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Figures and figure supplements
Cofilin-induced unidirectional cooperative conformational changes in actin filaments
revealed by high-speed atomic force microscopy
Kien Xuan Ngo, et al.

Ngo et al. eLife 2015;4:e04806. DOI: 10.7554/eLife.04806

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Figure 1. 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) Histograms of peak heights (D) and lengths of half helical pitches (E) incontrol actin filaments and cofilin-decorated actin segments. N was between 1722 and 2536. (F) Half helical pitchesof actin paracrystals (N = 1009). Solid lines show Gaussian fittings with confidence intervals of 99.73%. For comparison, the dark line in (F) shows the Gaussian fitting of control actin filaments. Measurements were made in F buffer
containing 1 mM ATP (A), 1 mM ATP, and 75 nM cofilin (B) or 1 mM ATP and 30 mM MgCl2 (C). Student's t-test
comparing control and cofilin-decorated actin segments showed that the differences in peak heights and half
helical pitches are statistically significant at p ≤ 0.00001. The mean of the half helical pitches of control actin
filaments and paracrystals did not differ significantly. Models of control actin filaments and cofilin-decorated actin
filaments with two different orientations on substrates are shown in Figure 1—figure supplement 1.
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Figure 1—figure supplement 1. Models of control actin filaments and cofilin-decorated filaments on a flatsubstrate. (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)). These images weregenerated using Chimera software. When the true crossover point coincides with the position of an actin subunit(A) or a bound cofilin molecule (C), the position of the peak is equal to that of the crossover point, and its height isthe tallest of the various possible orientations of the filament on the substrate. When the true crossover point is at
the middle of two actin subunits (B) or two bound cofilin molecules (D), the peak height is the shortest of thepossible orientations. In this latter case, the observed peak position is away from the true crossover point by asmuch as ∼2.5 nm. Depending on the orientation of the filament, therefore, the distance between the observed
peak position and the true crossover point fluctuates between 0 and 2.5 nm. Random distribution of distances
between the true crossover points and the highest points of the closest actin subunits would result in ±1.7 nm oferror in the estimation of the positions of the true crossover points. If the peak positions at both ends of a half helix
have this much error, the half helical pitch would have ±2.3 nm (±1.7 nm × √2) error. This is somewhat smaller thanthe ±3.0 nm standard deviation of the half helical pitches of actin filament paracrystals, and the difference should
derive from small structural fluctuations in the paracrystals and/or measurement error from various other sources. Ineither case, it is safe to use the 3.0 nm standard deviation as the upper limit of the combined measurement errors
and to estimate the lower limit of the structural fluctuations of control and cofilin-bound filaments.
DOI: 10.7554/eLife.04806.004

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Figure 2. 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). Measurements were made in F buffer containing 20 nM S1, 75 nM cofilin, 1mM ADP, and 0.1 mM ATP. Bar: 25 nm; Z-scale: 0–12 nm. (C and D) Time-dependent changes in the heights of theindicated peaks (white arrowheads) and half helical pitches between the indicated peaks. (E and F) Histograms of
the lengths of the half helical pitches of bare actin segments immediately neighboring cofilin clusters. Filaments
were incubated in F buffer containing 1 mM ADP and 0.1 mM ATP for 5 min before observation (E) as in (A and B)
or were incubated in F buffer containing 1 mM ADP for 30 min before observation (F). Pitches of the half helices ofthe first immediate neighbor on each side of cofilin clusters were measured. These values, together with those forthe second neighbors, are summarized in Table 1.
DOI: 10.7554/eLife.04806.005

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Figure 3. Actin filaments with bound cofilin or cofilin-rod fusion protein. (A–C) are electron micrographs ofnegatively stained samples, and (D) is a HS-AFM image of a sample similar to that shown in (B). (A) Actin filamentsbound with cofilin-rod. Arrowheads show crossover points in clusters of cofilin-rod. The rod portions of the fusion
proteins are not readily visible, which may be due to alignment of the rods along the cofilin clusters. Severing
activity and stoichiometric binding of cofilin-rod to actin filaments were confirmed by HS-AFM (Video 1) andco-sedimentation assays (Figure 3—figure supplement 3), respectively. (B) Cofilin-rod molecules sparsely boundto actin filaments, identified by the rod-like structures (black arrowheads). (C) Actin filaments with bound cofilinmolecules (without rod fusion). Arrowheads show crossover points in clusters. Actin filaments and cofilin orcofilin-rod were mixed at a 2:1 (A and C) or 1:1 (B) molar ratio in F buffer containing 1 mM ATP. Bars: 25 nm. (D)HS-AFM image of an actin filament and an apparently singly bound cofilin-rod molecule (blue arrowhead in theupper image) near P2 (white arrowhead). Conditions: F buffer containing 1 mM ATP and 75 nM cofilin-rod (withoutHis-tag). Bar: 25 nm. See Video 2. (E) shows heights of the three peaks and (F) shows spacing between them.
DOI: 10.7554/eLife.04806.007

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Figure 3—figure supplement 1. Co-sedimentation ofcofilin (with or without His-tag) with actin filaments. (Aand 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, respectively. Acrylamide
concentration of the gel was 12%. His-tag wasremoved by incubation with 1/4 (wt/wt) of His-tagged
TEV protease overnight at 5°C and then passed
through a Ni-column in the presence of 10 mM
imidazole. Lanes 1 to 4 in (C) show His-TEV protease,mixture of His-cofilin and His-TEV after reaction overnight, His-cofilin, and the reaction mixture that flowed
through a Ni-NTA column (i.e., cofilin without His-tag).
Co-sedimentation experiments were performed asfollows. Frozen aliquots of cofilin (±His-tag) and rabbit
skeletal actin were thawed on ice for 1 hr and werethen clarified by ultracentrifugation at 80,000 rpm for 5min at 5°C. The supernatant was collected, andprotein concentrations were measured using
Advanced Protein Assay reagent (Cytoskeleton). Actin
filaments were prepared by polymerization of G-actin(20 µM) in F buffer containing 1 mM ATP on ice for 30
min. The filaments formed were diluted to 1 µM andgently mixed with the cofilin (±His-tag) of various
concentrations (i.e., 0.1, 0.5, 1, and 2 µM) in F buffer
containing 1 mM ATP. These protein mixtures were
further incubated for 3–5 min at room temperature,
followed by ultracentrifugation at 80,000 rpm for 5 minat 25°C. Supernatant and pellet fractions wereseparately run on a SDS-polyacrylamide gel andstained with Coomassie Blue. Control experiments
were performed in the same manner using either 1 µM
actin filament only or 2 µM cofilin or its derivative only.
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Figure 3—figure supplement 2. Actin binding curvesof cofilin and cofilin without His tag. Band intensities ofgels 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 freecofilin concentration ([cofilin]sup). Mean ± SD are shown(N = 3).
DOI: 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 andwithout His-tag to actin filaments, respectively.
Experiments were carried out in F buffer containing 1mM ATP, and protein concentrations used are shown
above each gel. Cleavage of His-tag by TEV protease
was performed as described in the legend to Figure
3—figure supplement 1. Acrylamide concentration of
the gel was 10%.
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Figure 3—figure supplement 4. Representative still
images from Video 1, demonstrating cluster formationand severing function of cofilin-rod without His-tag.
Red arrowheads: severing points in end half helices incofilin clusters; white arrowheads: cofilin-rod clusters.
Z-scale: 0–12 nm. Scale bar: 25 nm.
DOI: 10.7554/eLife.04806.011

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Figure 4. 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 ADP, hexokinase, and glucose at room
temperature for 30 min (B), and along actin filaments carrying ADP and Pi, prepared by incubating filaments in 1 mM ADP and 10 mM Pi for 10 min atroom temperature (C). The concentrations of cofilin were 75 nM (A and B) or 900 nM (C) and those of S1 were 20 nM (A and B) or 150 nM (C). Each panel
consists of four sequential snapshots (top), a figure showing the heights of the indicated peaks (middle), and the half helical pitches between theindicated peaks (bottom). Yellow arrowheads show the transient association of S1. Note that P1 in (A) and P3 in (B) rose in two substeps. Bars: 25 nm;
Z-scale: 0–12 nm.
DOI: 10.7554/eLife.04806.014

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Figure 5. Directional preference of the growth ofcofilin clusters. The growth of cofilin clusters wasobserved under three buffer conditions: in thepresence 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. Growth of a cluster by
one-half helix was counted as one growth event. The
total number of observed growth events was 37, 46,and 188 for each condition. We speculate that at least
some of the cluster growth events in the barbed-end
direction were actually growth in the preferred
direction from invisibly small clusters on the barbed
side of a visible cluster.
DOI: 10.7554/eLife.04806.018

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Figure 6. Severing of actin filaments near cofilin clusters. (A–C) Typical cases of filament severing (red arrowheads)
within or near cofilin clusters (white arrowheads). The observation buffers were F-buffer containing 1 mM ADP (Aand C) and 1 mM ATP (B). Concentration of cofilin was 40 nM (A and C) and 75 nM. The first break in (A) was inside
a cluster, while the second was at or near the junction between a bare zone and a cluster. Red, blue, and green
arrowheads show severing points within cofilin clusters, in bare zone close to cofilin clusters, and in bare zones
more than half a helix away from a cofilin cluster, while white arrowheads show cofilin clusters. Bars: 25 nm; Z-scale:0–12 nm. See Videos 6–8. (D) Classification of severing sites into four categories: (1) in ‘far’ bare zone (between atall and a short black arrow or between two short black arrows, indicated by green bars in (E)); (2) in bare zone half
helices immediately neighboring a cofilin cluster (between a tall black and a tall orange arrow, indicated by blue
bars in (E)); (3) in ‘end’ cofilin cluster half helices immediately neighboring bare zones (between a tall and a shortorange arrow, indicated by red bars in (E)); and (4) in ‘inner’ cofilin cluster half helices (between two short orange
arrows, indicated by a red bar in (E)). Comparison of the last two categories demonstrates that severing within
cofilin clusters occurs preferentially near the ends. Note, however, that this comparison does not necessarily show aquantitative difference in the susceptibility to severing between end and inner half helices, since the number of end
and inner helices examined are not the same. (E) A schematic summary of the proposed distributions of boundcofilin molecules (red spheres), segments of normal (yellow) and shortened (orange) helical pitch, and normal (blackarrows) and tall (orange arrows) crossover points. Free cofilin molecules tend to bind to the supertwisted bare zone
on the pointed-end side of the cluster (gray arrows), driving the growth of the cluster in the pointed-end direction.
This is most certainly an oversimplification, ignoring a number of complex issues, some of which are discussed in
the main text.
DOI: 10.7554/eLife.04806.019

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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 cofilinclusters, in a bare half helix immediately neighboring acofilin cluster, and in bare zones more than half a helix
away from cofilin clusters, respectively. Whitearrowheads: cofilin clusters. Numbers (1–10) refer tothe sequence number in the video. Z-scale: 0–12 nm.
Scale bar: 25 nm.
DOI: 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, andgreen arrowheads indicate severing in half helices in
cofilin clusters, in a bare half helix immediately
neighboring a cofilin cluster, and in bare zones morethan half a helix away from cofilin clusters, respectively.
White arrowheads: cofilin clusters. Numbers (1–7) referto the sequence number in the video. Z-scale: 0–12
nm. Scale bar: 25 nm.
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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 abare half helix immediately neighboring a cofilincluster; green arrowheads: severing in bare zones
more than half a helix away from cofilin clusters; whitearrowheads: cofilin clusters. Z-scale: 0–12 nm. Scalebar: 25 nm.
DOI: 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 ofcofilin. Red arrowheads: severing in half helices in
cofilin clusters; blue arrowheads: severing in a barehalf helix immediately neighboring a cofilin cluster;white arrowheads: cofilin clusters. Z-scale: 0–12 nm.
Scale bar: 25 nm.
DOI: 10.7554/eLife.04806.023

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