Developmental Cell

Article Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres Elena Kremneva,1 Maarit H. Makkonen,1 Aneta Skwarek-Maruszewska,1 Gergana Gateva,1 Alphee Michelot,2 Roberto Dominguez,3 and Pekka Lappalainen1,* 1Institute

of Biotechnology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland de Physiologie Cellulaire et Ve´ge´tale, Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV, CNRS/CEA/INRA/UJF, 38054 Grenoble, France 3Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2014.09.002 2Laboratoire

SUMMARY

ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘‘aged’’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here, we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘‘actin nucleotide-state sensor’’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties that allow it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies.

INTRODUCTION The myofibrils of striated muscle cells are composed of contractile units called sarcomeres, where contractile force is generated by the sliding of thin actin filaments past thick, bipolar myosin filaments. The actin filaments in sarcomeres are uniform in length and precisely oriented with their barbed ends facing the Z-disc and their pointed ends directed toward the M-band (Huxley, 1963). Whereas actin filaments in nonmuscle cells typically undergo rapid turnover, the paracrystalline actin filament arrays of muscle sarcomeres were traditionally considered to be very stable. However, recent studies have provided evidence that also sarcomeric actin filaments undergo dynamic turnover, involving at least two distinct mechanisms. A subset of actin filaments in sarcomeres was shown to undergo rapid turnover as a result of myosin contractility (Skwarek-Maruszewska et al., 2009). Additionally, the length of actin filaments in mature sarcomeres is precisely regulated by actin subunit exchange at fila-

ment barbed and pointed ends (Littlefield et al., 2001; Gokhin and Fowler, 2013). Because, tropomodulin that caps the pointed end is more dynamically associated with the filament than CapZ that caps the barbed end, subunit exchange in sarcomeres is faster at pointed ends (Littlefield et al., 2001; Cooper and Sept, 2008). This contrasts with nonmuscle cells where actin dynamics occur mainly via addition of actin monomers to free barbed ends and severing of ‘‘aged’’ filaments toward their pointed ends (Pollard and Cooper, 2009). Actin filaments in sarcomeres are additionally decorated along their length by tropomyosin (Yamashiro et al., 2012) and a large number of actin-binding proteins, which contribute to maintaining sarcomere structure and organization. These include the giant protein titin that provides an elastic link between Z-discs and M-bands, nebulin, which may regulate the length of sarcomeric actin filaments and the actin nucleating protein leiomodin (Labeit and Kolmerer, 1995; Agarkova and Perriard, 2005; Chereau et al., 2008; Lange et al., 2006; Pappas et al., 2010; Sanger and Sanger, 2008). Additionally, many ubiquitous actin-binding proteins, which regulate actin dynamics in nonmuscle cells, are also expressed in muscles. In most cases, muscle-specific isoforms of these proteins have evolved to fulfill the specific requirements of actin dynamics in muscle cells. In mammals, these include the muscle-specific formin (FHOD3) (Taniguchi et al., 2009; Iskratsch et al., 2010), cyclase-associated protein (CAP2) (Bertling et al., 2004), twinfilin-2b (Nevalainen et al., 2009), and cofilin-2 (Ono, 2010). However, the specific biochemical properties distinguishing muscle from nonmuscle cytoskeletal protein isoforms are largely unknown. Defects in the regulation of the length or organization of actin filaments in sarcomeres due to mutations or misregulated expression of cytoskeletal proteins are a hallmark of many heart and skeletal muscle disorders, including several myopathies (Kho et al., 2012). ADF/cofilins are small (15–20 kDa), ubiquitous actin-binding proteins, which are essential regulators of actin dynamics in all eukaryotes from yeasts to animals (Bernstein and Bamburg, 2010). ADF/cofilins bind preferentially to ‘‘aged’’ ADP-actin filaments and promote actin dynamics by severing the actin filaments, thereby replenishing the pool of monomers for new rounds of filament assembly (Andrianantoandro and Pollard, 2006; Michelot et al., 2007). Cooperative binding of ADF/cofilins alters the conformation of ADP-actin filaments (McGough et al., 1997), resulting in increased fragmentation at the boundaries between cofilin-decorated and bare filaments (Andrianantoandro

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Figure 1. Localization of Cofilin-1 and Cofilin-2 in Cardiomyocytes (A) Western blot analysis showing the specificity of cofilin-1 and cofilin-2 antibodies generated in guinea pig and rabbit, respectively. One pmol:s of recombinant cofilin-1 and cofilin-2 proteins were run on gel, blotted, and detected with cofilin-1 (left) and cofilin-2 (right) antibody. In addition to the protein of expected mobility on SDS-gel, cofilin-1 antibody also cross-reacts with a protein of an apparent molecular weight of 70 kDa. (B) Western blot analysis of cofilin-1 and cofilin-2 expression during cardiomyocyte maturation. Cell lysates containing 60 mg of proteins were run on gel, blotted and detected with cofilin-1 and cofilin2 antibody. The blots were probed with antitubulin antibody as a loading control. (C) Localization of cofilin-1 and cofilin-2 in mature cardiomyocytes plated for 4 days on fibronectin. Cofilin-1 displays punctuate cytoplasmic localization, which does not overlap with myofibrils. Cofilin-2 concentrates on a striated pattern between a-actinin-2 rich stripes in cardiomyocyte sarcomeres. Bar represents 10 mm. (D) Line profile quantification of cofilin-1 (in red), cofilin-2 (in green), and a-actinin-2 (in blue) localization in myofibrils from the zoomed regions of (C). Cofilin-1 does not display a regular localization pattern, whereas cofilin-2 and a-actinin-2 display regular alternative localization pattern along myofibrils.

and Pollard, 2006; Elam et al., 2013; Suarez et al., 2011). ADF/ cofilins also bind actin monomers (G-actin), displaying higher affinity for ADP-G-actin, and inhibit nucleotide exchange on G-actin (Carlier et al., 1997; Hawkins et al., 1993; Hayden et al., 1993). Mammals express three ADF/cofilin isoforms: cofilin-1 ubiquitously, ADF in epithelial and neuronal tissues, and cofilin-2 mainly in muscles. Cofilin-2 is the only isoform present in mature skeletal muscles (Ono et al., 1994; Vartiainen et al., 2002). Despite partially overlapping expression patterns, ADF/cofilin

isoforms cannot functionally compensate for each other in animals; cofilin-1-deficient mice display early embryonic lethality and defects in actin-dependent morphogenic processes, whereas ADF inactivation leads to corneal defects (Bellenchi et al., 2007; Gurniak et al., 2005; Ikeda et al., 2003). Mutations in the cofilin-2 gene lead to myopathies in humans (Agrawal et al., 2007; Ockeloen et al., 2012), and cofilin-2-deficient mice show progressive disruption of the sarcomeric architecture and abnormal accumulation of F-actin in skeletal muscles. Primary myofibril assembly and muscle development are unaffected in these mice, but postnatal animals display defects in sarcomeric actin exchange and muscle maintenance (Agrawal et al., 2012; Gurniak et al., 2014). Cofilin-2 was reported to partially localize to myofibrils in muscle cells, suggesting that it may be involved in regulation of actin dynamics in sarcomeres (Nakashima et al., 2005; Gurniak et al., 2014). However, the mechanism by which cofilin-2 contributes to the assembly and/or maintenance of actin filament structures in myofibrils is unknown. Furthermore, apart from its higher affinity for ATP-actin monomers as compared to other ADF/cofilin isoforms (Vartiainen et al., 2002), the biochemical properties that account for the specific function of cofilin-2 in muscle cells are unknown. Thus, the

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Figure 2. Cofilin-2 Depletion by RNAi Disrupts the Periodic Actin Filament Pattern of Cardiomyocyte Myofibrils (A) F-actin and a-actinin-2 localization in 4-day-old cardiomyocytes incubated for 72 hr with control (upper panel) or cofilin-2-specific (lower panel) RNAi oligonucleotides. Actin filaments were visualized with fluorescently-labeled phalloidin, and a-actinin-2 antibody was used as a Z-disk marker. Insets show zoomed areas of the cells. Control cells display regular, periodic F-actin staining in myofibrils, whereas the ‘‘F-actin free’’ regions at the M-band are lost in majority of myofibrils in cofilin-2 knockdown cells. Bars represent 10 mm. Line profile quantification of actin (in red) and a-actinin-2 (in blue) localization in myofibrils from zoomed regions of (A). (B) Western blot analysis of cardiomyocyte lysates after incubation with control (line 1) or cofilin-2specific RNAi (line 2). GAPDH antibody was used as a control for equivalent loading. (C) Quantification of cofilin-2 levels of cardiomyocyte lysates from control and cofilin-2 siRNA transfected cells. Cofilin-2 levels were >50% lower in knockdown compared control cells. The data are from four and five independent experiments for control and cofilin-2-specific RNAi, respectively. The error bars represent SEM. See also Figure S1.

functional and mechanistic principles for why tissue-specific ADF/cofilin isoforms evolved in vertebrates have not been studied in detail. Here, we report that cofilin-2 localizes between Z-discs in cardiomyocytes, close to the pointed ends of the actin filaments, and its depletion leads to uncontrolled actin filament growth in sarcomeres. We further show that cofilin-2 disassembles ADP,BeFx-F-actin more efficiently than other ADF/cofilin isoforms and identified a cluster of residues at the surface of cofilin-2 responsible for high-affinity binding to ATP-G-actin. Based on our findings, we propose that cofilin-2 specifically evolved to control the length of actin filaments in sarcomeres, which undergo faster monomer exchange at their pointed ends. RESULTS Cofilin-2 Localizes near M-Bands in Myofibrils To study the role of cofilin-2 in muscle sarcomeres, we examined its localization and function in cultured neonatal rat cardiomyocytes, which provide a good model system to explore the func-

tion of muscle-specific actin-binding proteins (McElhinny et al., 2005; SkwarekMaruszewska et al., 2010). Because none of the available cofilin-2 antibodies, including those used in an earlier study (Nakashima et al., 2005), displayed specific localization in cardiomyocytes, we generated isoform-specific antibodies against rat cofilin-1 and cofilin-2, which share 80% sequence identity. We used two peptides, corresponding to surfaceexposed regions that differ between the two isoforms (Experimental Procedures). Immunization of guinea pigs and rabbits with these peptides yielded antibodies that specifically recognized cofilin-1 or cofilin-2 (Figure 1A) and displayed good specificity in western blots of cell lysates (Figure 1B). Using these antibodies, we found that cofilin-1 was present at equal levels in both newly plated cardiomyocytes, lacking properly organized sarcomeres (Skwarek-Maruszewska et al., 2009), and in day 3 cardiomyocytes having mature sarcomeres. In contrast, cofilin2 protein levels increased steadily during sarcomere maturation (Figure 1B). Immunofluorescence microscopy of day 4 cardiomyocytes displaying striated sarcomeric organization, revealed mostly irregular, punctuate localization of cofilin-1 in the cytoplasm. While cofilin-2 also displayed irregular, punctuate localization, a regular striated pattern was also observed (Figure 1C). Line scans along myofibrils demonstrated that cofilin-2 concentrated between the a-actinin-2 peaks (Figure 1D). These results show that cofilin-2 expression increases during cardiomyocyte maturation and that it localizes between Z-discs in sarcomeres.

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nofluorescence microscopy confirmed that cofilin-2 levels were strongly reduced, especially in fluorescently conjugated RNAitransfected cells, where the oligonucleotides displayed punctuate cytoplasmic localization, although 20%–40% of the cells still expressed detectable levels of cofilin-2 (Figure S1A available online). The depletion of cofilin-2 was accompanied by an increase in the intensity of a-actinin-2 staining (Figure S1A), consistent with previous results that cofilin-2-deficient mice display a significant increase in the expression levels of other cytoskeletal proteins, including a-actinin-2 (Agrawal et al., 2012). All three RNAi oligonucleotides resulted in a similar phenotype, suggesting that the observed alterations in actin organization do not result from off-target effects (Figure S1B). Cardiomyocytes transfected with scrambled control oligonucleotides showed a characteristic striated pattern of phalloidin-staining, with clearly defined M-bands devoid of F-actin (Figure 2A). Depletion of cofilin-2 led to an abnormal F-actin pattern, characterized by the lack of visible actin-free zones along M-bands. Quantification of three independent experiments showed that 75% of control cells displayed clear periodic F-actin staining versus 35% in cofilin-2 depleted cells. Even in knockdown cells that displayed periodic actin staining, only a fraction of the myofibrils exhibited actin-free M-bands and showed less uniform periodicity than control cells. Furthermore, the typical striated pattern of the pointed end capping protein tropomodulin 1 (Tmod1) observed in control cells was severely diminished in cofilin-2 knockdown cells (Figure 3B), further suggesting that in the absence of cofilin-2 the actin filaments have irregular lengths. Alternatively, the loss of striated Tmod1 pattern may result from a more complex interplay of Tmod1 and cofilin-2. Yet, a-actinin-2 in Z-discs and myomesin in M-bands showed normal striated localization (Figures 2A and 3A), suggesting that sarcomere organization is mostly preserved in cofilin-2 knockdown cells.

Figure 3. Cofilin-2 Depletion Results in Diffuse Localization of Pointed End Capping Protein Tmod1 (A) F-actin and myomesin localization in 4-day-old control and cofilin-2 knockdown cardiomyocytes. The M-band protein myomesin displayed regular striated localization pattern also in knockdown cells lacking the F-actin free region at the M-band. (B) Tmod1 displays regular striated localization pattern in cells transfected with control RNAi oligonucleotides, whereas it is mostly diffuse in cofilin-2 knockdown cells. Bars represent 10 mm.

Cofilin-2 Regulates Actin Filament Length in Cardiomyocyte Sarcomeres To address the role of cofilin-2 in sarcomeres, we transfected day 1 cardiomyocytes with three different cofilin-2-specific RNAi oligonucleotides, resulting in >50% reduction of cofilin-2 protein levels 72 hr after transfection (Figures 2B and 2C). Immu-

Cofilin-2 Binds and Disassembles ATP-Actin Filaments To understand the biochemical features of cofilin-2 responsible for its function in sarcomeres, we compared the interactions of cofilin-1 and cofilin-2 with actin filaments. Cosedimentation assays revealed that cofilin-1 and cofilin-2 bind ADP-F-actin with similar affinities (Figure 4A). TIRF microscopy using GFP-cofilin2 and mCherry-cofilin-1 showed that the two isoforms can bind to the same filament, suggesting that their distinct localization patterns in sarcomeres do not arise from incompatible cooperative interaction with the actin filament (Figure S2). Furthermore, both cofilin isoforms competed with tropomyosin for F-actin interactions and did not display isoform-specific binding toward skeletal muscle a-actin or nonmuscle b/g-actin (Figure S3). However, two observations led us to examine the interactions of the two cofilin isoforms with ATP-F-actin. First, earlier studies demonstrated that cofilin-2 interacts with ATP-actin monomers with higher affinity compared to other ADF/cofilin isoforms (Vartiainen et al., 2002). Second, due to continuous exchange of actin subunits, filament pointed ends in muscle sarcomeres might contain a mixture of ATP/ADP-Pi- and ADP-actin subunits (Littlefield et al., 2001). Due to rapid nucleotide hydrolysis and phosphate release, we could not determine the binding of cofilin isoforms to ATP-actin filaments. Therefore, filaments were saturated with the Pi analog

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Figure 4. Interactions of Cofilin-1 and Cofilin-2 with ADP- and ADP,BeFx-Actin Filaments (A and B) Equilibrium binding of mouse cofilin-1 and cofilin-2 to ADP- (A) and ADP,BeFx-actin filaments (B). Cofilins (1 mM) were mixed with 0, 0.2, 0.5, 1, 2, 3, 4, 6, and 8 mM of prepolymerized actin (A) or 0, 0.5, 1, 2, 4, 7, 10, 15, 20, and 25 mM ADP,BeFx-actin filaments (B). Actin filaments were sedimented, and the amounts of cofilins in the supernatant and pellet fractions were quantified from six independent experiments. (C–F) Disassembly of ADP- and ADP,BeFx-actin filaments in the presence (E and F) and absence (C and D) of Tmod1 (0.25 mM) and CapZ (0.05 mM). Cofilin-1 or cofilin-2 (0.5 mM in C and E or 2.5 mM in D and F) were incubated for 5 min with 2.5 mM of prepolymerized ADP- (C and E) or ADP,BeFx- (D and F) pyrene-actin filaments, followed by addition of actin monomer sequestering vitamin D-binding protein (4 mM). Error bars represent SEM from four to six independent experiments. See also Figures S2 and S3. Assays were carried out in 5 mM Tris (pH 7.5) in the presence of 100 mM KCl and other components specified in the Experimental Procedures.

BeFx to mimic the ADP-Pi-F-actin state (Combeau and Carlier, 1988; Muhlrad et al., 2006). Based on cosedimentation assays, a small fraction (20%) of both cofilin-1 and cofilin-2 appeared to bind ADP,BeFx-F-actin with 1 mM affinity. Importantly, a majority of cofilin-1 bound ADP,BeFx-F-actin only with low affinity (Kd > 10 mM), whereas a majority of cofilin-2 bound ADP,BeFx-F-actin with higher affinity (Kd 5 mM) (Figure 4B). To estimate the effects of cofilin-1 and cofilin-2 on actin filament turnover, we monitored the decrease of pyrene-actin fluorescence resulting from filament disassembly in the presence of the two cofilin isoforms and vitamin D-binding protein (DBP) (Gandhi et al., 2009). DBP binds in the hydrophobic cleft between actin subdomains 1 and 3, competing with cofilin binding (Lees et al., 1984; Otterbein et al., 2002), and was used in this experiment to sequester the actin monomers and impede their reincorporation into filaments. While the binding of cofilin alters the fluorescence of pyrene-actin (Carlier et al., 1997) (Figure S4A), this effect does interfere with the interpretation of the data, because cofilin was incubated with F-actin for 5 min before addition of DBP. At an actin:cofilin molar ratio of 5:1, both cofilin isoforms displayed high ADP-actin filament disassembly activity (Figure 4C), albeit cofilin-1 had slightly higher rate than cofilin-2 (relative dissociation rates of 4.08 ± 0.13 versus 3.65 ± 0.03 ms1, respectively). Under identical concentrations, both isoforms displayed weak ADP,BeFx-F-actin disassembly activity

(data not shown). However, at an actin:cofilin molar ratio of 1:1, cofilin-2 increased the disassembly rate of ADP,BeFx-Factin by 10-fold, whereas cofilin-1 did not significantly promote the disassembly of ADP,BeFx-F-actin (Figure 4D). Importantly, cofilin-2 also promoted disassembly of ADP- and ADP,BeFx-actin filaments capped by CapZ and Tmod1 at their barbed and pointed ends, whereas cofilin-1 could only disassemble capped filaments in the ADP-bound state (Figures 4E and 4F). These data suggest that, unlike other ADF/cofilin isoforms characterized so far, cofilin-2 can disassemble of ADP-Pi-mimetic filaments. Identification of Cofilin-2 Residues Required for Binding ADP-Pi-Actin Filaments and ATP-Actin Monomers Sequence analysis reveals only a few clusters of residues conserved in cofilin-2 but not cofilin-1 (Figure 5A). Among these residues only Thr6, Asn8, Glu10, Trp135, Val137, Asp141, Asp142, and Ile143 are located on the G/F- and F-actin-binding surface (Figure 5B). To identify cofilin-2-specific residues responsible for its higher affinity for ATP-actin, we designed four cofilin-1 mutants by replacing clusters of surface-exposed residues with their cofilin-2 counterparts and testing the ability of the hybrid mutants to disassemble ADP,BeFx-F-actin. Out of the four mutants analyzed, mutant cof-1/cof-2141,142,143, in which residues 141–143 of cofilin-1 were replaced by cofilin-2 residues Asp141, Asp142, Ile143, disassembled ADP,BeFx-actin filaments significantly faster than wild-type cofilin-1 (Figures 5C and S4B). The inverse mutant, cof-2/cof-1141,142,143, in which the same residues in cofilin-2 were replaced by their cofilin-1 counterparts, displayed significantly lower ADP,BeFx-actin filament disassembly activity compared to cofilin-2 (Figure 5C).

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Cofilin-2 also binds ATP-actin monomers with higher affinity compared to cofilin-1 (Vartiainen et al., 2002). To examine whether the same residues that increase the affinity of cofilin-2 for ADP,BeFx-F-actin also increase its affinity for ATP-G-actin, we used a fluorometric assay with NBD-labeled actin. The binding of cofilin-1 and cofilin-2 to NBD-G-actin resulted in a fluorescence increase, with saturation at higher cofilin concentrations, enabling us to measure the Kd of the interactions (Figure 5D). Mutant cof-1/cof-2141,142,143 displayed 2.5-fold higher affinity for ATP-G-actin than wild-type cofilin-1 (Figures 5D and S5). The affinity was further increased by introducing the cofilin-2specific residue Val137 into this mutant. The inverse mutant, cof-2/cof-1141,142,143 had significantly lower affinity for ATPG-actin compared to cofilin-2 (Figure 5D). ATP-Actin Binding Residues Are Essential for Cellular Function of Cofilin-2 To assess the importance of cofilin-20 s ability to bind ATP-actin for its function in cardiomyocyte sarcomeres, we performed RNAi-rescue experiments with wild-type and mutant cofilins. HA-tagged cofilin-2 displayed mainly diffuse cytoplasmic localization when expressed in cardiomyocytes, but also accumulated in the M-band and Z-disc regions as detected by costaining with myomesin, Tmod1 and a-actinin antibodies (Figure S6 and data not shown). Moreover, HA-cofilin-2 expression rescued the loss of a striated F-actin pattern resulting from depletion of endogenous cofilin-2. In contrast, neither HA-cofilin-1 nor HA-cof-2/cof-1141,142,143, which cannot efficiently disassemble ADP,BeFx-F-actin or bind ATP-G-actin with high affinity, rescued the cofilin-2 depletion phenotype. Whereas the majority of control and cofilin-2 rescue cells displayed a periodic F-actin staining pattern, most cofilin-1 and cof-2/cof-1141,142,143 expressing cofilin-2 knockout cells lost the striated F-actin pattern along myofibrils (Figures 6A and 6B). DISCUSSION Three tissue-specific ADF/cofilin isoforms are expressed in mammals, but the biochemical properties and cellular functions distinguishing these isoforms have remained elusive. Here, we demonstrate that the muscle-specific isoform cofilin-2 localizes between Z-discs in myofibrils and is an essential regulator of proper actin filament length in sarcomeres. We provide evidence that, unlike other ADF/cofilin isoforms, cofilin-2 also disassembles ADP,BeFx-actin filaments. Finally, we identify a cluster of residues on the surface of cofilin-2 that is responsible for its high affinity for ATP-actin and propose that this region functions as a nucleotide state sensor in ADF/cofilin.

The ability of cofilin-2 to bind and disassemble both ADP- and ADP-Pi-F-actin specifically adapts it for the disassembly of actin filaments near M-bands in sarcomeres. In nonmuscle cells, actin monomer addition occurs primarily at the barbed ends due to rapid association of ATP-G-actin subunits to free barbed ends. This and the differences in the affinities of the two ends for Pi increase the likelihood that barbed ends will contain ATP- or ADPPi-actin, whereas ADP-actin accumulates toward the pointed end (Fujiwara et al., 2007; Pollard, 2007; Bugyi and Carlier, 2010). In sarcomeres, the majority of the barbed ends are tightly capped by CapZ, and subunit exchange occurs primarily at the pointed end (Littlefield and Fowler, 2008). Because of this reversal of monomer exchange kinetics in sarcomeres, rapidly elongating filaments (at least uncapped filaments) may contain a mixture of ADP- and ATP/ADP-Pi-actin at their pointed ends. This might create the necessity for a muscle-specific ADF/cofilin isoform with the ability to promote the disassembly of both ADPand ATP/ADP-Pi-actin filaments. Filament disassembly by cofilin-2 may be particularly necessary if rapidly elongating filament extensions do not contain tropomyosin. While not addressed here, cofilin-2 may collaborate with tropomodulin for this activity (Yamashiro et al., 2012). Our results (Figure S3) and an earlier study (Nakashima et al., 2005) also suggest that cofilin-2 is slightly more effective than cofilin-1 at competing with tropomyosin for F-actin binding/disassembly. It is also unclear what the exact mechanism is by which cofilin-2 localizes to the M-band region. We suggest that interactions with M-band-specific proteins may be responsible for cofilin-2 localization to this region. In this regard, it is interesting to note that in nonmuscle cells ADF/cofilins interact with CAP1 (Makkonen et al., 2013; Moriyama and Yahara, 2002), whose muscle-specific isoform CAP2 localizes to M-bands (Peche et al., 2007). The proposed role of cofilin-2 as a regulator of actin filament length in mature sarcomeres is consistent with its spatial and temporal expression pattern in cardiomyocytes and mouse tissues and with published genetic data in mice and humans. In mice, cofilin-2 mRNA expression increases during myogenesis and is the only ADF/cofilin isoform expressed in mature skeletal muscles (Ono et al., 1994; Vartiainen et al., 2002). Here, we also demonstrate maturation-dependent expression of cofilin-2 at the protein level. In agreement with its specific function in mature myofibrils, cofilin-2 depletion in mice did not severely affect muscle development, but resulted instead in progressive sarcomeric disruption and accumulation of abnormal F-actin structures (Agrawal et al., 2012; Gurniak et al., 2014). Similarly, mutations in the human cofilin-2 gene that decrease the expression/stability of cofilin-2 result in nemaline myopathy, which is characterized by the abnormal organization of actin filaments in striated

Figure 5. Identification of Cofilin-2 Residues Responsible for High-Affinity Binding to ADP,BeFx-F-Actin (A) Sequence alignment of mammalian cofilin-1 and cofilin-2 isoforms. A blue background highlights conservation, with darker blue representing higher conservation. Colored frames highlight cofilin-2 residues introduced into cofilin-1 (magenta: cof-1/cof-26,8,10, red: cof-1/cof-2135, cyan: cof-1/cof-2137, and orange: cof-1/cof-2141,142,143). (B) Location of mutated residues on the surface of the cofilin-1 structure (Pope et al., 2004) color-coded as in (A). Ovals highlight surfaces participating in binding to G-actin (green) and F-actin (blue and green). (C) Disassembly of 2.5 mM ADP,BeFx-F-actin in the presence of 4 mM DBP and 2.5 mM of cofilin-1, cofilin-2, cof-2/cof-1141,142,143, or cof-1/cof-2141,142,143 mutants. The error bars represent SEM from four to six independent experiments. (D) Bar diagram showing the dissociation constants of wild-type and mutant cofilin-1 and cofilin-2 for ATP-G-actin. Error bars indicate SEM from three independent experiments. See also Figures S4 and S5. Assays were carried out in 5 mM Tris (pH 7.5) in the presence of 100 mM KCl and other components specified in the Supplemental Experimental Procedures.

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Figure 6. Rescue of Cofilin-2 Depletion (A) Distributions of HA-tagged cofilins (left) and F-actin in 4-day-old cardiomyocytes incubated for 72 hr with cofilin-2-specific RNAi oligonucleotides and transfected with plasmids expressing RNAi-refractory HA-cofilin-1 (upper panel), HA-cofilin-2 (middle panel) or HA-cof2/cof1141,142,143 DNA (lower panel). Expression of HA-cofilin-2 rescued the loss of striated F-actin pattern resulting from coflin-2 depletion, whereas majority of HA-cofilin-1 and HAcof2/cof1141,142,143 expressing cofilin-2 knockdown cells did not display periodic F-actin pattern along their myofibrils. (B) Quantification of percentage of control, cofilin-2 knockdown and cofilin-2 knockdown-rescue cells displaying periodic F-actin pattern. Error bars indicate SEM from four to five independent experiments. See also Figure S6.

muscle myofibrils (Agrawal et al., 2007; Wallgren-Pettersson et al., 2011). We propose that compromised cofilin-2 activity leads to uncontrolled elongation of sarcomeric actin filaments, which in turn impacts the maintenance and correct organization of the sarcomeric apparatus in mature myofibrils. Cofilin-1 and cofilin-2 diverged late in evolution, after the emergence of vertebrates, and thus the invertebrate musclespecific ADF/cofilin isoforms are not functionally equivalent to mammalian cofilin-2 (Poukkula et al., 2011). For example, in Caenorhabditis elegans the muscle-specific ADF/cofilin isoform UNC-60B appears to be involved in muscle development, not maintenance (Ono et al., 2003), and the biochemical differences between the muscle and nonmuscle isoforms are distinct from those revealed here for mammalian cofilin-1 and cofilin-2 (Yamashiro et al., 2005). By introducing cofilin-2-specific residues in cofilin-1, we were able to increase its affinity for ATP-actin monomers and its efficiency to promote the disassembly of ADP,BeFx-F-actin. Reciprocally, replacing the same residues in cofilin-2 by their cofilin-1 equivalents diminished the affinity of cofilin-2 for ATP-actin monomers. These results point to a specific cluster of residues (Asp141, Asp142, and Ile143) on the surface of cofilin-2 responsible for its higher affinity for ATP-actin monomers. However, because these mutants only partially increased and decreased the ATPactin monomer affinity of cofilin-1 and cofilin-2, respectively (Figure 5), other regions of cofilin-2 are also likely to contribute to ATP-actin monomer interactions. Importantly, the higher affinity of cofilin-2 for skeletal muscle ATP-G-actin does not result from actin isoform specificity, because cofilin-2 also binds b/g -ATPactin monomers with higher affinity compared to cofilin-1 (Figure S3). The residues required for high-affinity ATP-actin binding in cofilin-2 are located at the barbed end region of actin monomer that is sensitive to the bound nucleotide. The actin monomer consists of two major domains on each side from the nucleotide-binding cleft that, according to their relative position in the filament, are referred to as the outer (subdomains 1 and 2) and inner (subdomains 3 and 4) domains (Dominguez and Holmes, 2011). The main difference between monomeric ATP-actin and ADP-actin subunits in the filament is a propeller-twist rotation of the outer domain relative to the inner domain, which renders the actin subunits in the filament flatter compared to monomeric actin (Fujii et al., 2010; Oda et al., 2009) (Figure 7A). In the actin monomer, nucleotide-dependent conformational changes are subtler, but they also involve a propeller-twist rotation, albeit far less pronounced than in the filament (Otterbein et al., 2001). Opposite to the nucleotide-binding cleft, actin has a second cleft, known as the hydrophobic target-binding cleft or the barbed end groove, where most actin-binding proteins interact (Dominguez, 2004). This cleft also mediates intersubunit contacts in the filament (Dominguez and Holmes, 2011; Fujii et al., 2010; Oda et al., 2009). In this way, nucleotide- and polymerization-depended conformational changes in actin are transmitted to the target-binding cleft, such that proteins that bind there can sense the state of the nucleotide. Structural studies have shown that cofilin family members interact in this cleft of actin, both in the monomeric (Paavilainen et al., 2008) and filamentous (Galkin et al., 2011) states (Figures 7B–7D). The cofilin surface involved in interactions with the actin monomer is also implicated in

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Figure 7. The Mechanism of Cofilin-2 Interaction with ATP-Actin (A) Actin in the filament (red) undergoes a propeller-twist rotation, resulting in a flatter conformation compared to monomeric actin (blue) (Dominguez and Holmes, 2011; Fujii et al., 2010; Oda et al., 2009). A similar, but less pronounced propeller-twist rotation occurs between the ATP- and ADP-bound conformations of the actin monomer (Otterbein et al., 2001). (B) Illustration of the nuclear magnetic resonance (NMR) structure of human cofilin-1 (Pope et al., 2004), showing the side chains of all the amino acids that differ between cofilin-1 and cofilin-2 (Figure 5A). (C) Model of cofilin-1 bound to G-actin obtained by superimposing its NMR structure (Pope et al., 2004) onto the crystal structure of the C-terminal ADF-homology domain 2 of twinfilin in complex with actin (Paavilainen et al., 2008). (D) A ribbon diagram and space-filling model of cofilin-1 bound to the actin filament made by fitting atomic structures into a relatively low resolution reconstruction from EM structure (Galkin et al., 2011), showing only two actin subunits of the long-pitch helix. The Ca backbone of cofilin-1 is shown in gray, except for regions implicated in binding to G-actin (green Ca backbone) and F-actin (green and cyan Ca backbones). Side chains with yellow Ca atoms correspond to a cluster of cofilin-2-specific residues that when introduced into cofilin-1 (mutant Cof-1141,142,143) increase its affinity for ATP-actin. These residues face Lys291, Lys326, and Lys328 in subdomain 3 of actin (actin subdomains are numbered 1–4) and are expected to form salt bridges that would be affected in different ways for cofilin-1 and cofilin-2 by the propeller-twist rotation in actin (see main text).

interactions with an actin subunit in the filament, while additional contacts are established through other surfaces with a second actin subunit of the long pitch filament helix (Figures 5B and 7B–7D). In this way, cofilin can sense nucleotide-dependent conformational changes in actin, both in the monomer and in the filament. The cofilin-2 residues implicated here in high-affinity binding to ATP-actin are all located on the surface that contacts subdomain 3 of actin. Specifically, cofilin-2 residues Asp141 and Asp142 face actin residues Lys291, Lys326, and Lys328, likely forming salt bridges. In cofilin-1 these two positions are occupied by longer glutamic acid side chains (Figure 5A), which probably also form salt bridges with the three lysine residues on actin in the ADP state (following the propeller-twist rotation), but would be too close to actin in the ATP state for favorable interactions (i.e., their longer side chains may sterically hinder the interaction with ATP-actin). In summary, we have identified residues on the surface of cofilin-2 that account for its higher affinity for ATP-actin monomers than cofilin-1. Although cofilin-2 may display also other biochemical differences to cofilin-1 and ADF, our RNAi-rescue experiments provided evidence that the residues responsible for ATP-G-actin-binding and ADP,BeFx-actin filament disassembly are essential for the function of cofilin-2 in regulating the length and/or organization of actin filaments in muscle sarcomeres.

EXPERIMENTAL PROCEDURES Generation of Cofilin-1 and Cofilin-2 Antibodies Two guinea pigs and two rabbits were immunized with appropriate mixture of two peptides conjugated to Keyhole Limpet Hemocyanin. Peptide synthesis and immunizations were performed in Inbiolabs. The peptides corresponding to surface exposed regions were (NHCOCH3)CASGVAVSDGVIK(CONH2) and (NHCOCH3)CLSEDKKNIILEEGK(CONH2), for cofilin-1 and (NHCOCH3)CASG VTVNDEVIK(CONH2) and (NHCOCH3)CWQVNGLDDIKDRSTLG(CONH2) for cofilin-2. The serums were collected after four immunizations, and the antibodies were affinity purified as described previously (Vartiainen et al., 2000). Isolation of Neonatal Rat Cardiomyocytes Neonatal rat cardiomyocytes were isolated and cultured as described before (Skwarek-Maruszewska et al., 2009). Briefly, neonatal (1–3 days) rat hearts were dissected and enzymatically digested. After plating for 70 min to discard fibroblasts, the cardiomyocytes were replated on fibronectin-coated dishes. After culturing for 24 hr, the plating medium was exchanged for ‘‘maintaining medium’’ (Skwarek-Maruszewska et al., 2009). Cell Biological Methods Western blotting, immunofluorescence microscopy, siRNA treatment, and rescue experiments were performed as described in the Supplemental Experimental Procedures. Protein Expression and Purification Recombinant wild-type and mutant cofilin-1, cofilin-2, CapZ, and Tmod1 were produced as described in the Supplemental Experimental Procedures. Rabbit muscle actin was prepared from acetone powder as described in Spudich and

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Watt (1971), and pyrene-actin was from AP05, Cytoskeleton). Rabbit muscle actin and human platelet actin (APHL99-E, Cytoskeleton) were labeled with NBD (7-chloro-4-nitrobenz-2-oxa-1,3-diazole-Cl) as described previously (Detmers et al., 1981). ADP- and BeFx-Actin Disassembly Assays The steady-state rate of actin filament disassembly was monitored by the decrease in the pyrene fluorescence with excitation at 365 nm and emission at 407 nm after addition of 4 mM vitamin D binding protein (DBP) (Human DBP, G8764, Sigma) using modified protocol (Gandhi et al., 2009). To prepare ADP-pyrene actin, a mixture of G-actin and 5% pyrene-actin was polymerized in G-buffer by the addition of 5 mM MgCl2 in the presence of 100 mM KCl and 1 mM EGTA. To prepare BeFx-pyrene actin, polymerization was performed as described above in the presence of 0.2 mM ADP, 5 mM NaF, 600 mM BeCl2, 100 mM KCl, and 1 mM EGTA. Samples of ADP-pyrene or BeFx-pyrene actin (2.5 mM) were preincubated in the presence of 0.5 or 2.5 mM cofilin-1/2 for 5 min and the reaction was initialized by the addition of DBP. To analyze the effect of Tmod1 and CapZ on disassembly activity of cofilins ADP-pyrene or BeFx-pyrene actin were additionally incubated with 0.25 mM Tmod1 and 0.05 mM CapZ for 5–6 min at room temperature before mixing with cofilins. During the experiments, salt conditions were constant: 100 mM KCl, 2 mM MgCl2, and 1 mM EGTA. All measurements were carried out using Perkin Elmer fluorometer. Origin 7.5 software (OriginLab) was used to analyze the data. To calculate initial relative actin filament disassembly rates for cofilin-1 and cofilin-2, first 80 s of each curve was fitted with linear equation y = a 3 x + b, where coefficient ‘‘a’’ represents speed of the process. Actin-Binding Assays The protocols for actin monomer binding and actin filament binding assays are available in the Supplemental Experimental Procedures.

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Statistical Analysis Mann-Whitney nonparametric test was used to check significance of the observed effects.

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SUPPLEMENTAL INFORMATION

Dominguez, R. (2004). Actin-binding proteins—a unifying hypothesis. Trends Biochem. Sci. 29, 572–578.

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Cofilin-2 controls actin filament length in muscle sarcomeres.

ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of "aged" ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but...
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