Redox Modification of Nuclear Actin by
MICAL-2 Regulates SRF Signaling
Mark R. Lundquist,1Andrew J. Storaska,2Ting-Chun Liu,3Scott D. Larsen,4Todd Evans,3Richard R. Neubig,2,5
and Samie R. Jaffrey1,*
1Department of Pharmacology, Weill Cornell Medical College, Cornell University, New York, NY 10065, USA
2Department of Pharmacology, University of Michigan, Ann Arbor, MI, 48109, USA
3Department of Surgery, Weill Cornell Medical College, Cornell University, New York, NY 10065, USA
4Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA
5Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824, USA
The serum response factor (SRF) binds to coactiva-
tors, such as myocardin-related transcription factor-
A (MRTF-A), and mediates gene transcription elicited
by diverse signaling pathways. SRF/MRTF-A-depen-
dent gene transcription is activated when nuclear
MRTF-A levels increase, enabling the formation of
transcriptionally active SRF/MRTF-A complexes.
The level of nuclear MRTF-A is regulated by nuclear
G-actin, which binds to MRTF-A and promotes its
nuclear export. However, pathways that regulate nu-
clear actin levels are poorly understood. Here, we
show that MICAL-2, an atypical actin-regulatory pro-
tein, mediates SRF/MRTF-A-dependent gene tran-
scription elicited by nerve growth factor and serum.
increases MRTF-A in the nucleus. Furthermore, we
show that MICAL-2 is a target of CCG-1423, a small
molecule inhibitor of SRF/MRTF-A-dependent tran-
scription that exhibits efficacy in various preclinical
disease models. These data identify redox modifica-
tion of nuclear actin as a regulatory switch that medi-
ates SRF/MRTF-A-dependent gene transcription.
Serum response factor (SRF) mediates gene transcription
induced by serum, various growth factors, and G-protein-
coupled receptor signaling pathways (Posern and Treisman,
2006). SRF-dependent gene transcription is modulated by SRF
coactivators, including ternary complex factor (TCF) and myo-
cardin-related transcription factor A (MRTF-A) (Shaw et al.,
1989; Wang et al., 2002). MRTF-A binds to SRF, forming a com-
plex that influences SRF binding to the CArG box promoter
element, which is found in SRF target genes (Miralles et al.,
2003; Treisman, 1986). SRF/MRTF-A-dependent gene tran-
scription mediates diverse cellular processes including cellular
migration (Leitner et al., 2011), cancer cell metastasis (Brandt
et al., 2009; Medjkane et al., 2009), mammary myoepithelium
development (Li et al., 2006), and neurite formation (Kno ¨ll and
Nordheim, 2009; Wickramasinghe et al., 2008).
SRF/MRTF-A-dependent gene transcription is induced when
MRTF-A localizes to the nucleus (Posern and Treisman, 2006).
MRTF-A is found in both the cytosol and the nucleus but exhibits
increased nuclear localization in response to various signaling
pathways. The nuclear localization of MRTF-A enables it to
form complexes with SRF, resulting in transcription of genes
that contain promoter elements that bind the SRF/MRTF-A
complex (Posern and Treisman, 2006). Thus, SRF/MRTF-A-
dependent gene transcription is highly influenced by the levels
of nuclear MRTF-A.
Recent studies have shown that MRTF-A localization is
regulated by actin dynamics in the nucleus (Baarlink et al.,
2013; Vartiainen et al., 2007). G-actin in the nucleus binds to
MRTF-A, enabling it to be exported to the cytosol (Vartiainen
et al., 2007). Thus, high levels of G-actin in the nucleus seen
during serum deprivation lead to low levels of nuclear MRTF-A.
when signaling pathways reduce nuclear G-actin, which pre-
vents MRTF-A export, resulting in accumulation of MRTF-A in
the nucleus (Vartiainen et al., 2007).
G-actin levels in the nucleus can be regulated by F-actin for-
mation in the cytosol. When actin polymerization is induced in
the cytosol, for example following RhoA-induced stress fiber for-
mation, cellular actin becomes sequestered in cytosolic stress
fibers, leading to the depletion of G-actin throughout the cell
(Vartiainen et al., 2007). RhoA-dependent depletion of G-actin
in the nucleus subsequently activates SRF/MRTF-A-dependent
gene transcription in NIH 3T3 cells (Vartiainen et al., 2007).
The depletion of monomeric actin by cytosolic stress fibers
is unlikely to mediate SRF/MRTF-A signaling in all cell types.
For example, SRF/MRTF-A signaling regulates axon growth (Lu
and Ramanan, 2011) and other neuronal functions (Kno ¨ll and
Nordheim, 2009; Wickramasinghe et al., 2008), but stress fiber
formation is not typically seen in neurons. Therefore, additional
pathways that induce SRF/MRTF-A signaling remain to be
Here, we describe a mechanism that regulates SRF/MRTF-A-
dependent gene expression which involves depolymerization of
Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc. 563
Figure 1. MICAL-2 Is a Nuclear Protein that Depolymerizes Actin
(A) Cherry-MICAL-2 and -3 expressed in HEK293T cells are localized to the nucleus (blue), while Cherry-MICAL-1 and NLS mutant Cherry-MICALs (M2NLSMut
and M3NLSMut) exhibited cytosolic localization. Scale bar, 10 mm.
(legend continued on next page)
564 Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc.
nuclear actin by MICAL-2, a member of a family of recently
described atypical actin-regulatory proteins (Terman et al.,
2002). MICAL-2 is homologous to MICAL-1, an enzyme that
binds to F-actin in the cytosol and triggers its depolymerization
through a redox modification of methionine (Hung et al., 2011;
2010). We show that MICAL-2 is enriched in the nucleus and
induces depolymerization of F-actin in the nucleus. Expression
of MICAL-2 reduces nuclear actin, resulting in nuclear retention
of MRTF-A and subsequent activation of SRF/MRTF-A-depen-
dent gene transcription. We find that MICAL-2 promotes SRF/
MRTF-A-dependent gene expression in several cell types, and
mediates NGF-dependent neurite growth in neuronal cells.
Furthermore, CCG-1423,a small molecule
pathway inhibitor that exhibits efficacy in various preclinical
disease models, directly binds MICAL-2 and inhibits its activity.
Together, these data show that SRF/MRTF-A signaling is regu-
lated by MICAL-2-dependent redox regulation of nuclear actin.
MICAL-2 Is Enriched in the Nucleus
Because of the important roles of MICAL-1 in depolymerizing
actin in axonal growth cones (Hung et al., 2010), we sought to
understand if MICAL-2 and -3 have related functions. In order
to understand the roles of MICAL-2 and -3, we examined the
subcellular localization of each MICAL isoform. Cherry-tagged
MICAL-1 was cytoplasmic in HEK293T cells; however, Cherry-
MICAL-2 and Cherry-MICAL-3 were nuclear (Figure 1A). To
confirm that these localizations are not due to the presence of
the Cherry tag, we monitored the localization of endogenous
MICALs by subcellular fractionation using isoform-specific anti-
bodies (Figures S1A and S1B available online). MICAL-1 was
cytoplasmic, while MICAL-2 and -3 were enriched in the nuclear
fraction in HEK293T cells (Figure 1B). The nuclear localization
of MICAL-2 was also confirmed by immunofluorescence in
HEK293T, COS7, and HeLa cells (Figures S1C and S1D).
The PredictProtein algorithm (Rost et al., 2004) revealed a
putative bipartite nuclear localization sequence (NLS) in both
MICAL-2 and -3, which was not found in MICAL-1 (Figure 1C).
the nuclear localization of MICAL-2 and -3 (Figure 1A), confirm-
ing that these sequences constitute a NLS domain in these
MICAL-2 Redox Activity InducesActin Depolymerization
Because MICAL-2 and -3 share 60% and 63% identity, respec-
domain (Terman et al., 2002), we asked if they also depolymerize
F-actin. To test this, we purified the bacterially expressed cata-
lytic domain and the adjacent actin-binding calponin homology
(CH) domain (designated ‘‘redoxCH’’) for each MICAL isoform
(Hung et al., 2010). To measure actin depolymerization, we
usedapreviouslydescribedassayfor MICAL-1-dependent actin
depolymerization using pyrene-labeled actin (Hung et al., 2010).
As expected, MICAL-1redoxCH-induced actin depolymerization in
an NADPH-dependent manner (Hung et al., 2010) (Figure 1D).
Similarly, MICAL-2redoxCHand -3redoxCH-induced actin depoly-
merization in an NADPH-dependent manner. The rate of NADPH
consumption by MICAL-2 and -3 was markedly enhanced by
F-actin, suggesting that F-actin is a specific substrate of these
enzymes (Figure S1E).
MICAL-2 Regulates Nuclear Actin Polymerization
The ability of MICAL-2 and -3 to depolymerize F-actin in vitro,
combined with their nuclear localization, suggests that these iso-
forms may regulate actin polymerization within the nucleus. We
therefore examined the effect of inhibiting MICAL-2 activity on
F-actin levels within the nucleus in various cell lines. Expression
of either of two dominant-negative MICAL-2 constructs, MICAL-
and MICAL-2GV, which contains a glycine to valine mutation at
amino acid 95 that blocks FAD-binding in flavoprotein monooxy-
actin into linear filaments that span large portions of the nucleus
(Figures 1E and S1F). Similar mutations in MICAL-3 expression
constructs did not appear to alter nuclear actin (data not shown).
MICAL-2 knockdown (Figure S1B) resulted in the appearance
of nuclear F-actin as detected by phalloidin staining in fixed cells
(Figure S1G).Similarly, expression of GFP-tagged LifeAct, which
binds to F-actin (Riedl et al., 2008), revealed nuclear F-actin in
live cells expressing MICAL-2-specific shRNA (Figure S1H).
The MICAL-2 knockdown constructs did not affect cellular
viability (Figures S1I and S1J).
To validate that these actin filaments are localized within the
nucleus, we costained cells with an antibody to lamin A/C, a
marker of the inner nuclear membrane (Figure S1K). Actin fila-
ments were indeed within the nucleus, rather than surrounding
(B) Subcellular fractionation of MICAL isoforms. Endogenous MICAL-2 and -3 are localized to the nucleus while MICAL-1 was detected in the cytosol. MEK1/2,
VDAC, Calnexin, and PCNA immunoblotting was used to confirm the purity of the cytosolic (Cyto), mitochondrial (Mito), microsomal (Micro), and nuclear (Nuc)
(C) MICAL-2 and -3 each contain a putative bipartite NLS. The domain structure of MICAL-2 is indicated above. The enzymatic domain contains a GXGXXG,
aspartate-glycine (DG), and a glycine-aspartate (GD) motif, which is the characteristic FAD-binding motif. The F-actin-binding CH domain and LIM domain
are indicated. A potential bipartite NLS (red box) in MICAL-2 and MICAL-3 is not found in MICAL-1. Shown in blue letters are the amino acids that constitute the
NLS in MICAL-2 and -3. Shown in red are the amino acids that are mutated in the M2NLSMut and M3NLSMut. Uppercase letters are amino acids conserved in
MICAL-1, -2 and -3.
(D) MICAL-2 and -3 depolymerize F-actin similarly to MICAL-1. MICAL-1redoxCH, MICAL-2redoxCHand MICAL-3redoxCHwere incubated with pyrene-labeled actin
and NADPH as indicated. These data indicate that MICAL-2 and MICAL-3, like MICAL-1, induce NADPH-dependent F-actin depolymerization. Statistical sig-
(E) Dominant-negative inhibition of MICAL-2 lead to increased nuclear F-actin. Expression of dominant-negative MICAL2 (MICAL-2CT or MICAL-2GV, green)
resulted in the appearance of filaments (yellow arrows) throughout the nucleus (blue), as seen by phalloidin labeling (red staining, blue arrows). The dominant-
negative constructs colocalized with the F-actin (yellow). Scale bar, 5 mm.
See also Figure S1
Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc. 565
shRNA MICAL-2 #2 shRNA MICAL-2 #1
Nuclear/Cytosolic MRTF-A Ratio
Nuclear/Cytosolic MRTF-A Ratio
Nuclear/Cytosolic MRTF-A Ratio
shRNA MICAL-2 #2
shRNA MICAL-2 #1
shRNA MICAL-2 #1
shRNA MICAL-2 #2
shRNA MICAL-2 #1
shRNA MICAL-2 #2
(legend on next page)
566 Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc.
the nuclear membrane (Figures S1L–S1O). Collectively, these
data indicate that MICAL-2 enzymatic activity inhibits polymeri-
zation of nuclear actin in cells.
MICAL-2 Expression Induces Nuclear Localization of
Since MICAL-2 regulates F-actin within the nucleus, and since
nuclear G-actin mediates nuclear export of MRTF-A (Vartiainen
et al., 2007), we considered the possibility that MICAL-2 reduces
nuclear MRTF-A by promoting its export. To test this, HEK293T
cells were infected with a virus expressing either GFP or
GFP-MICAL-2 and serum-starved for 18 hr under low-serum
conditions (0.3% fetal bovine serum [FBS]) (Miralles et al.,
2003) (Figures 2A and 2B). Contrary to our expectation, overex-
pression of MICAL-2 under these conditions caused a marked
increase in nuclear localization of MRTF-A (Figures 2A and 2B).
We next asked whether the nuclear localization of MRTF-A
induced by serum is mediated by MICAL-2. Treatment of
serum-starved HEK293T cells with 10% FBS for 10 min altered
MICAL-2 phosphorylation levels, suggesting that serum may
regulate the function of MICAL-2 (Figures S2A and S2B). To
determine whether MICAL-2 is required for serum-mediated
accumulation of MRTF-A in the nucleus, we monitored MRTF-A
localization following knockdown of MICAL-2. In HEK293T cells
expressing either of two MICAL-2-specific shRNAs, but not
control shRNA, serum-induced localization of MRTF-A to the
nucleus was markedly reduced (Figures S2C and S2D). Thus,
MICAL-2 induces nuclear localization of MRTF-A in the absence
of serum stimulation and is required for the increase in nuclear
MRTF-A levels after serum stimulation.
We considered the possibility that MICAL-2 could increase
nuclear MRTF-A by increasing expression levels of MRTF-A.
However, MICAL-2 expression has no discernable effect on
global levels of MRTF-A (Figure S2E). Additionally, the nuclear
accumulation of MRTF-A does not appear to reflect binding of
MRTF-A to MICAL-2 (Figures S2F and S2G). Lastly, expression
of MICAL-2 did not induce stress fibers or increase cytosolic
F-actin levels in HEK293T cells (Figures S2I–S2L). Thus,
MICAL-2 does not increase nuclear MRTF-A by increasing cyto-
MICAL-2 Mediates NGF-Dependent Nuclear
Localization of MRTF-A
We next examined whether MICAL-2 is required for nuclear
localization of MRTF-A after growth factor stimulation. MRTF-A
was readily detectable in the cytosol in untreated PC12 cells
and exhibited increased nuclear localization in response to
NGF treatment (Figures 2C and 2D). However, MRTF-A nuclear
localization in response to NGF was markedly reduced by
expression of either of two MICAL-2-specific shRNAs, but not
by a control shRNA (Figures 2C and 2D).
We next asked whether MICAL-2 promotes the nuclear local-
used rat embryonic day (E) 14 dorsal root ganglia (DRG) sensory
neurons, which are typically cultured in NGF. Because these
neurons require NGF for survival (Chun and Patterson, 1977),
we did not test culture conditions lacking NGF. In these neurons,
MRTF-A was readily detectable in both the nucleus and cyto-
plasm (Figures 2E and 2F). Infection of these cells with lentivirus
expressing either of two MICAL-2-specific shRNAs resulted in a
marked decrease in nuclear MRTF-A (Figures 2E and 2F). Taken
together, these data indicate that MICAL-2 is required for effi-
cient nuclear localization of MRTF-A.
MICAL-2 Expression Induces the SRF/MRTF-A-
Because MICAL-2 induces nuclear localization of MRTF-A, we
asked whether MICAL-2 activates SRF/MRTF-A-dependent
gene transcription. To test this, we determined the effect of
MICAL-2 expression on a transcriptional reporter containing
luciferase under the control of a CArG [CC(A/T)6GG] element.
In serum-starved HEK293T cells, minimal levels of luciferase
were detected (Figure 3A). However, expression of MICAL-2
This effect was not seen following expression of MICAL-2
mutants that were catalytically inactive or which contain NLS
mutations (Figure 3A). MICAL-2 did not induce a transcriptional
reporter containing the highly related TCF/ETS SRE element,
which requires SRF, but not MRTF-A (Buchwalter et al., 2004)
(Figure 3B). Collectively, these data indicate that MICAL-2 in-
duces SRF/MRTF-A-dependent reporter gene expression.
MICAL-1 and MICAL-3 overexpression did not activate the
reporter, indicating that activation of SRF/MRTF-A-dependent
gene expression is limited to MICAL-2 in HEK293T cells (Fig-
ure 3C, S3A, and S3B).
To confirm that MICAL-2-mediated activation of SRF/MRTF-A
gene transcription requires MRTF-A, we expressed MRTF-A-
DTAD, a dominant-negative MRTF-A mutant. This protein lacks
the transcription activation domain (TAD) that is necessary for
an active SRF/MRTF-A complex (Wang et al., 2003). Expression
Figure 2. MICAL-2 Induces the Nuclear Localization of MRTF-A
(A) MICAL-2 expression (green) induces nuclear localization of MRTF-A (red) under serum-starvation. Nuclei are outlined in white, based on DAPI staining (blue).
Scale bar, 5 mm.
(B)Quantification of(A).Theaverage intensityof MRTF-Aimmunofluorescence wasquantified inthenucleusand thecytoplasm. Thenucleus:cytoplasmic ratioof
MRTF-A increased 10.76-fold in GFP-MICAL-2-expressing cells compared to GFP-expressing cells. ***p < 0.0005, Student’s t test, n R 30.
(C) MICAL-2 is required for NGF-induced nuclear localization of MRTF-A in PC12 cells. NGF treatment induces nuclear localization of MRTF-A in LacZ shRNA-
to uninfected cells (blue arrows). Nuclear border indicated by dotted white lines. Scale bar, 10 mm.
(D) Quantification of (C). ***p < 0.0005, ANOVA (***p < 0.0001) with Dunnett posttest n R 30. All data in this figure are mean ± SEM.
(E) The nuclear localization of MRTF-A in dissociated E14 DRG neurons cultured in the presence of NGF is dependent upon MICAL-2. Infection of DRG neurons
with MICAL-2-specific shRNA results in a substantial nuclear depletion of MRTF-A (red). Scale bar, 5 mm.
(F) Quantification of (E). ANOVA (***p < 0.0005) with Dunnett posttest. n R 25.
See also Figure S2.
Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc. 567
of MRTF-A-DTAD blocked the effect of MICAL-2 on the SRF/
MRTF-A reporter (Figure 3D). Thus, MICAL-2 induces the re-
porter in an MRTF-A-dependent manner.
MICAL-2 Expression Induces Endogenous SRF/MRTF-A
Target Gene Expression
We next asked whether MICAL-2 induces endogenous SRF/
MRTF-A target genes. SRF/MRTF-A target genes have been
validated in numerous cell lines (Medjkane et al., 2009; Selvaraj
and Prywes, 2004) and neuronal cells (Kno ¨ll and Nordheim,
2009). Expression of MICAL-2 in serum-starved HEK293T cells
resulted in increased levels of several known SRF/MRTF-A
target genes in these cells by 30%–50%, including Acta2,
Cyr61, SRF, and VCL (Figure S4A). Expression of MICAL-2
had a negligible effect on MRTF-A-independent SRF-dependent
genes that contain the TCF/ETS SRE element in their promoter
TSP1, PTGS1, and Egr2.
Expression of MICAL-2 in PC12 cells similarly induced SRF/
MRTF-A target genes Acta2, Cyr61, and SRF by 100%–200%
(Figure S4C). MICAL-2 expression did not induce SRF-
PTGS1, and Egr2 (Figure S4C). These data indicate that
dent gene expression.
Relative Luciferase Activity (a.u.)
Relative Luciferase Activity (a.u.)
Relative Luciferase Activity (a.u.)
Relative Luciferase Activity (a.u.)
esa r e f i cu l FCT / FRSes a r e f i cu l A-FTRM / FRS
esa r e f i c u l A-FTRM / FRSes a r e f i cu l A-FTRM/ FRS
Figure 3. MICAL-2 Induces SRF/MRTF-A-Mediated Luciferase Expression
(A) MICAL-2 induces the SRF/MRTF-A transcriptional reporter in serum-starved (0.3% FBS)-treated cells. Catalytically inactive MICAL-2 mutants (MICAL-2GV
and MICAL-2CT), as well asaMICAL-2mutant that does not localize tothe nucleus (M2NLSMUT)does notinduce the reporter. ANOVA (**p < 0.005) withDunnett
posttest. n = 18. All data in this figure are mean ± SEM.
(B) MICAL-2 does not activate the MRTF-A-independent SRF/TCF luciferase promoter. ANOVA (***p < 0.0001) with Dunnett posttest. n = 18.
(C) MICAL-2, but not MICAL-1 or MICAL-3, activates the SRF/MRTF-A luciferase reporter. ANOVA (***p < 0.0005) with Dunnett posttest. n = 18.
(D) MICAL-2-dependent induction of the SRF/MRTF-A reporter requires MRTF-A. Coexpression of dominant-negative MRTF-ADTAD blocked MICAL-2-
dependent induction of luciferase. ANOVA (***p < 0.0001) with Dunnett posttest. n = 18.
See also Figure S3.
568 Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc.
MICAL-2 Is Required for Serum and NGF-Induced SRF/
MRTF-A Target Gene Expression
We next examined whether MICAL-2 mediates the induction of
SRF/MRTF-A target genes in response to serum and NGF. In
fibroblasts, serum induces the expression of Acta2, Cyr61,
VCL, and SRF in an SRF- and MRTF-A-dependent manner
(Descot et al., 2009; Lee et al., 2010) (Figure S4B). However,
expression of either of two MICAL-2-specific shRNAs blocked
this effect (Figure S4B). MICAL-2-specific shRNAs did not affect
the induction of MRTF-A-independent SRF-dependent genes
TSP1, PTGS1, and Egr2. In PC12 cells, NGF induces the expres-
sion of Acta2, Cyr61, VCL, and SRF in an SRF- and MRTF-A-
dependent manner (Figure S4D). MICAL-2 shRNAs impaired
NGF-induced expression of these SRF/MRTF-A target genes
(Figure S4D) but did not affect NGF-mediated induction of
PTGS1. Similarly, rat E14-15 DRG neurons cultured in NGF
exhibited a reduction in Acta2, Cyr61, and VCL expression
upon infection with lentivirus expressing either of two MICAL-2
shRNAs, while MRTF-A-independent SRF-dependent genes
TSP1, PTGS1, and Egr2 were unchanged (Figure S4E). Thus,
MICAL-2 promotes serum- and NGF-dependent increases in
SRF/MRTF-A-dependent gene expression.
MICAL-2 Is Required for NGF-Dependent Neurite
To further address whether MICAL-2 mediates NGF-induced
SRF/MRTF-A-dependent transcription, we monitored the effect
of MICAL-2 knockdown on NGF-dependent neurite outgrowth in
PC12 cells and embryonic DRG sensory neurons. In PC12 cells
expressing MICAL-2-specific shRNA, neurite length 4 days after
NGF treatment was reduced by ?40% compared to LacZ
shRNA-infected PC12 cells (Figures 4A and 4B). Knockdown of
MICAL-2 in differentiated PC12 cells also decreases neurite for-
mation after NGF treatment (Figure 4C). Similar to PC12 cells,
DRG neurons expressing MICAL-2-specific shRNA exhibited a
?45% decrease in axon growth rate at DIV5 compared to
LacZ-shRNA expressing neurons (Figures 4D and 4E). Taken
together, these data indicate that MICAL-2 mediates NGF-
induced SRF/MRTF-A signaling in both PC12 cells and primary
embryonic sensory neurons.
Embryonic MICAL-2 Knockdown Impairs SRF/MRTF-A-
Dependent Gene Expression
We next asked whether MICAL-2 mediates SRF/MRTF-A-
dependent gene transcription induced by physiologic signals.
SRF-dependent gene transcription is required for heart develop-
ment in zebrafish (Chong et al., 2012). MICAL isoforms have
been characterized in zebrafish (Xue et al., 2010), with the
cardiac-enriched mical2b exhibiting highest sequence identity
to mouse MICAL-2.
We therefore knocked down mical2b during embryonic
development using antisense morpholinos (Figures S5A and
S5C). We used either a splice-blocking morpholino or a transla-
tion-blocking morpholino and injected them into developing
embryos at the 1-cell stage. Depletion of mical2b transcripts
by the splice-blocking morpholino was validated by qRT-PCR
(Figure S5B). Knockdown of mical2b resulted in small hearts
that failed to undergo normal looping at 24 hpf (Figures 5A and
5B), with thin, linear morphology compared to wild-type hearts
at 48 hpf (Figures 5C and 5D). Additionally, cardiomyocytes in
morphant hearts were spatially disorganized, rather than
displaying the relatively even distribution seen in wild-type
hearts, as shown at 52 hpf in the atria (Figures 5E–5G). Taken
together, these data confirm that mical2b expression was effec-
tively knocked down in zebrafish.
We next asked whether mical2b-deficient animals exhibited
We found that many cardiac muscle-specific SRF/MRTF-A
target genes, such as cardiac alpha actin, smooth muscle alpha
actin, cardiac actin smooth muscle, skeletal alpha actin, smooth
muscle 22a, srf, and calponin (Chong et al., 2012; Davis et al.,
2008; Descot et al., 2009) were markedly reduced in mical2b
knockdown animals (Figure 5H). The control genes vascular
endothelial growth factor (vegf), fibroblast growth factor 4
(fgf4), and ETS translocation variant 4 (pea3), which regulate
MRTF-A-independent cardiac development in zebrafish, were
not affected by mical2b knockdown (Figure 5H) (Liang et al.,
2001; Znosko et al., 2010). Coinjection of the morpholinos with
RNA encoding full-length human MICAL-2, but not MICAL-
2GV, normalized expression of the SRF/MRTF-A-dependent
genes (Figure 5H). Thus, mical2b regulates SRF/MRTF-A-
dependent gene transcription in response to physiological stim-
uli during zebrafish development.
MICAL-2 Does Not Activate SRF/MRTF-A through RhoA
We considered the possibility that MICAL-2 activates SRF/
MRTF-A signaling by increasing RhoA activity. However, appli-
cation of cell-permeable C3-transferase, an inhibitor of RhoA,
or Y-27632, a ROCK inhibitor, did not block MICAL-2-induced
activation of the SRF/MRTF-A reporter in serum-starved
HEK293T cells (Figure 6A) or MICAL-2-dependent MRTF-A
nuclear accumulation (Figure 6B). These data, along with our
ures S2I–S2L), indicate that MICAL-2 does not activate SRF/
MRTF-A reporter expression through the RhoA pathway.
MICAL-2 Induces Depletion of Nuclear Actin
We next asked if MICAL-2 activates SRF/MRTF-A-dependent
gene transcription by depleting nuclear G-actin. To test this,
we measured nuclear actin levels in HEK293T cells with Alexa
594-DNase I, which specifically binds G-actin (Suck et al.,
1981). Consistent with previous studies (Mouilleron et al., 2011;
Vartiainen et al., 2007), we found that serum-starvation was
associated with higher G-actin levels in the nucleus than in the
cytoplasm (Figures 6C and 6D). MICAL-2 overexpression
caused a significant reduction in nuclear G-actin, even in the
absence of serum (Figures 6C and 6D). The reduction in nuclear
G-actin induced by MICAL-2 was similar to the reduction
induced by serum stimulation (Figures 6C and 6D). These data
suggest that MICAL-2 induces nuclear localization of MRTF-A
by reducing nuclear G-actin levels.
We next asked if MICAL-2 promotes the reduction in nuclear
G-actin seen after NGF stimulation. In differentiated PC12 cells,
NGF induced a significant decrease in nuclear G-actin (Figures
S6A and S6B). Expression of GFP-MICAL-2 in unstimulated
Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc. 569
Projection length (μm)
shRNA MICAL-2 #2 shRNA MICAL-2 #1
shRNA MICAL-2 #2
shRNA MICAL-2 #1
Growth rate (μm/h)
shRNA MICAL-2 #1
shRNA MICAL-2 #2
shRNA MICAL-2 #1
shRNA MICAL-2 #2
shRNA MICAL-2 #1
shRNA MICAL-2 #2
Figure 4. MICAL-2 Is Required for NGF-Dependent Neurite Outgrowth in PC12 Cells and DRG Neurons
(A) MICAL-2 is required for NGF-induced neurite outgrowth in PC12 cells. NGF treatment (50 ng/ml) for 48 hr resulted in prominent neurite outgrowth in LacZ
shRNA (green) control as measured by Alex568-phalloidin staining (red). Knockdown of MICAL-2 with either of two shRNA significantly reduces neurite
outgrowth. Scale bar, 10 mm.
ANOVA (***p < 0.0001) with Dunnett posttest. *p < 0.05, n R 40. All data in this figure are mean ± SEM.
(C) Quantification of neurite number in (A). Knockdown of MICAL-2 significantly reduced the average number of neurites induced by NGF treatment. ANOVA
(***p < 0.0001) with Dunnett posttest. ***p < 0.0005, **p < 0.005, n R 40.
(D) MICAL-2 knockdown decreases axon growth rates in DRG neurons. DRG neurons expressing either LacZ-specific or MICAL-2-specific shRNA were imaged
at 0 (blue arrow) and 60 min (yellow arrow). Scale bar, 10 mm.
(E) Quantification of rates in D. ANOVA (***p < 0.0004) with Dunnett posttest. ***p < 0.0005, **p < 0.005, n R 20.
See also Figure S4 and Table S1.
570 Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc.
Figure 5. MICAL-2 Regulates SRF/MRTF-A-Dependent Gene Transcription during Zebrafish Development
(A,B) Representativecontrolembryo derived from transgenic myl7:egfp reporterfish at24hpf, showing thesizeand positionof anormal looping hearttube. By 48
hpf the control heart is fully looped. The myl7:egfp transgene directs eGFP expression to myocardial cells and facilitates detection of alterations in heart
(C,D) mical2b regulates cardiogenesis during zebrafish development. The mical2b splice-blocking morphant embryo at 24 hpf shows a smaller heart tube that
fails to loop normally and thus is positionally displaced and leads to a significant pericardial edema (PE). By 48 hpf the morphant heart tube is linear and
dysmorphic. Similar results were obtained with the translation blocking morpholino. For A-D n > 100.
(E) Higher magnification view of the normal control heart at 52 hfp highlights the evenly spaced cardiomyocytes (white arrows) focused on the atrial chamber.
injected with 2 or 4 ng of the splice-blocking morpholino, respectively (n > 50).
(legend continued on next page)
Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc. 571
PC12 cells also induced a significant decrease in the ratio of
nuclear:cytosolic G-actin (Figures S6A and S6B). Furthermore,
in PC12 cells expressing either of two MICAL-2-specific
shRNAs, NGF-dependent reduction in nuclear G-actin levels
was significantly impaired (Figures S6C and S6D). Consistent
with these findings, DRG neurons expressing either of two
MICAL-2-specific shRNAs exhibited significantly increased
nuclear G-actin levels (Figures S6E and S6F). These data sug-
gest that MICAL-2 mediates NGF-induced reductions in nuclear
We next asked if the effects of MICAL-2 were due to a direct
effect on actin. MICAL-1 oxidizes methionine 44 in actin to
form methionine sulfoxide (Hung et al., 2011). We therefore
asked whether a M44Q substitution, which mimics features of
the oxidized form of actin, alters actin localization in cells.
Wild-type GFP-actin waslargelycytosolic thoughasmallportion
was foundinthe nucleus(FiguresS6GandS6H).However, GFP-
actin M44Q was significantly excluded from the nucleus. On the
other hand, GFP-actin M44L, which is not oxidizable, exhibited
higher nuclear levels than wild-type actin (Figures S6G and
S6H). These data suggest that oxidation of Met44 promotes
exclusion of actin from the nucleus. GFP-actin and GFP-actin
M44L colocalized with phalloidin staining at similar levels;
however, GFP-actin M44Q colocalization with phalloidin was
significantly reduced (Figure S6I). The lack of incorporation of
GFP-actin M44Q in F-actin is consistent with the known depoly-
merizing effect of MICAL-2 on actin. We next asked whether
oxidation of actin methionine 44 is required for MICAL-2-depen-
dent reduction in nuclear G-actin. To test this, we monitored the
effect of MICAL-2 on nuclear localization of GFP-actin and GFP-
actin M44L. In these experiments we found that MICAL-2
coexpression decreased nuclear GFP-actin, but not GFP-actin
M44L (Figures 6E and 6F). These data suggest that MICAL-2-
dependent oxidation of methionine 44 leads to a reduction in
The SRF/MRTF-A Pathway Inhibitor CCG-1423 Is a
CCG-1423 is a small molecule inhibitor of SRF/MRTF-A-depen-
a SRF/MRTF-A reporter (Evelyn et al., 2007). While CCG-1423
has shown utility in preclinical disease models, its specific mo-
lecular target is not known.
CCG-1423 treatment reduces nuclear MRTF-A levels (Jin
et al., 2011), which is similar to the effect of MICAL-2 knockdown
(Figures 2C–2F and Figures S2C and S2D). Therefore, we
wondered whether the effects of CCG-1423 on SRF/MRTF-A
signaling are mediated by inhibition of MICAL-2. As a first test,
we asked whether CCG-1423 blocks MICAL-2 signaling in cells.
In this experiment, we monitored the effect of CCG-1423 on
MICAL-2-dependent induction of the SRF/MRTF-A reporter in
serum-starved HEK293T cells. Expression of MICAL-2-induced
luciferase expression, while treatment with 5 mM CCG-1423
for 4 hr reduced it by 75% (p < 0.001, Figure 7A). These data
indicate that CCG-1423 blocks MICAL-2-mediated induction of
the SRF/MRTF-A reporter.
We next asked whether CCG-1423 binds MICAL-2. To detect
CCG-1423 binding to MICAL-2, we used a thermal denaturation
assay. In this assay, the melting temperature (Tm) of a protein is
measured by increasing the temperature of a sample in the
presence of a fluorescent dye, such as 1-anilinonaphthalene-8-
sulfonic acid (1,8-ANS) (Pantoliano et al., 2001). As the protein
unfolds, 1,8-ANS binds the exposed hydrophobic domains,
increasing the dye’s fluorescence. Ligand binding is detected
by observing an increase or decrease in the Tm. Incubation of
MICAL-2 with CCG-1423 decreased the Tmby ?0.3?C with an
EC50of 3.8 mM. A structurally similar compound with markedly
reduced potency at inhibiting SRF/MRTF-A signaling (CCG-
100594) (Evelyn et al., 2010) had no significant effect on Tm(Fig-
ure 7B, and S7A–S7C), indicating that these effects are specific
to CCG-1423. Taken together, these data indicate that CCG-
1423 directly binds MICAL-2.
We next asked whether CCG-1423 binding affects MICAL-2
catalytic activity. To test MICAL-2 activity, we used an NADPH
depletion assay. Recombinant MICAL-2, comprising the enzy-
matic domain and the CH domain (MICAL-2redoxCH), was
incubated with NADPH in the presence of F-actin, and the con-
sumption of NADPH was monitored by UV absorbance. Addition
of CCG-1423 (10 mM) markedly reduced NADPH consumption
by MICAL-2 compared to CCG-100594 (Figure 7C). CCG-1423
increases the Kmof NADPH and exhibited a Kiof 1.57 mM (Fig-
ure 7D). Taken together, these data indicate that CCG-1423
binds MICAL-2 and inhibits its enzymatic activity.
Our studies identify redox modification of nuclear actin as a
dent gene transcription. We find that MICAL-2 catalyzes the
disassembly of nuclear actin polymers, which leads to a reduc-
tion in nuclear G-actin levels. This reduction in nuclear G-actin
allows MRTF-A to accumulate in the nucleus by impairing its
nuclear export, which in turn facilitates SRF/MRTF-A-dependent
gene induction. Indeed, expression of MICAL-2 is sufficient to
induce SRF/MRTF-A-dependent gene expression. Additionally,
knockdown of MICAL-2 markedly reduces NGF-stimulated
SRF/MRTF-A-dependent expression and neurite growth. Our
findings identify MICAL-2 as a novel regulator of SRF/MRTF-A
signaling that acts by regulating nuclear actin.
SRF/MRTF-A-dependent gene transcription can be induced
by RhoA-dependent pathways that lead to the sequestration of
actin in stress fibers (Miralles et al., 2003; Stern et al., 2009).
Our studies identify a mechanism for activation of SRF/MRTF-
A-dependent gene transcription which does not involve
(H) mical2b knockdown impairs SRF/MRTF-A-dependent, but not SRF/MRTF-A-independent gene transcription. Levels are compared to control wild-type (WT)
embryos for the mical2b morphants (2B), and in embryos rescued by coinjected RNA encoding the full-length wild-type murine MICAL-2 or the MICAL-2GV
mutant protein. Experimental gene levels were normalized by comparison to housekeeping gene EF1a. ANOVA (***p < 0.0005) with Dunnett posttest. ***p <
0.0005, **p < 0.005, **p < 0.05, n R 6. Mean ± SEM.
See also Figure S5 and Table S2.
572 Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc.
Nuclear / Cytosolic
Nuclear / Cytosolic
Figure 6. MICAL-2 Regulates Nuclear Actin Independent of RhoA
(A) MICAL-2 induces the SRF/MRTF-A luciferase reporter in a ROCK- and RhoA-independent manner. Treatment with either inhibitor reduced luciferase
expression induced by serum-stimulation, but not by MICAL-2 expression. ANOVA (***p < 0.0001) with Dunnett posttest. n = 8. All data in this figure are
mean ± SEM.
(B) MICAL-2 induces nuclear MRTF-A in a ROCK- and RhoA-independent manner. HEK293T cells were infected with either GFP or GFP-MICAL-2. Nuclear
accumulation of MRTF-A was unaffected in MICAL-2 expressing, serum-starved HEK293T cells treated with either 2 mg/ml C3-transferase, a RhoA inhibitor, or
100 mM Y27632, a ROCK inhibitor. ANOVA (***p < 0.0001) with Dunnett posttest.
(C) MICAL-2 expression decreases the nuclear to cytosolic ratio of G-actin in HEK293T cells. In starved GFP-expressing cells, G-actin is readily detectable in
the nucleus (blue), as measured by DNase I staining (red). Serum stimulation and GFP-MICAL-2 significantly reduced the amount of G-actin in the nucleus. Scale
bar, 5 mm.
(D) Quantification of the nuclear:cytosolic DNase I staining seen in C. ANOVA (***p < 0.0001) with Dunnett posttest. ***p < 0.0005, n R 30.
(E) MICAL-2 expression reduces nuclear levels of GFP-Actin, but not GFP-actin M44L. When overexpressed, GFP-actin M44L is enriched in the nucleus (blue)
whencomparedtowild-typeGFP-Actin. MICAL-2(red)coexpressionfurtherdecreasesthelevelsofnuclearwild-typeGFP-actin(green) whilehavingnoeffecton
the nuclear levels of GFP-actin M44L. Scale bar, 5 mm.
(F) Quantification of the nuclear:cytosolic GFP-actin staining seen in G. ANOVA (***p < 0.0001) with Dunnett posttest. ***p < 0.0005, n R 25.
See also Figure S6.
Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc. 573
regulation of cytosolic actin pathways but which involves regula-
tion of nuclear actin levels. The significance of MICAL-2-depen-
dent activation of SRF/MRTF-A-dependent gene transcription
is supported by the impaired nuclear localization of MRTF-A
and SRF/MRTF-A-dependent gene transcription in MICAL-2
knockdown PC12 cells, DRG sensory neurons, and HEK293T
cells. SRF/MRTF-A-dependent gene transcription in vivo is
impaired by knockdown of the zebrafish MICAL-2 ortholog.
These studies identify MICAL-2 as a physiologic mediator of
SRF/MRTF-A signaling in diverse cell types.
The MICAL-2-mediated increase in nuclear MRTF-A levels
appears to be caused by a reduction in nuclear G-actin levels.
This is the same mechanism by which stress fiber induction
leads to nuclear retention of MRTF-A (Vartiainen et al., 2007).
G-actin binds MRTF-A to induce its nuclear export (Miralles
et al., 2003; Mouilleron et al., 2011). Byreducing nuclear G-actin,
MRTF-A loses the cofactor needed for its export, resulting in
nuclear accumulation. The ability of MICAL-2 to depolymerize
nuclear F-actin originally suggested to us that MICAL-2 would
increase nuclear G-actin levels and therefore decrease MRTF-A
levels in the nucleus. However, the opposite effect was seen:
MICAL-2 expression led to increased nuclear levels of MRTF-A.
Furthermore, MICAL-2 expression reduced overall nuclear
G-actin levels, to the same degree that is seen following serum
and NGF stimulation. Several mechanisms may explain how
MICAL-2 activity leads to reduced G-actin in nuclei. Oxidation
of actin by MICAL-2 may affect the kinetics of actin import
M44L and M44Q. Additionally, MICAL-2-mediated oxidation of
actin may alter the binding between MRTF-A and actin. The pre-
cise mechanisms by which MICAL-2 activity reduces nuclear
actin levels remains to be established.
While MICAL-1 is cytosolic, MICAL-2 and -3 appear to be
enriched in the nucleus. MICAL-2 has also been detected in
both HeLa cytosol and nucleus by another group; however,
this localization was seen using a MICAL-2 overexpression
construct (Giridharan et al., 2012). The effects of MICAL-2 that
we see in HEK293T cells, PC12 cells, and DRG neurons are
unlikely to reflect cytosolic functions of MICAL-2 because
MICAL-2 is predominantly nuclear in these cell types and
because the effects of MICAL-2 on SRF/MRTF-A-dependent
gene transcription are not seen following expression of MICAL-2
constructs that contain mutations in the nuclear-localization
lymerize nuclear F-actin.
The proteins that regulate nuclear actin polymerization are
poorly understood. In addition to its role in mediating export of
transcription of ribosomal RNA genes (rDNA) (Philimonenko
et al., 2004), preinitiation complexes necessary for RNA Pol II
transcription (Hofmann et al., 2004), and nuclear morphology
(Krauss et al., 2003). Therefore, MICAL-2 could influence other
actin-mediated pathways as well. MICAL-3, may also have
nuclear roles. Although we did not find an effect of MICAL-3 on
SRF/MRTF-A-dependent signaling, MICAL-3 may regulate
pools of nuclear actin linked to other processes.
Relative NADPH absorbance (a.u.)
0.5 μM CCG-1423
5.0 μM CCG-1423
10 μM CCG-100594
10 μM CCG-1423
Ki = 1.57 μM
Figure 7. MICAL-2 Is Targeted by CCG-1423
(A)CCG-1423inhibits MICAL-2-induced activation
of the SRF/MRTF-A transcriptional reporter by
both serum and MICAL-2 expression. ANOVA
n = 24. All data in this figure are mean ± SEM.
(B) CCG-1423 exhibits concentration-dependent
thermal destabilization of MICAL2-EN. Recombi-
nant MICAL2-EN, which comprises the enzymatic
increasing concentrations of CCG-1423, or the
control compound, CCG-100594, and the Tmwas
calculated in a thermal denaturation assay. Incu-
bation of CCG-1423 exhibited thermal destabili-
zation of MICAL-2 with an IC50of 3.8 mM and Hill
coefficient -1.1. ANOVA, Bonferroni’s multiple
comparisons test *p < 0.05 10 mM 1423 versus
DMSO; **p < 0.01 25 mM 1423 versus DMSO; ###,
at n = 12 and 25 mM at n = 7.
(C) CCG-1423 inhibits MICAL-2 enzymatic activity.
We monitored MICAL-2 activity using an NADPH
consumption assay. MICAL-2 was incubated
with either 5 mM CCG-1423 or the inactive control
compound CCG-100594 in the presence of 2 mM
F-actin and 10 mM NADPH. ***p < 0.0005, Stu-
dent’s t test, n = 9.
(D) Enzyme kinetics of MICAL-2. Velocity of
recombinant MICAL-2 activity as a function of
NADPH and CCG-1423. MICAL-2redoxCH
incubated with DMSO, 0.5 mM or 5 mM CCG-1423.
CCG-1423 inhibits MICAL-2 with a Kiof 1.57 mM.
See also Figure S7.
574 Cell 156, 563–576, January 30, 2014 ª2014 Elsevier Inc.
The pathways that regulate MICAL-2 activity are not known.
MICAL-2 phosphorylation is affected by serum, suggesting
that phosphorylation could alter aspects of MICAL-2 function.
Additionally, because MICAL-2 uses NADPH to induce F-actin
depolymerization, NADPH levels may influence MICAL-2 activ-
ity. Indeed, cellular NADPH levels are markedly altered by onco-
genic mutations and affect cancer cell growth and metastasis
through poorly understood pathways (Dang, 2012). It will be
interesting to determine whether these metabolic alterations in-
fluence MICAL-2 activity and SRF/MRTF-dependent gene
Our findings point to MICAL-2 as a target of CCG-1423, an
inhibitor of SRF/MRTF-A signaling. CCG-1423 was originally
identified in a screen for compounds that inhibit an SRF/
MRTF-A transcriptional reporter (Evelyn et al., 2007). However,
the molecular target of CCG-1423 has been elusive thus far.
We find that CCG-1423 exhibits effects on MRTF-A localization
and SRF/MRTF-A reporter expression that resemble the effects
seen with MICAL-2 inhibition. Our data suggest that the effects
of CCG-1423 are due to inhibition of MICAL-2. Indeed, CCG-
SRF/MRTF-A signaling in cells (Evelyn et al., 2007; Minami et al.,
2012). Importantly, a closely related analog devoid of activity
against SRF/MRTF-A signaling shows negligible binding to
Recent studies have shown that CCG-1423 improves glyce-
mic control in insulin-resistant mice (Jin et al., 2011), prevents
fibrosis (Sakai et al., 2013), and reduces metastatic behavior of
prostate and melanoma cancer cell lines (Evelyn et al., 2010).
Although CCG-1423 has promising effects in preclinical studies,
improvements in the potency of CCG-1423 have been chal-
lenging (Evelyn et al., 2010), in large part because the specific
CCG-1423 target was unknown. Thus, our identification of
MICAL-2 as a target of CCG-1423 will facilitate the development
of novel inhibitors of SRF/MRTF-A-dependent gene transcrip-
tion that function by inhibiting MICAL-2 enzymatic activity.
Cell Culture and Reagents
Primers, shRNA, plasmids, cell lines, and antibodies used in this study are
listed in Tables S1 and S2 and the Extended Experimental Procedures.
TotalRNAwaspreparedusingTRIzol (Invitrogen). WesynthesizedcDNA using
Superscript III (Invitrogen) and performed qRT-PCR utilizing iQ SYBR Green
Supermix (Bio-rad) and the Realplex Mastercycler ep s (Eppendorf). For a
list of primers used see Table S1 and the Extended Experimental Procedures.
Luciferase and MICAL Assays
Activation of SRF/TCF and SRF/MRTF-A-dependent gene transcription was
measured using the pGL4.33 and pGL4.34 reporter plasmids, respectively
(Promega). Luciferase levels were measured using ONE-glo (Promega) and
with a Spectramax L luminometer (Molecular Devices).
Recombinant MICAL-1-, -2- and -3-induced actin depolymerization was
measured by monitoring the loss of fluorescence of pyrene-labeled actin
(Cytoskeleton) as described previously (Hung et al., 2010). MICAL-1, -2
and -3 activity assays were measured by in an NADPH depletion assay as
previously described (Hung et al., 2010). Further details can be found in the
Extended Experimental Procedures.
Wild-type zebrafish(AB/Tu hybrid strain) were maintained as described (West-
erfield, 1993). Transgenic reporter strain tg(myl7:egfp) was obtained from
ZIRC, and tg(myl7:actn3b-egfp) was kindly provided by D. Yelon (UCSD).
Morpholinos were purchased from Genetools. Sequences and experimental
details can be found in the Extended Experimental Procedures and Table S2.
We thank A. Deglincerti and members of the Jaffrey lab for helpful comments
and suggestions. Thiswork was supported byNIHgrants F32AT4340(M.R.L.),
HL111400 (T.E.), and NS56306 (S.R.J.).
Received: December 31, 2012
Revised: September 23, 2013
Accepted: November 12, 2013
Published: January 16, 2014
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