Cofilin and Aip1 Promote the Rapid Disassembly of Yeast Actin Cables
Actin disassembly-promoting factors such as cofilin have not been detected on actin cables in wild-type cells. However, aip1Δ
cells accumulate aberrantly thick actin cables decorated with cofilin (Rodal et al., 1999
). This led us to explore the possible roles of cofilin and Aip1 in regulating cable dynamics. We first tested the role of cofilin, encoded by the essential gene COF1
(Iida et al., 1993
; Moon et al., 1993
). We used two mutant alleles of cofilin, cof1-19
. Cof1-19 has normal actin filament–severing activity in vitro (Ojala et al., 2001
) but disrupts Aip1 capping and net disassembly of cofilin-decorated filaments (Balcer et al., 2003
). Further, cof1-19
disrupts localization of Aip1 to patches, indicating that cofilin-Aip1 interactions are required to maintain Aip1 localization (Rodal et al., 1999
). Interestingly, Cof1-19 localizes to both patches and cables (whereas wild-type cofilin is only detected on patches). Thus, normal functional interactions between cofilin and Aip1 may result in a highly dynamic decoration of cables by cofilin (i.e., short-lived), not detected in wild-type cells, but detected in cof1-19
cells where the interaction is impaired. Cof1-22 has partially impaired actin filament disassembly activity in vitro and greatly reduced rates of patch turnover in vivo (Lappalainen et al., 1997
). Cof1-22 does not disrupt Aip1 capping of filaments (Balcer et al., 2003
) and consequently does not accumulate thickened cables or stably decorate cables.
To investigate the role of cofilin in cable turnover, we treated wild-type, cof1-19
, and cof1-22
cells with Lat-A. Lat-A is an actin monomer sequestering agent that blocks new actin assembly, thus allowing measurement of actin turnover rate, which correlates with the rate of disappearance of F-actin structures. The actual half-life of patches and cables is likely to be <20 s, as measured in the presence of high concentrations of Lat-A (Yang and Pon, 2002
; Kaksonen et al., 2003
). However, lower concentrations of Lat-A can be used to compare strains for relative
rates of actin turnover. In fact, in the presence of higher concentrations of Lat-A, actin structures disappear so rapidly that mutant effects (e.g., 2–5-fold reduced rates of turnover) can be difficult to assess. For this reason, we used concentrations of Lat-A optimized for detecting differences between wild-type and mutant strains (20 μM for cable turnover and 50 μM for patch turnover).
After cells were treated with 20 μM Lat A, cable disappearance was examined at different time points by immunofluorescence microscopy using anti-actin antibodies (representative staining in A). Quantified results (, B and C) show that cof1-22 markedly reduces the rate of cable disassembly, increasing by ~20-fold the time in Lat A required for 50% of cells to lose visible cable staining (wild-type cells, 1 min; cof1-22 cells, 20 min). Thus, the filament severing and disassembly activity of cofilin is required for rapid turnover of cables. cof1-19 cells also showed significantly reduced rates of cable turnover, ~10-fold slower than wild-type cells, indicating that rapid cable turnover also requires functional interactions between cofilin and Aip1. Consistent with this view, similar cable turnover defects were observed for aip1Δ cells (see below). These observations led us to dissect the cofilin-dependent function of Aip1 in promoting actin turnover.
Figure 1. Reduced rates of actin cable turnover in cof1 mutant cells. Wild-type (COF1) and mutant (cof1-19, cof1-22) yeast cells were incubated with 20 μM Latrunculin A (Lat-A). Samples of cells were removed at the indicated time points, fixed, stained (more ...)
Identification of aip1 Alleles with Actin Cable Defects
The cellular function of Aip1 has remained undefined, in part because point mutations have not been introduced into Aip1 to disrupt its specific molecular interactions and activities. Therefore, we performed a systematic mutagenesis of Aip1 surface residues. Using an alignment of Aip1 sequences from diverse organisms (Supplementary Figure S1) and the crystal structures of S. cerevisiae
Aip1 (Voetgli et al., 2003
) and Caenorhabditis elegans
UNC-78/Aip1 (Mohri et al., 2004
), we targeted all residues that are both conserved and solvent exposed. The Aip1 structure resembles an open clamshell and consists of two β-propellers, each formed by seven WD-blades, connected at an angle of ~110°. A total of 31 surface residues were mutated within 18 different alleles ( and Supplementary Figure S1). The residues mutated are found primarily on one face of Aip1, designated as the “front” face (), distributed between the N- and C-terminal lobes. The “back” face of Aip1 is not well conserved. In addition, we generated one substitution allele at a yeast-specific insertion located in the C-terminal lobe (aip1-119
) and two truncation alleles that express the N- and C-terminal propellers of Aip1 alone: N-aip1
(Δ326–615) and C-aip1
(Δ14–319). All 21 resulting alleles were integrated at the AIP1
locus. Immunoblotting demonstrated that mutant protein levels are similar to Aip1 levels in wild-type cells (A). Expression of N-aip1
was detected by immunofluorescence (cytoplasmic localization, E), but not by immunoblotting (B).
Data compilation for aip1 mutants
Figure 2. Surfaces on Aip1 required for its in vivo function. Mutated residues were modeled on the crystal structure of S. cerevisiae Aip1 (1PI6) with mutant allele numbers indicated (). The majority of conserved surface residues map to the “front” (more ...)
Figure 3. Actin cytoskeleton organization and Aip1 and cofilin localization in wild-type and mutant aip1 cells. (A–C) Aip1 protein levels in wild-type and mutant aip1 cells. Whole cell extracts from integrated wild-type and mutant strains were immunoblotted (more ...)
Each aip1 strain was examined for actin organization and localization of cofilin and Aip1. Four alleles showed thickened actin cables, similar to the aip1Δ mutations: aip1-107, aip1-108, aip1-109, and aip1-119 (D). The remaining aip1 alleles did not show striking phenotypes compared with wild-type cells (). The thickened cables observed in specific aip1 alleles as well as in aip1Δ and cof1-19 are not actin bars, because they are detected by Alexa-488 phalloidin (Supplementary Figure S2). Of the four alleles that showed actin cable defects, clear decoration of thickened cables by cofilin was observed only for aip1-107 (D, right panels). In addition, the severity of these defects correlated with loss of Aip1 localization from actin patches (D, left panels). aip1-107 showed a complete loss of Aip1 from patches (cytoplasm only), whereas aip1-108, aip1-109, and aip1-119 caused partial mislocalization of Aip1. These data suggest that thickened cables arise from partial loss of Aip1 function, but detectable cofilin-decoration on cables only occurs with more severe loss of Aip1 function. Consistent with this hypothesis, N-aip1 and C-aip1 alleles, which behave genetically like null mutants, showed thick cables with cofilin decoration similar to aip1Δ (E). Further, biochemical data suggest that Aip1-107 is more severely impaired than Aip1-108 or Aip1-109 (see below).
Genetic Interactions of aip1 Alleles with cof1-22
The complete loss of AIP1
) is synthetic lethal with cof1-22
(Rodal et al., 1999
). Similarly, the complete loss of SRV2
function is synthetic lethal with cof1-22
(Balcer et al., 2003
). This suggests that when the essential function of COF1
in promoting actin turnover is partially impaired by the cof1-22
mutation, cell viability becomes dependent on the activities of cofilin cofactors Aip1 and Srv2 (Rodal et al., 1999
; Balcer et al., 2003
). In these cases, synthetic lethality does not reflect two independent and parallel pathways leading to one essential function, but rather the functionally linked contributions of multiple actin-binding proteins driving actin turnover. We tested genetic interactions of our aip1
alleles with cof1-22
to evaluate loss of function. The results are summarized in . Similar to an aip1
Δ mutation, N-aip1
were synthetic lethal with cof1-22,
demonstrating that each half of Aip1 is required for its normal cellular function. No single aip1
allele carrying point mutations was synthetic lethal with cof1-22
. However, a number of aip1
alleles displayed synthetic sick interactions with cof1-22
; that is, the double mutants grew more slowly than either single mutant alone at 25°C (Supplementary Figure S3). These included the alleles described above with thickened cable phenotypes (aip1-107, aip1-108, aip1-109, aip1-119
) and one allele with a moderate cable phenotype (aip1-106
). This suggests that these mutations each cause partial but not complete loss of Aip1 function.
Highlighting the mutated residues on the S. cerevisiae Aip1 structure reveals three key functional surfaces (, dotted circles). The largest functional surface we identified (thick dotted circle) is located on the front face of the N-terminal lobe of Aip1 and encompasses residues mutated in aip1-107 and aip1-108, as well as aip1-104 and aip1-105. The two other functional surfaces are located along the rim that separates the front and back faces, one on the rim of the N-terminal lobe, marked by aip1-109 (thin dotted circle), and the other on the rim of the C-terminal lobe, marked by aip1-119 (thick dotted circle).
Because no single allele with point mutations showed complete loss of Aip1 function, we next tested the additive effects of combining partial loss of function alleles in pairs on a single molecule: aip1-107/108, aip1-108/109, aip1-107/119, aip1-108/119, and aip1-109/119. Aip1 protein levels in aip1-107/108, aip1-108/109, and aip1-108/119 cells were similar to wild-type cells (C). Aip1 expression in aip1-107/119 and aip1-109/119 cells was detected by immunofluorescence (E, left panel), but not by immunoblotting (B). Both of the double aip1 mutants that include aip1-107 showed cofilin decoration on thickened cables (E, right panel) and were synthetic lethal with cof1-22 (), suggesting that they cause a more complete loss of Aip1 function than the single mutants from which they are derived. In contrast, the phenotypes of aip1-108/109 and aip1-108/119 double mutants were not significantly different from their respective single mutants in terms of cable thickness, Aip1 localization, and genetic interactions with cof1-22. Effects of the aip1-109/119 double mutant were intermediate, displaying a stronger genetic interaction with cof1-22 than the single mutants, but not complete synthetic lethality (). Together, these data suggest that aip1-107 represents a critical site for Aip1 function in vivo and that the other surfaces we identified as important (aip1-108, aip1-109, aip1-119) play supportive roles, in some cases additive (particularly when combined with aip1-107).
Biochemical Defects of Mutant Aip1 Proteins
To understand better the cause of the more severe mutant phenotypes, we investigated the underlying biochemical and molecular defects of aip1-107, -108, -109, -119, N-aip,1 and C-aip1. Wild-type and mutant Aip1 proteins were overexpressed in yeast as GST-fusion proteins under control of the GAL1 promoter and purified by glutathione affinity. GST tags were removed by thrombin digestion, and the released Aip1 proteins were purified further by ion exchange chromatography (A). Purified wild-type and mutant Aip1 proteins were compared for 1) binding to actin filaments in the presence and absence of cofilin (, B and C), 2) cofilin-dependent net disassembly of filaments (, A and B), and 3) cofilin-dependent capping of filaments (, C and D). Attempts to purify N-Aip1 and C-Aip1 were unsuccessful.
In most studies thus far, biochemical interactions of Aip1 with F-actin have been demonstrated using RMA. Aip1 exhibits cofilin-enhanced association with RMA F-actin and a moderate net disassembly activity on filaments, shifting a fraction of the actin at steady state into the supernatant (Aizawa et al., 1999
; Okada et al., 1999
; Mohri et al., 2003
). By comparison, using S. cerevisiae
actin, yeast Aip1 and cofilin induce almost a complete shift of actin into the supernatant fraction (Rodal et al., 1999
; also A). This represents the strongest net disassembly activity observed for Aip1 and precludes the use of yeast actin for testing cofilin-dependent association of Aip1 with F-actin by cosedimentation. Thus, the employment of different actins can uncouple Aip1’s cofilin-dependent functions in binding filaments versus promoting filament disassembly.
Using RMA, we compared F-actin association of wild-type and mutant Aip1 proteins in the presence and absence of cofilin. Wild-type Aip1 showed modest association with F-actin in the absence of cofilin (32% bound), with increased binding in the presence of cofilin (52% bound, B). Aip1-107 showed weaker association than wild-type Aip1 both in the presence and absence of cofilin (C). Aip1-108 showed a similar pattern, though slightly more modest defects. Despite having weaker overall associations with F-actin, Aip1-107 and Aip1-108 both showed improved F-actin association in the presence of cofilin, similar to wild-type Aip1. In contrast, Aip1-119 had relatively normal F-actin associations in the absence of cofilin (72% of wild type), but displayed only a negligible increase in binding in the presence of cofilin, making this mutant unique. Aip1-109 associated with F-actin, similar to wild-type Aip1 levels.
We also compared wild-type and mutant Aip1 proteins for two-hybrid interactions with actin and cofilin (D). In biochemical assays using purified proteins, Aip1 binds specifically to actin filaments and not monomers (Okada et al., 1999
; Rodal et al., 1999
). However, Aip1 also interacts with actin by the two-hybrid assay, suggesting that the actin-AD fusion protein has a conformation that may partially mimic F-actin. Our results from two-hybrid analysis showed a similar pattern to our biochemical results above: aip1-107
had the most severe defects, failing to interact with actin; aip1-108
showed intermediate weakened interactions with actin; and aip1-109
interacted normally with actin. On the other hand, none of these aip1
mutants were defective in two-hybrid interactions with cofilin. However, N-aip1
mutants (the expression of which were verified by immunoblotting) failed to interact with cofilin, suggesting that cofilin binding requires contributions from each half of Aip1. An important conclusion we draw from these data is that the actin and cofilin interactions of Aip1 are separable, because Aip1 mutants defective in actin interactions (e.g., aip1-107
) still interact normally with cofilin.
We next compared the ability of wild-type and mutant Aip1 proteins to induce net disassembly of actin filaments (assembled from purified yeast actin) in the presence of cofilin. In this assay, filaments disassemble into fragments, but do not necessarily depolymerize into monomers. High concentrations of Aip1 and cofilin cause the formation of very short fragments of F-actin that fail to sediment by high-speed ultracentrifugation and are not recognized as monomeric actin in DNase I–binding assays (Mohri et al., 2003
). Formation of these short fragments depends on two distinct activities of cofilin and Aip1, severing and capping (Balcer et al., 2003
). Disassembly, as measured by sedimentation, depends on high concentrations of cofilin, possibly because severing into very short fragments requires heavy decoration of filaments by cofilin. On the other hand, cofilin-dependent capping of filaments by Aip1 requires much lower concentrations of cofilin (Balcer et al., 2003
; and below). As shown in , A and B, wild-type Aip1 induces concentration-dependent net disassembly of filaments specifically in the presence of cofilin. Aip1-107 was significantly impaired in this activity, whereas Aip1-108 showed a subtle defect (B). These results are in good agreement with differences in the severity of their in vivo phenotypes () and their actin binding defects in biochemical and two-hybrid assays (, C and D).
The data also suggest that direct interactions with F-actin are critical for Aip1-mediated filament disassembly in vitro and Aip1 function in vivo. Further, the analysis with Aip1-119 indicates that net filament disassembly requires cofilin-dependent association of Aip1 with filaments. Aip1-109 showed no defects in filament disassembly, consistent with its normal binding to actin.
Aip1 has been shown to cap the barbed ends of actin filaments in a cofilin-dependent manner (Okada et al., 2002
; Balcer et al., 2003
). Capping can be measured using a modified net disassembly assay that contains low concentrations of cofilin and Aip1 and high concentrations of F-actin and profilin. Low concentrations of cofilin and Aip1 are sufficient to sever and cap filaments but are not sufficient to induce net disassembly of filaments. Further addition of high concentrations of profilin blocks monomer addition to the pointed ends of filaments and thereby leads to filament net disassembly specifically when barbed ends are capped (by Aip1 or Cap1/2; Balcer et al., 2003
). As expected, omission of Aip1 from the reaction ingredients restores filaments, because barbed ends are no longer capped (C). Using this assay, we compared the capping activities of Aip1 mutant proteins with variable phenotypes (pseudowild type, moderately defective, or severely defective; ). As shown in D, loss of capping activity in vitro correlates closely with loss of function in vivo (). However, there are two mutants (Aip1-109 and Aip1-111) that show defects in vivo but are not obviously impaired in capping in vitro. Because Aip1-111 defects in vivo are subtle, this may explain its lack of biochemical defects. On the other hand, Aip1-109 has strong in vivo defects. As mentioned above, Aip1-109 interacts normally with actin and cofilin in the two-hybrid assay and biochemical tests. Because this mutant is expressed at normal levels in vivo, the basis of its loss of function in vivo remains a mystery.
Aip1 Promotes Actin Patch and Cable Turnover In Vivo
Although it has been postulated that Aip1 promotes actin disassembly based on in vitro activities and genetic interactions with cofilin, this has never been demonstrated in living cells. Therefore, we compared rates of actin turnover in vivo for wild-type and aip1
mutant strains, focusing first on patch turnover. Isogenic wild-type, aip1Δ, aip1-107, aip1-107/108,
cells were treated with 50 μM Lat-A to block new actin assembly. Samples were removed at different time points after Lat-A treatment, and cells were stained with fluorescently labeled phalloidin to visualize F-actin structures (A). All of the aip1
mutant cells exhibited a delayed loss of F-actin staining compared with wild-type cells, indicating defects in actin turnover. In addition, three other alleles, aip1-108, aip1-109,
showed delayed actin turnover (Supplementary Figure S4). cof1-22
cells had an even more pronounced delay in patch turnover (unpublished data) as previously reported (Lappalainen and Drubin, 1997
). To quantify these effects, we scored cells for visible F-actin structures at each time point (B). The graphs reveal significant differences between wild-type and aip1
mutant cells after 5 min of Lat-A treatment. We also note that our scoring system provides a conservative measure of turnover defects, because cells scored as defective do not include those that have lost some or most of their actin staining (e.g., compare the actin staining of wild-type and aip1
mutant cells after Lat-A treatment in A). Thus, mutant defects may be even more severe than suggested. Importantly, these data provide the first demonstration that Aip1 activity in vivo promotes turnover of a cellular actin structure.
Figure 6. Defects in actin patch turnover in aip1 mutant cells. (A) Wild-type (AIP1) and mutant (aip1Δ, aip1-107, aip1-107/108, aip1-107/119) yeast cells were treated with 50 μM latrunculin A (Lat-A). Samples of cells were removed at the indicated (more ...)
Given our new observations that cofilin promotes rapid turnover of cables () in addition to patches, we next tested Aip1 involvement in cable turnover. There are conflicting data in the literature as to whether patches or cables are more sensitive to Lat-A treatment. We determined that in our strain background cables are more sensitive to Lat-A treatment than patches. Therefore, we used a low concentration of Lat-A (20 μM) to visualize loss of cables compared with patches. Four mutant aip1 alleles were selected for these analyses based on the severity of their phenotypes and their strong genetic interactions with cof1-22: aip1Δ, aip1-107, aip1-107/108, and aip1-107/119. Each of these aip1 strains showed greatly reduced rates of cable turnover compared with the isogenic wild-type strain (). These data, combined with those in and 6, demonstrate that Aip1 and cofilin contribute to the rapid turnover of both patches and cables. Further, our data show that specific defects in F-actin binding and capping by Aip1 lead to reduced turnover of these actin structures in vivo.
Figure 7. Defects in actin cable turnover in aip1 mutant cells. (A) Wild-type (AIP1) and mutant (aip1Δ, aip1-107, aip1-107/108, aip1-107/119) yeast cells were treated with 20 μM latrunculin A (Lat-A). Samples of cells were removed at the indicated (more ...)
Deletion of AIP1 Rescues Loss of Actin Cables in tpm1 Mutants
The data above indicate that Aip1 and cofilin cooperatively disassemble actin filaments. In contrast, tropomyosin is known to decorate and stabilize cables (Liu and Bretscher, 1989
; Pruyne et al., 1998
). Thus these two sets of proteins may have an antagonistic relationship in regulating cable stability, with normal cable morphology resulting from their opposing activities. To test this hypothesis, we deleted the AIP1
gene in a tpm1
Δ mutant background. tpm1Δ
cells lack obvious actin cables and exhibit temperature-sensitive growth at 37°C (Liu and Bretscher, 1989
). Deletion of AIP1
rescued both the temperature-sensitive growth and the actin cable defects of this strain (, A and B). Similar results were obtained for aip1Δ tpm1-2
mutants (unpublished data). Further, the cables in aip1Δtpm1Δ
cells were decorated with cofilin, which supports the view that cofilin and tropomyosin compete for binding to actin filaments. Also note that cap2Δ
mutations failed to rescue tpm1Δ
phenotypes (unpublished data and Adams et al., 1993
), suggesting that Aip1 plays a specific cellular role, distinct from capping protein.
Figure 8. (A) Haploid strains were grown to log phase (OD600 = 0.5) and then cells were serially diluted, plated on YPD medium, and grown for 2 d at 25 or 37°C. (B) Cells were fixed and labeled with rabbit anti-actin and chicken anti-cofilin antibodies (more ...)