Coordination of the filament stabilizing versus destabilizing activities of cofilin through its secondary binding site on actin

Abstract

Cofilin is a ubiquitous modulator of actin cytoskeleton dynamics that can both stabilize and destabilize actin filaments depending on its concentration and/or the presence of regulatory co-factors. Three charge-reversal mutants of yeast cofilin, located in cofilin's filament-specific secondary binding site, were characterized in order to understand why disruption of this site leads to enhanced filament disassembly. Crystal structures of the mutants showed that the mutations specifically affect the secondary actin-binding interface, leaving the primary binding site unaltered. The mutant cofilins show enhanced activity compared to wild-type cofilin in severing and disassembling actin filaments. Electron microscopy and image analysis revealed long actin filaments in the presence of wild-type cofilin, while the mutants induced many short filaments, consistent with enhanced severing. Real-time fluorescence microscopy of labeled actin filaments confirmed that the mutants, unlike wild-type cofilin, were functioning as constitutively active severing proteins. In cells, the mutant cofilins delayed endocytosis, which depends on rapid actin turnover. We conclude that mutating cofilin's secondary actin-binding site increases cofilin's ability to sever and de-polymerize actin filaments. We hypothesize that activators of cofilin severing, like Aip1p, may act by disrupting the interface between cofilin's secondary actin-binding site and the actin filament.

Keywords: S. cerevisiae; actin cytoskeleton; cofilin; endocytosis; severing.

Figure 1. Crystal structures of cofilin mutants

Structure of cof1-157p (A), cof1-158p (B), and cof1-159p (C) are shown in ribbon diagram. Structural elements as defined by (Federov et al., 1997) are notated in (B) for clarity. Regions mentioned in the text, residues 1-5 and the β4/5 turn are indicated with an asterisk and arrow, respectively.

Figure 2. Effect of mutations on cofilin structure

The structure of wild type cofilin (1COF; (Federov et al., 1997)) is rendered in gold and superimposed with cof1-157p in blue (A), cof1-158p in cyan (B) and cof1-159p in magenta (C). Mutations are shown in stick representation alongside corresponding wild type residues.

Figure 3. Structural modeling predicts the cofilin mutants affect the filament specific binding interface on actin

A trimeric model for cofilin bound to two consecutive subunits in an actin filament is shown for rabbit muscle actin and human cofilin. The model (pdb file 3JOS) was derived by cryo-electron miscroscopy and image averaging (Galkin et al., 2011). The actin subunit nearer the pointed end of the filament is rendered in cyan, the actin subunit nearest the barbed end is space filled in grey, and the cofilin subunit is in yellow. K152 of human cofilin-2, equivalent to R135 of yeast cofilin, is space filled in blue. K96 of human cofilin-2, equivalent to R80 of yeast cofilin, is space filled in cyan. D98 of human cofilin-2, equivalent to K82 of yeast cofilin, is space filled in magenta. The primary (1°) and secondary (2°) binding interfaces are indicated with arrows.

Figure 4. Mutation of the secondary actin-binding interface in cof1-158p affects actin filament binding

Pyrene quenching of actin filaments by wild-type cofilin (A) and cof1-158p (B) was followed at 384nm over time and plotted against fluorescence arbitrary units (FAU). At time 0, different ratios of cofilin-to-actin were added to pre-polymerized actin filaments (7.4% pyrene-iodoacetamide labeled, final concentration 2 μM). Buffer alone was added to the control reactions.

Figure 5. The secondary actin cofilin binding interface is required for cofilin-assisted actin polymerization/nucleation

2±M pyrene-labeled yeast actin was polymerized in the presence of different concentrations of wild type cofilin (A), cof1-157p (B), cof1-158p (C), and cof1-159p (D). Pyrene fluorescence at 384 nm is plotted versus time.

Figure 6. cof1-158p and cof1-159p affect actin filament structure leading to filament fragmentation

Electron micrographs and image averaging (insets) of negatively stained (1% uranyl-acetate) actin filaments in the absence of cofilin (A), and presence of wild-type cofilin (B), cof1-158p (C), or cof1-159p (D). Cofilin was added to 3 ±M pre-polymerized actin shortly before it was spotted on a carbon-coated copper grid at cofilin:actin ratios of 1:1 (B and C) and 1:2 (D). Insets are of the best class sums after several rounds of multi-reference alignment/classification using the EMAN1 software package (Ludtke et al., 1999). The insets are the averages of 20 (A), 90 (B) and 77 (C) raw images. Scale bar=100 nm. Arrows indicate sites of possible filament severing. (E) Crossover lengths were measured for representative class sums of images of undecorated and wild-type cofilin-decorated filament segments. Image analysis was done using Imagic5 (van Heel et al., 1996) (Dube et al., 1993) using the same electron micrographs as in Panels A and B. The class sums have 45 (undecorated) and 43 (decorated) members.

Figure 7. Yeast cofilin induces a conformational change in neighboring, undecorated filament segments

Representative class sums of incomplete wild-type cofilin-decorated filament segments after several rounds of multi-reference alignment/classification using the EMAN1 software package (Ludtke et al., 1999); averages of 241 (B) and 208 (C) raw images are shown. These class sums originated from the same set of micrographs and presumably represent cofilin-decorated (C) and undecorated (B) segments that both have the shorter crossover length. Class sums for undecorated (A) and wild type cofilin decorated (D) filament segments are shown for comparison (same as the insets Figure 6A and B).

Figure 8. Microscopic visualization of filament disassembly by cof1-157p, cof1-158p, and cof1-159p

1μM Oregon green-labeled yeast actin filaments were visualized by fluorescence microscopy after either the addition of control buffer, or after the addition of wild type cofilin, cof1-157p, cof1-158p, or cof1-159p at the indicated ratios of cofilin to actin.

Figure 9. Microscopic visualization of filament severing by cof1-157p, cof1-158p, and cof1-159p

2 μM Oregon green-labeled actin filaments were placed under a coverslip and visualized by time-lapse (1 frame per 200 ms) fluorescence microscopy following the diffusion of 2μM cofilin under the coverslip. Three consecutive frames are displayed from the movies shown in the supplemental data (movies S1–S4). Arrows point at filaments before and after (white) a severing event (A). Panel B shows enlarged regions from Panel A where severing events were captured; severing sites are marked before severing with black arrows and after severing with white arrows.

Figure 9. Microscopic visualization of filament severing by cof1-157p, cof1-158p, and cof1-159p

2 μM Oregon green-labeled actin filaments were placed under a coverslip and visualized by time-lapse (1 frame per 200 ms) fluorescence microscopy following the diffusion of 2μM cofilin under the coverslip. Three consecutive frames are displayed from the movies shown in the supplemental data (movies S1–S4). Arrows point at filaments before and after (white) a severing event (A). Panel B shows enlarged regions from Panel A where severing events were captured; severing sites are marked before severing with black arrows and after severing with white arrows.

Figure 10. Quantitative motion analysis of Abp1-GFP-labeled actin patches in wild-type, cof1-157 and cof1-159 strains

Mean-squared displacement (MSD) plots are shown for patches aligned at the start (left) or end (right) of their lifetimes (A). For patches aligned at the start (left), the curves are truncated at the median lifetime. Average total lifetime of patches, defined as the time from the appearance of a patch until its disappearance (B). Percentage of patches that leave the membrane, defined as traveling >200 nm from their point of origin (C). Average time spent by patches within 200 nm from their point of origin (Phases I and II; D). Average lifetime of patches after they travel 200 nm from their point of origin until the time they disappear (Phase III; E). MSD plot of Phase III patch movement (F). For each patch, only data from patches that travelled more than 200 nm from the point of origin were included. Means ± s.e. (standard error of the mean) of three segregants are shown. Student’s t-tests for statistical significance were performed for B, D and E as indicated. The yeast strains used are described in Table 1. The numbers of patches analyzed were 196 for COF1^wt, 212 for cof1-157, and 47 for cof1-159.

Figure 11. Model for filament destabilization by cof1-158p and cof1-159p

Actin subunits are rendered in green and are shown in either the average, filament-specific conformer found in naked actin filaments or in the cofilin-stabilized tilted conformer found in cofilin-decorated filaments. Wild-type cofilin is rendered in white as are the cofilin mutants with the mutated secondary binding site shaded in pink. Severing events are indicated with lightening bolts and sites where the cofilin mutants block polymerization are marked with black X’s. The filament in the upper left has no cofilin bound and the actin subunits are shown in the normal, average F-actin conformer. The filament in the middle right is decorated with wild-type cofilin, shows that cofilin has stabilized the actin subunits in the tilted conformer, and indicates that severing is most probable at boundaries between regions decorated with cofilin and regions lacking cofilin. The model on the lower right shows a filament interacting with either cof1-158p or cof1-159p. The mutants are shown to induce the tilted conformer but are unable to compensate for destabilization of actin-actin contacts through the secondary binding site resulting very high probabilities of severing at nearest neighbor sites. In addition, the mutants cannot support subunit addition at the barbed end and sequester actin subunits in the monomer pool resulting in net filament disassembly.