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Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
Developmental Cell
Article
Cofilin-2 Controls Actin Filament Lengthin Muscle SarcomeresElena 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, Finland2Laboratoire de Physiologie Cellulaire et Vegetale, Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV,CNRS/CEA/INRA/UJF, 38054 Grenoble, France3Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
*Correspondence: pekka.lappalainen@helsinki.fi
http://dx.doi.org/10.1016/j.devcel.2014.09.002
SUMMARY
ADF/cofilins drive cytoskeletal dynamics by promot-ing the disassembly of ‘‘aged’’ ADP-actin filaments.Mammals express several ADF/cofilin isoforms, buttheir specific biochemical activities and cellularfunctions have not been studied in detail. Here, wedemonstrate that the muscle-specific isoform cofi-lin-2 promotes actin filament disassembly in sarco-meres to control the precise length of thin filamentsin the contractile apparatus. In contrast to other iso-forms, cofilin-2 efficiently binds and disassemblesbothADP- andATP/ADP-Pi-actin filaments.Wemap-ped surface-exposed cofilin-2-specific residuesrequired for ATP-actin binding and propose thatthese residues function as an ‘‘actin nucleotide-statesensor’’ among ADF/cofilins. The results suggestthat cofilin-2 evolved specific biochemical andcellular properties that allow it to control actin dy-namics in sarcomeres, where filament pointed endsmay contain amixture of ADP- andATP/ADP-Pi-actinsubunits. Our findings also offer a rationale for whycofilin-2 mutations in humans lead to myopathies.
INTRODUCTION
The myofibrils of striated muscle cells are composed of contrac-
tile units called sarcomeres, where contractile force is generated
by the sliding of thin actin filaments past thick, bipolar myosin fil-
aments. 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 un-
dergo 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 fil-
aments 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 sarco-
meres is precisely regulated by actin subunit exchange at fila-
De
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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 be-
tween 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 ubiq-
uitous 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-associ-
ated protein (CAP2) (Bertling et al., 2004), twinfilin-2b (Neva-
lainen 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 fil-
aments in sarcomeres due to mutations or misregulated expres-
sion 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 fila-
ments and promote actin dynamics by severing the actin fila-
ments, 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 be-
tween cofilin-decorated and bare filaments (Andrianantoandro
velopmental Cell 31, 1–12, October 27, 2014 ª2014 Elsevier Inc. 1
3086
Figure 1. Localization of Cofilin-1 and Cofi-
lin-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 cofilin-
2 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 localiza-
tion, which does not overlap with myofibrils. Cofi-
lin-2 concentrates on a striated pattern between
a-actinin-2 rich stripes in cardiomyocyte sarco-
meres. Bar represents 10 mm.
(D) Line profile quantification of cofilin-1 (in red),
cofilin-2 (in green), and a-actinin-2 (in blue) locali-
zation 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 myo-
fibrils.
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Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
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 ubiqui-
tously, 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
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2 Developmental Cell 31, 1–12, October 27, 2014 ª2014 Elsevier Inc.
isoforms cannot functionally compensate
for each other in animals; cofilin-1-defi-
cient mice display early embryonic
lethality and defects in actin-dependent
morphogenic processes, whereas ADF
inactivation leads to corneal defects (Bel-
lenchi et al., 2007; Gurniak et al., 2005;
Ikeda et al., 2003). Mutations in the cofi-
lin-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 develop-
ment are unaffected in these mice, but
postnatal animals display defects in sar-
comeric actin exchange and muscle
maintenance (Agrawal et al., 2012; Gur-
niak 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
Figure 2. Cofilin-2 Depletion by RNAi Dis-
rupts 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 visu-
alized 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-2-
specific RNAi (line 2). GAPDH antibody was used
as a control for equivalent loading.
(C) Quantification of cofilin-2 levels of car-
diomyocyte 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.
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Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
functional and mechanistic principles for why tissue-specific
ADF/cofilin isoforms evolved in vertebrates have not been stud-
ied in detail.
Here, we report that cofilin-2 localizes between Z-discs in car-
diomyocytes, 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 iso-
forms and identified a cluster of residues at the surface of cofi-
lin-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 un-
dergo faster monomer exchange at their pointed ends.
RESULTS
Cofilin-2 Localizes near M-Bands in MyofibrilsTo study the role of cofilin-2 in muscle sarcomeres, we examined
its localization and function in cultured neonatal rat cardiomyo-
cytes, which provide a good model system to explore the func-
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Developmental Cell 31, 1–
tion of muscle-specific actin-binding pro-
teins (McElhinny et al., 2005; Skwarek-
Maruszewska et al., 2010). Because
none of the available cofilin-2 antibodies,
including those used in an earlier study
(Nakashima et al., 2005), displayed spe-
cific 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 surface-
exposed regions that differ between the two isoforms (Experi-
mental Procedures). Immunization of guinea pigs and rabbits
with these peptides yielded antibodies that specifically recog-
nized cofilin-1 or cofilin-2 (Figure 1A) and displayed good spec-
ificity 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, cofilin-
2 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 matu-
ration and that it localizes between Z-discs in sarcomeres.
12, October 27, 2014 ª2014 Elsevier Inc. 3
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. TheM-band proteinmyomesin 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 knock-
down cells. Bars represent 10 mm.
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Cofilin-2 Promotes Actin Dynamics in Sarcomeres
Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
Cofilin-2 Regulates Actin Filament Length inCardiomyocyte SarcomeresTo 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-
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4 Developmental Cell 31, 1–12, October 27, 2014 ª2014 Elsevier Inc
nofluorescence microscopy confirmed that cofilin-2 levels were
strongly reduced, especially in fluorescently conjugated RNAi-
transfected cells, where the oligonucleotides displayed punc-
tuate 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 in-
crease 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 or-
ganization do not result from off-target effects (Figure S1B).
Cardiomyocytes transfected with scrambled control oligo-
nucleotides showed a characteristic striated pattern of phalloi-
din-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 experi-
ments showed that �75% of control cells displayed clear peri-
odic 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. Further-
more, the typical striated pattern of the pointed end capping pro-
tein 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 fila-
ments 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 pre-
served in cofilin-2 knockdown cells.
Cofilin-2 Binds and Disassembles ATP-Actin FilamentsTo 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 as-
says revealed that cofilin-1 and cofilin-2 bind ADP-F-actin with
similar affinities (Figure 4A). TIRF microscopy using GFP-cofilin-
2 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 coopera-
tive interaction with the actin filament (Figure S2). Furthermore,
both cofilin isoforms competed with tropomyosin for F-actin in-
teractions and did not display isoform-specific binding toward
skeletal muscle a-actin or nonmuscle b/g-actin (Figure S3). How-
ever, 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 (Var-
tiainen 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 (Little-
field 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
6
.
Figure 4. Interactions of Cofilin-1 and Cofi-
lin-2 with ADP- and ADP,BeFx-Actin Fila-
ments
(A and B) Equilibrium binding of mouse cofilin-1
and cofilin-2 to ADP- (A) and ADP,BeFx-actin fil-
aments (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 quanti-
fied 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 Pro-
cedures.
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Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
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 affin-
ity (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 fila-
ment turnover, we monitored the decrease of pyrene-actin fluo-
rescence 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 ex-
periment to sequester the actin monomers and impede their rein-
corporation 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 addi-
tion of DBP. At an actin:cofilin molar ratio of 5:1, both cofilin iso-
forms 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 ms�1, respectively). Under identical concentrations, both
isoforms displayed weak ADP,BeFx-F-actin disassembly activity
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Developmental Cell 31, 1–1
(data not shown). However, at an actin:co-
filin molar ratio of 1:1, cofilin-2 increased
the disassembly rate of ADP,BeFx-F-actin by �10-fold, whereas cofilin-1 did
not significantly promote the disassembly
of ADP,BeFx-F-actin (Figure 4D). Impor-
tantly, cofilin-2 also promoted disas-
sembly of ADP- and ADP,BeFx-actin fila-
ments 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, cofi-
lin-2 can disassemble of ADP-Pi-mimetic filaments.
Identification of Cofilin-2 Residues Required for BindingADP-Pi-Actin Filaments and ATP-Actin MonomersSequence 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-1mu-
tants 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 mu-
tants 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 signifi-
cantly faster than wild-type cofilin-1 (Figures 5C and S4B). The
inverse mutant, cof-2/cof-1141,142,143, in which the same resi-
dues in cofilin-2 were replaced by their cofilin-1 counterparts,
displayed significantly lower ADP,BeFx-actin filament disas-
sembly activity compared to cofilin-2 (Figure 5C).
2, October 27, 2014 ª2014 Elsevier Inc. 5
(legend on next page)
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Cofilin-2 Promotes Actin Dynamics in Sarcomeres
6 Developmental Cell 31, 1–12, October 27, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
Developmental Cell
Cofilin-2 Promotes Actin Dynamics in Sarcomeres
Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
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 bind-
ing of cofilin-1 and cofilin-2 to NBD-G-actin resulted in a fluores-
cence 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 affin-
ity for ATP-G-actin than wild-type cofilin-1 (Figures 5D and S5).
The affinity was further increased by introducing the cofilin-2-
specific residue Val137 into this mutant. The inverse mutant,
cof-2/cof-1141,142,143 had significantly lower affinity for ATP-
G-actin compared to cofilin-2 (Figure 5D).
ATP-Actin Binding Residues Are Essential for CellularFunction of Cofilin-2To assess the importance of cofilin-20s 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 locali-
zation when expressed in cardiomyocytes, but also accumu-
lated in the M-band and Z-disc regions as detected by costain-
ing 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 disas-
semble 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 ex-
pressing 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 disassem-
bles 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.
Figure 5. Identification of Cofilin-2 Residues Responsible for High-Affi
(A) Sequence alignment of mammalian cofilin-1 and cofilin-2 isoforms. A blue b
servation. Colored frames highlight cofilin-2 residues introduced into cofilin-1 (m
orange: cof-1/cof-2141,142,143).
(B) Location of mutated residues on the surface of the cofilin-1 structure (Pope et
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
mutants. The error bars represent SEM from four to six independent experiment
(D) Bar diagram showing the dissociation constants of wild-type and mutant co
pendent experiments. See also Figures S4 and S5. Assays were carried out in 5 m
in the Supplemental Experimental Procedures.
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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 in-
crease the likelihood that barbed ends will contain ATP- or ADP-
Pi-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 ADP-
and ATP/ADP-Pi-actin filaments. Filament disassembly by cofi-
lin-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 tropomy-
osin 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 pro-
teins 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; Mor-
iyama 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 tis-
sues 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 inmature
myofibrils, cofilin-2 depletion inmice did not severely affectmus-
cle 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/stabil-
ity of cofilin-2 result in nemaline myopathy, which is character-
ized by the abnormal organization of actin filaments in striated
nity Binding to ADP,BeFx-F-Actin
ackground highlights conservation, with darker blue representing higher con-
agenta: cof-1/cof-26,8,10, red: cof-1/cof-2135, cyan: cof-1/cof-2137, and
al., 2004) color-coded as in (A). Ovals highlight surfaces participating in binding
2.5 mM of cofilin-1, cofilin-2, cof-2/cof-1141,142,143, or cof-1/cof-2141,142,143s.
filin-1 and cofilin-2 for ATP-G-actin. Error bars indicate SEM from three inde-
M Tris (pH 7.5) in the presence of 100 mMKCl and other components specified
3086
velopmental Cell 31, 1–12, October 27, 2014 ª2014 Elsevier Inc. 7
Figure 6. Rescue of Cofilin-2 Depletion
(A) Distributions of HA-tagged cofilins (left) and F-actin in 4-day-old car-
diomyocytes 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 HA-
cof2/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 indi-
cate SEM from four to five independent experiments.
See also Figure S6.
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Developmental Cell
Cofilin-2 Promotes Actin Dynamics in Sarcomeres
8 Developmental Cell 31, 1–12, October 27, 2014 ª2014 Elsevier Inc
Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
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 muscle-
specific ADF/cofilin isoforms are not functionally equivalent to
mammalian cofilin-2 (Poukkula et al., 2011). For example, inCae-
norhabditis 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 (Yama-
shiro 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 effi-
ciency to promote the disassembly of ADP,BeFx-F-actin. Recip-rocally, 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 ATP-
actinmonomer affinity of cofilin-1 and cofilin-2, respectively (Fig-
ure 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 -ATP-
actin monomers with higher affinity compared to cofilin-1
(Figure S3).
The residues required for high-affinity ATP-actin binding in co-
filin-2 are located at the barbed end region of actinmonomer 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 (subdo-
mains 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 sub-
units 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 pro-
nounced 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 fila-
ment (Dominguez and Holmes, 2011; Fujii et al., 2010; Oda
et al., 2009). In this way, nucleotide- and polymerization-de-
pended 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
6
.
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 tomonomeric actin (blue) (Dominguez andHolmes,
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 toG-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-fillingmodel of cofilin-1 bound to the actin filamentmade 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).
Developmental Cell
Cofilin-2 Promotes Actin Dynamics in Sarcomeres
Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
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-affin-
ity 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 mono-
mers 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 disas-
sembly are essential for the function of cofilin-2 in regulating
the length and/or organization of actin filaments in muscle
sarcomeres.
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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 anti-
bodies 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 ‘‘main-
taining 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 Exper-
imental 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
3086
velopmental Cell 31, 1–12, October 27, 2014 ª2014 Elsevier Inc. 9
Developmental Cell
Cofilin-2 Promotes Actin Dynamics in Sarcomeres
Please cite this article in press as: Kremneva et al., Cofilin-2 Controls Actin Filament Length in Muscle Sarcomeres, Developmental Cell (2014), http://dx.doi.org/10.1016/j.devcel.2014.09.002
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 mMMgCl2 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.
Statistical Analysis
Mann-Whitney nonparametric test was used to check significance of the
observed effects.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures
and six figures and can be found with this article online at http://dx.doi.org/
10.1016/j.devcel.2014.09.002.
ACKNOWLEDGMENTS
We thank Carol Gregorio for Tmod1 antibody and Minna Poukkula, Stanislav
Rozov, and Maria Vartiainen for critical reading of the manuscript. Sari
Tojkander is acknowledged for help in cardiomyocyte isolation. This study
was supported by grants from the Academy of Finland and Finnish Foundation
for Cardiovascular Research to (P.L). M.H.M. and G.G. were supported by fel-
lowships from GPBM and VGSB graduate programs. R.D. was supported by
NIH grant R01 GM073791.
Received: June 7, 2014
Revised: July 3, 2014
Accepted: September 3, 2014
Published: October 27, 2014
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