Video 2
Sparse binding of individual cofilin-rod molecules to actin filaments.

Conditions: F buffer containing 1 mM ATP and 75 nM cofilin-rod without His-tag, imaging rate: 4 frames/s, and playing rate: 5 frames/s. Transient binding of cofilin-rod to and dissociation from an …

https://doi.org/10.7554/eLife.04806.013
Video 3
Growth of a cofilin cluster toward the pointed end of a filament in F buffer containing 1 mM ADP, 0.1 mM ATP, 20 nM S1, and 75 nM cofilin.

Imaged at 2 frames/s and played at 5 frames/s. White arrowheads show growth of the cofilin cluster, and yellow and magenta arrowheads show binding of S1. Magenta arrowheads indicate S1 molecules …

https://doi.org/10.7554/eLife.04806.015
Video 4
Growth of a cofilin cluster toward the pointed end of a filament in F buffer containing 1 mM ADP, 20 nM S1, and 75 nM cofilin.

Imaged at 2 frames/s and played at 5 frames/s. For color codes of arrowheads, see the legend to Video 3. Z-scale was 0–12 nm. For magnifications and polarity of the analyzed filaments, refer to Figur…

https://doi.org/10.7554/eLife.04806.016
Video 5
Growth of a cofilin cluster toward the pointed end of a filament in F buffer containing 1 mM ADP, 10 mM Pi, 150 nM S1, and 900 nM cofilin (without His-tag).

For color codes of arrowheads, see the legend to Video 3. For magnifications and polarity of the analyzed filaments, refer to Figure 4 in the main text. Under this condition, binding of S1 was so …

https://doi.org/10.7554/eLife.04806.017
Video 6
Severing of actin filaments in a cofilin cluster.

Conditions: F buffer containing 1 mM ADP and 40 nM cofilin. Severing of actin filaments occurred within a cofilin cluster (at 53 s) and then at or near the boundary between a bare zone and another …

https://doi.org/10.7554/eLife.04806.024
Video 7
Severing of actin filaments at or near a boundary between a bare zone and a cofilin cluster.

Conditions: F buffer containing 1 mM ATP and 75 nM cofilin. Severing occurred at 42 s. Imaged at 2 frames/s and played at 5 frames/s. For color codes of the arrowheads, see the legend to Video 6. …

https://doi.org/10.7554/eLife.04806.025
Video 8
Severing of actin filaments in a bare zone more than one half helix away from a cofilin cluster.

Conditions: F buffer containing 1 mM ADP and 40 nM cofilin. Severing occurred at 28 s. Imaged at 2 frames/s and played at 5 frames/s. For color codes of the arrowheads, see the legend to Video 6. …

https://doi.org/10.7554/eLife.04806.026
Video 9
Severing of actin filaments by cofilin.

To show more general view of severing events, in addition to the small number of representative cases shown in Videos 6–8, 10 different image sequences from different experiments were merged. …

https://doi.org/10.7554/eLife.04806.027
Video 10
Severing of actin filaments by cofilin without His-tag.

In this video, seven different image sequences from different filaments and experiments were merged. Conditions: F buffer containing 1 mM ATP and 75 nM cofilin, except in sequence 3 in which cofilin …

https://doi.org/10.7554/eLife.04806.028
Video 11
Severing of actin filaments in the absence or presence of low concentrations of cofilin.

Four independent image sequences are merged. Conditions: F buffer containing 1 mM ATP and 0, 10 or 40 nM cofilin without His-tag. In this video, three data sets which represent three cases of the …

https://doi.org/10.7554/eLife.04806.029
Video 12
Actin filaments decorated with high concentrations of cofilin.

The first three quarter of this video were taken in F buffer containing 1 mM ATP and 1 µM cofilin, and the last one quarter was taken in the presence of 650 nM cofilin. In the presence of 1 µM …

https://doi.org/10.7554/eLife.04806.030

Tables

Table 1

Peak heights and lengths of half helical pitches in bare actin segments neighboring cofilin clusters

https://doi.org/10.7554/eLife.04806.006
1 mM ADP + 0.1 mM ATP1 mM ADP
Peak height (nm)Half helix (nm)Peak height (nm)Half helix (nm)
First neighbor on the P-end side9.2 ± 1.028.8 ± 4.58.9 ± 1.028.7 ± 4.7
Second neighbor on the P-end side9.1 ± 1.036.5 ± 4.18.8 ± 1.136.9 ± 4.9
First neighbor on the B-end side9.0 ± 0.937.3 ± 4.68.7 ± 0.938.4 ± 4.3
Second neighbor on the B-end side9.2 ± 0.635.4 ± 3.98.7 ± 0.836.7 ± 4.2
  1. Actin filaments were incubated in F buffer containing 1 mM ADP and 0.1 mM ATP for 5 min or in F buffer containing 1 mM ADP for 30 min prior to the addition of cofilin. Filaments under the latter condition were shorter than those under the former condition and were apparently in the process of spontaneous depolymerization.

  2. Each mean and SD were calculated from 423 to 446 data.

Additional files

Source code 1

The Kodec4.4.7.39 for HS-AFM image viewing and analysis software is coded in Visual C# (Visual Studio 2010, Microsoft, USA).

https://doi.org/10.7554/eLife.04806.031

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