alpha-Skeletal muscle actin mutants causing different congenital myopathies induce similar cytoskeletal defects in cell line cultures.

Drieke Vandamme, Heidi Rommelaere, Ellen Lambert, Davy Waterschoot, Joël Vandekerckhove, Bruno Constantin, Christophe Ampe

► To cite this version:

Drieke Vandamme, Heidi Rommelaere, Ellen Lambert, Davy Waterschoot, Joël Vandekerckhove, et al. .. alpha-Skeletal muscle actin mutants causing different congenital myopathies induce similar cytoskeletal defects in cell line cultures.. Cell Motility and the Cytoskeleton, 2009, 66 (4), pp.179-192.
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# α-Skeletal Muscle Actin Mutants Causing Different Congenital Myopathies Induce Similar Cytoskeletal Defects in Cell Line Cultures

Drieke Vandamme,¹,² Heidi Rommelaere,¹,² Ellen Lambert,¹,²

Davy Waterschoot,¹,² Joël Vandekerckhove,¹,²

Bruno Constantin,³ and Christophe Ampe¹,²*

¹Department for Medical Protein Research, VIB, Gent University, A. Baertsoenkaai 3, Gent, Belgium

²Department of Biochemistry, Faculty of Medicine and Health Sciences, Gent University, A. Baertsoenkaai 3, Gent, Belgium

³Institut de Physiologie et Biologie Cellulaire, UMR CNRS/Université de Poitiers 6187, Pôle Biologie Santé, 86022 Poitiers Cedex, France

Central core disease (CCD), congenital fibre type disproportion (CFTD), and nemaline myopathy (NM) are earlyonset clinically heterogeneous congenital myopathies, characterized by generalized muscle weakness and hypotonia. All three diseases are associated with α-skeletal muscle actin mutations. We biochemically characterized the CCD and CFTD causing actin mutants and show that all mutants fold correctly and are stable. Expression studies in fibroblasts, myoblasts, and myotubes show that these mutants incorporate in filamentous structures. However they do not intercalate between the nascent z-lines in differentiating muscle cell cultures. We also show that the distribution of mitochondria and of the ryanodine receptors, and calcium release properties from ryanodine receptors, are unchanged in myotubes expressing the CCD causing mutants. CFTD causing mutants induce partly similar phenotypes as NM associated ones, such as rods and thickened actin fibers in cell culture. Our results suggest that molecular mechanisms behind CFTD and NM may be partly related. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc.

**Key words:** α-actin mutation; central core disease; congenital fibre type disproportion; nemaline myopathy; muscle cells

Drieke Vandamme and Heidi Rommelaere contributed equally to this work.

Contract grant sponsor: CNRS; contract grant numbers: FWO-G.0133.06 and IWT-51185; Contract grant sponsor: French Ministry for Research; Contract grant sponsor: Faculty of Medicine and Health Sciences; contract grant number: 1.5.080.07.

Heidi Rommelaere's present address is Ablynx NV, Technologiepark 4, 9052 Zwijnaarde, Belgium.

Ellen Lambert's present address is Department of Plant Production, Faculty of Bioscience Engineering, Gent University, Coupure associations 653, Gent, Belgium.

*Correspondence to: Christophe Ampe, Department of Biochemistry, Ghent University, A. Baertsoenkaai 3, B-9000 Gent, Belgium. E-mail: christophe.ampe@ugent.be

Received 5 September 2008; Accepted 5 January 2009

Published online 00 Month 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cm.20340
INTRODUCTION

Central core disease (CCD) and congenital fibre
type disproportion (CFTD) are earlyonset, clinically het-
erogeneous congenital myopathies, characterized by
generalized muscle weakness and hypotonia. CCD is
associated with areas devoid of oxidative enzyme activ-
ity (core lesions) and shows type I fiber predominance.
CFTD is also associated with type I fiber predominance,
but the main hallmark in patients is that the mean diame-
ter of type I fibers (slow twitch) is at least 12% smaller
than of type II fibers (fast twitch) [Brooke, 1973;
Romero et al., 2005].

Ninety-percent of CCD patients have a mutation in the ryanodine I receptor (RyR1), which is a calcium-release channel of the sarcoplasmic reticulum with important functions for Ca²⁺ homeostasis [Zhang et al., 1993]. Next to that, mutations in selenoprotein N-1 (SEPN1) and myosin heavy chain-7 have also been described [Fananapazir et al., 1993; Ferreiro et al., 2002]. SEPN1 mutations are also associated with CFTD in recessive cases [Clarke et al., 2005], but for most of the CFTD cases, the genetic basis is still unknown. Remarkably, some dominant CCD and CFTD cases are associated with mutations in the α-skeletal muscle actin gene (ACTA1) [Kaindl et al., 2004; Laing et al., 2004].

Another congenital myopathy resulting from muta-
tions in α-skeletal muscle actin (further referred to as α-
actin) is nemaline myopathy (NM). This disease is char-
acterized by muscle weakness and hypotonia as well, but
is defined by the presence of nemaline bodies or rods in
muscle biopsies of patients. Over 100 ACTA1 mutations
leading to NM have been identified until now and they
account for about 20% of the NM cases [Wallgren-Pet-
tersson and Laing, 2006]. The other genetic loci associ-
ated with NM are also coding for components of skeletal
muscle thin filaments (e.g. troponin and tropomyosin, for
a review see [Clarkson et al., 2004]. The impact of this
disease on the patients can be very different, the types
range from mild, with minor muscle weakness and long-
time survival, to severe, with lethality soon after birth.
The clinical severity correlates poorly with the muscle
pathology of the patients [Ryan et al., 2003] of which
biopsies show various phenotypes. Based on this NM
caused by ACTA1 mutations is subdivided in classes
[Sparrow et al., 2003]; intranuclear rod myopathy is
characterized by intranuclear inclusions and NM by sar-
coplasmic nemaline bodies. Another muscle disease:
actin myopathy is distinguished by thin filamentous
inclusions in areas which normally would be part of the
myofibrillar lattice, but no nemaline bodies are found in
muscle biopsies. However, more than one phenotype can
be caused by the same ACTA1 mutation [Nowak et al.,
1999].

It is not surprising that mutations in α-actin are
causing diseases, since actin is an essential protein in
muscle contraction. It also matures via a special folding
pathway, involving the chaperones prefoldin and cyto-
solic chaperone containing TCP-1 (CCT). CCT assists in
the folding of actin, in an ATP-dependent manner and
releases it as an active monomer [Gao et al., 1992; Vain-
berg et al., 1998; Neirynck et al., 2006]. In addition actin
has an absolute requirement for ATP to remain stable
[Estes et al., 1992]. Once folded, actin can polymerize
into filaments, which in muscle form the thin filaments
that interact with several (muscle specific) actin-binding
proteins e.g. α-actinin, troponin, and myosin, thereby
contributing to contractile force generation [Gordon
et al., 2000].

In a previous analysis of 19 NM causing mutants of α-actin, we found that the mutations can lead to three different biochemical defects in actin: folding defects, defective ATP-binding resulting in unstable proteins and reduced copolymerisation capacity [Costa et al., 2004]. Additionally, we could reproduce the typical hallmarks of NM, rods and aggregates, in cultured fibroblasts. Here, we conducted similar assays with the α-actin mutants D1Y and E334A, associated with CCD, and L221P, D292V, and P332S, associated with CFTD. We show that all mutants fold correctly and are stable. Expression in cultured fibroblasts and myoblasts shows they are all capable of integrating in nonmuscle specific actin filaments, yet they behave aberrant in myotubes. Additionally, we observed similar cellular phenotypes as are induced by NM associated α-actin mutants, when expressed in fibroblasts [Costa et al., 2004], or in muscle cells [Bathe et al., 2007] (Rommelaere et al., in preparation). These data show that these diseases, which are very different at the histological level, may have related or common molecular causes.

MATERIALS AND METHODS

Construction and Biochemical Analysis of
the α-Actin CCD and CFTD Causing Mutants

ACTA1 mutations were made with the QuikChange
site directed mutagenesis kit (Stratagene) according to
the manufacturer's instructions, using the human ACTA1
cDNA sequence in pcDNA3.1 (Invitrogen) as a template
and appropriate primers. N-terminal myc-tagged wild
type actin and actin mutants were made by PCR using
wild type actin, respectively the actin mutants in the
pcDNA3.1 vector as template, a 5' primer containing the
myc-sequence preceded by a HindIII site and a 3' primer
containing an XbaI site. These fragments were ligated
into HindIII-XbaI digested pcDNA3.1. Constructs were
sequenced to verify the complete ACTA1 coding
sequence and correct introduction of the desired mutation. Expression of actin mutants, band shift assays with ABPs, copolymerization assays were performed as described in [Costa et al., 2004; Rommelaere et al., 2004]. Briefly, α-skeletal muscle actins were expressed as ³⁵S-labeled proteins by in vitro translation reactions in reticulocyte lysates (MP Biochemicals). Reaction products were analysed on denaturing tricine gels [Schagger and von Jagow, 1987] and on nondenaturing polyacrylamide gels [Safer, 1989] with ATP, followed by autoradiography. To monitor cyclase associated protein (CAP) binding, ATP was omitted from the native gels. The amount of ³⁵S-actin bound to CAP was quantified by phosphor imaging (Typhoon 9200 variable mode imager; Amersham Biosciences) and the Image Quant software package. As a reference the folding compromised NM mutant L94P and unstable NM mutant G182D were included. For the band-shift assays, a solution of actin binding proteins (ABP) was added. Final concentration of each ABP was 8 µM for DNaseI, 4,8 µM for Vitamin D binding protein (VDBP). Binding capacity was assessed qualitatively; reduced binding means only a part of the population of the mutant molecules shifted, and part remained unshifted.

## Cell Culture and Transfection

NIH3T3 fibroblasts were grown on glass coverslips, in Dulbecco's modified Eagle medium (DMEM) (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and 1% antibiotics (penicillin-G, 100 µg/ml and streptomycin, 100 µg/ml, Invitrogen). After 24 h cells were transfected with lipofectamin 2000 (Invitrogen), according to manufacturer's protocol. Coverslips were mounted for immunofluorescence after 24 h of transfection. Sol8 myogenic cells [Mulle et al., 1988] were seeded in 12 well plates containing thermanox coverslips (Nunc). Myoblasts were grown in DMEM/ HamF-12 medium (Invitrogen) (50:50) supplemented with 10% fetal calf serum (Invitrogen), 1% L-glutamine, and 1% antibiotics (penicillin-G, 100 i.u./ml and streptomycin, 100 µg/ml, Invitrogen). After 24 h the cells were transfected using jetPEI (Qbiogene) or nucleofected, using cell line nucleofector kit V (Amaxa biosystems) with the pcDNA3.1 vectors encoding N-terminally myc-tagged α-skeletal muscle actin (wild type or mutant). To study transfected myoblasts, coverslips were mounted for immunofluorescence after 48 h of transfection. To promote fusion (F) of myoblasts into myotubes, the growth medium was replaced by a differentiation medium (DMEM supplemented with 2% heat-inactivated horse serum (Invitrogen), 10 µg/ml insulin (Sigma), 1% L-glutamine and 1% antibiotics, following 2-3 days of proliferation (37°C, 5% CO₂, water saturated air) to

around 80% of confluence. Myotubes were immunostained on day F + 4 or F + 5.

## Immunological Staining

The cultured cells were fixed in TBS (20 mM Tris base, 154 mM NaCl, 2 mM EGTA, 2 mM MgCl₂, pH 7.5)/4% paraformaldehyde and permeabilized with TBS/ 0.5% Triton X-100. Samples were then incubated for 1 h with primary antibodies in TBS/1%BSA. To visualise the mutant actins we used a polyclonal anti-myc antibody (Abcam) or a mouse anti-myc antibody (Cell Signalling technologies). To visualise z-bodies we used a monoclonal antibody against α-actinin (Sigma), for visualisation of mitochondria a mouse anti-cytochrome C antibody (invitrogen), a mouse anti-apoptosis inducing factor (AIF) antibody (Santa Cruz) or rabbit anti-endonuclease G (Endo G) antibody (Abcam). Ryanodine receptors were visualized with a goat polyclonal anti RyR1 antibody (Sigma). After washing in TBS, the cells were incubated for 30 min in TBS/1% BSA with alexa 488-conjugated goat anti-mouse, alexa 594-conjugated anti-rabbit or alexa 594-donkey anti-goat secondary antibodies, or alexa 488-phalloidin (Molecular Probes). Samples were mounted using Vectashield mounting medium (Vector).

## Cytofluorescence Analysis

The immunolabelled samples were examined using an Olympus IX71 epifluorescence microscope and images were acquired using a cooled Spot Camera (Diagnostic Instruments) and Analysis software (Soft Imaging Systems). Confocal images were acquired by means of confocal laser scanning microscopy using an Olympus confocal system equipped with an argon-krypton gas laser. The excitation wavelength of 488 and 543 nm were respectively used for acquisition of images in green channel (520 nm) and in red channel (600 nm). For statistics of stress fibre incorporation and diffuse cytoplasmic myc-actin staining, pictures were visually inspected and blindly scored by a second person.

## Ratiometric Ca²⁺ Measurement

Myotubes, plated on glass coverslips, were briefly rinsed with a standard external solution (1.8 mM Ca²⁺ solution) (130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 10 mM Hepes, 5.6 mM D-glucose, pH 7.4 with NaOH), incubated for 30 min at room temperature and then 15 min at 37°C in the same solution supplemented with 3 µM (final concentration) Fura-2 AM (FluoProbes). After loading, cells were washed with standard external solution (1.8 mM Ca²⁺ solution), before measurement of calcium responses. Interrogated cells were perfused by means of a home-made gravity
microperfusion device. Fura-2 loaded cells were excited by alternating 340/380 nm light with a CAIRN monochromator (Cairn Research Limited) and emission fluorescence was monitored at 510 nm using a CCD camera (Photonic Science Limited) coupled to an Olympus IX70

inverted microscope (× 40 water immersion fluorescence objective). The variation of fluorescence ratio (340/380) was recorded with the Imaging Workbench 4.0 (IW 4.0) software (Indec BioSystems).

<figure><img src="image_2.png" /></figure>

b. -ATP

<figure><img src="image_3.png" /></figure>

+ATP

<figure><img src="image_4.png" /></figure>

RESULTS

CCD and CFTD Causing α-Actin Mutants are Correctly Folded and Stable

In this study, we investigated α-actin mutants causing either CCD (α-actin D1Y and E334A) or CFTD (α-actin L221P, D292V, and P332S). All five α-actin mutations lie on the surface of the actin monomer, but do not cluster together (Fig. 1a, [Kabsch et al., 1990]). Using similar approaches as in our previous studies on a set of α-actin mutants causing NM [Costa et al., 2004; Rommelaere et al., 2003, 2004], we monitored the folding capacity and stability of these mutant actin molecules. Analysis on native gels shows that they all fold properly as none of the mutants significantly accumulates on CCT (Fig. 1b). Additionally, in the absence of ATP in the native gels, none of the mutants shows an increased binding to CAP, compared to WT α-actin, suggesting ATP-binding is not compromised and that these mutants are stable. They appear correctly folded since they migrate at a position similar to monomeric actin in gels with ATP (Fig. 1b) and can bind to DNAseI and VDBP that contact opposing phases of the actin molecule [Kabsch et al., 1990; Otterbein et al., 2001] (Table I).

CCD and CFTD α-Actin Mutants Show Actin Stress Fiber Incorporation, but Act L221P and P332S also Induce Diffuse Cytoplasmic myc-actin Staining in Fibroblasts

Since expression of myc-tagged α-actin mutants associated with NM in NIH3T3 fibroblasts induces a range of defects in the actin cytoskeleton [Costa et al., 2004], we investigated if expression of myc-tagged α-actin mutants causing CCD and CFTD induce similar cytoskeletal defects. Therefore, we used fluorescent phalloidin to probe for actin filaments and to test for integration of myc-tagged α-actin mutants into cellular

Fig. 1. (a) Location of the studied α-actin mutations on the 3-Dimensional representation of the actin molecule (Kabsch et al., 1990). Mutants in blue are causing CFTD, mutants in red are causing CCD. I–IV indicate the actin subdomains. ATP is in yellow. The PDB structure 1ATN was displayed by ViewerLite. (b) Native gel analysis of the CCD and CFTD causing α-actin mutants. Autoradiograms of native gel analysis in the absence or presence of ATP of ³⁵S-labeled α-actin and α-actin mutants produced in in vitro transcription translation reactions in reticulocyte lysate, which endogenously contains CAP (McCormack et al., 2001; Neirynck et al., 2004) and prefoldin and CCT (Gao et al., 1992; Rommelaere et al., 1993; Vainberg et al., 1998).
<table><caption>TABLE I. Summary of the Biochemical Characterization of Seven Myopathy Causing α-Skeletal Muscle Actin Mutants</caption><thead><tr><th>Mutant</th><th>Phenotype</th><th>Folding</th><th>CAP</th><th>ABP</th><th>Copol</th><th>In actin structure</th></tr></thead><tbody><tr><td>WT</td><td></td><td>+</td><td>+</td><td>+</td><td>++</td><td></td></tr><tr><td>D1Y</td><td>CCD</td><td>+</td><td>+</td><td>+</td><td>++</td><td>N-terminus</td></tr><tr><td>L221P</td><td>CFTD†</td><td>+</td><td>+</td><td>+</td><td>++</td><td>Surface</td></tr><tr><td>D292V</td><td>CFTD†</td><td>+</td><td>+</td><td>+</td><td>++</td><td>Surface</td></tr><tr><td>P332S</td><td>CFTD</td><td>+</td><td>+</td><td>+</td><td>+</td><td>Surface</td></tr><tr><td>E334A</td><td>CCD</td><td>+</td><td>+</td><td>+</td><td>++</td><td>Surface</td></tr></tbody></table>

The properties investigated here are folding, binding to cyclase associated protein (CAP), binding to the actin binding proteins (ABP), DNAseI, and Vitamin D binding protein in band shift assays and copolymerisation with actin in cells (copol). The position in the actin structure according to Kabsch et al. (Kabsch et al., 1990) is also indicated, in addition to the patient's phenotype as presented in the reference.

† = lethal, CCD = Central core disease, CFTD = congenital fibre type disproportion.

F-actin containing structures. We use NIH3T3 fibroblasts because these cells show a pronounced staining of F-actin structures with fluorescent phalloidin. We did not observe many differences between WT and mutant α-actin with respect to incorporation in actin fibers. All CCD and CFTD causing α-actin mutants are able to incorporate in stress fibers in the majority of NIH3T3 fibroblasts (Figs. 2a and 2b and Table II). Intriguingly, next to proper incorporation in filamentous structures we also observed a diffuse cytoplasmic myc-actin staining in some of the mutant transfected cells. We counted these cells, to rule out the effect of different expression levels of the mutant, and noticed that the majority of the fibroblasts expressing the CFTD causing mutants α-actin L221P and P332S (resp. 90 and 85%) display this phenotype (Fig. 2b), whereas the other mutant and WT-expressing cells were similar. Additionally, we monitored the behavior of the mutants in the same way in myoblasts, which are physiologically more relevant. Myc-tagged WT α-actin incorporates into the actin cytoskeleton of myoblasts and myc staining of L221P, D292V, and P332S is comparable (Fig. 2c).

## Two CFTD Associated α-Actin Mutants D292V and L221P Induce Nemaline Rods and Aggregates in Fibroblasts

Two α-actin mutants induce phenotypes that are typical for NM. α-actin D292V induces long thin rods (Fig. 3) in a significant part of the transfected fibroblast population. These rods do not stain with phalloidin and are in a few cases intra- or perinuclear, but in most cases they are present in the periphery of cells. In addition, this mutant also induces cytoplasmic aggregate formation (Fig. 3). Similar phalloidin negative aggregates and rods are also present in fibroblasts expressing α-actin L221P, but only in a minority of the population (data not

shown). In myoblasts, however, these mutants did not induce these phenotypes (Fig. 2c).

## CCD and CFTD Causing α-Actin Mutants Exhibit Disturbed Localization in Nascent Myofilaments

To monitor the behavior of the CCD and CFTD causing α-actin mutant in differentiating muscle cells, we transfected myoblasts with the α-actin mutants and allowed them to fuse into myotubes during 5 days. The myotubes can be identified by their elongated shape and the presence of multiple nuclei. They contain premyofibrils, which have α-actinin rich z-bodies fused to short actin filaments. Later on, these z-bodies move towards the centre of the myotube, align and fuse with formation of the z-disc [Sanger et al., 2002; Lloyd et al., 2004]. Confocal images reveal the expected striated α-actinin staining or troponin-T pattern for WT actin expressing cells (Fig. 4a). We could see the α-actinin striations in all cells expressing mutants, yet α-actin D1Y, E334A (CFTD), and P332S (CCD) show few or no intercalation with the α-actinin striation in the differentiating sarcomers (Figs. 4b and 4c). Expression of L221P and D292V (CFTD) resulted in another phenotype; thick curved actin fibers, that were only partly decorated with α-actinin (Fig. 4c). This phenotype was also observed for NM mutants D154N (Fig. 4d) and G15R [Bathe et al., 2007]. Strikingly, expression of α-actin L221P and D292V induced α-actin rod formation, which contained α-actinin.

## Localization of Mitochondria and Ryanodine Receptor 1 Appears Normal in Myotubes Expressing CCD Causing α-Actin Mutants

The major histological phenotype of CCD in patient muscle biopsies is the existence of areas devoid of oxidative enzyme activity. The lack of oxidative enzyme activity suggests that in CCD patients, the location or activity of mitochondria in muscle cells may be affected.

We therefore investigated the distribution of mitochondria in myotubes, expressing CCD causing α-actin mutants D1Y and E334A. We performed stainings for cytochrome c, a marker for mitochondria, on differentiated myotubes, in combination with myc-staining to identify the transfected cells (Fig. 5). Differentiating muscle cells increase the number of mitochondria in order to cope with the high energy demands [Rochard et al., 2000; Hood et al., 2006] and this is reflected in a much more abundant dot-like cytoplasmic cytochrome C staining in myotubes, compared to myoblasts or fibroblasts. However, we observed no differences in staining pattern between transfected WT and mutant α-actin expressing myotubes. These results were confirmed by stainings with two other mitochondrial proteins, endonuclease G (Fig. 5) and AIF (not shown). In addition we did not observe dif-
a. fibroblast

CFTD

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

CCD

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

Fig. 2. α-actin mutants causing CCD and CFTD show normal actin stress fiber incorporation in fibroblasts and myoblasts, but two CFTD mutants show in addition diffuse cytoplasmic myc-staining. (a) Myc staining of NIH3T3 fibroblasts expressing CFTD causing myc-tagged mutants α-act L221P, D292V, and P332S and NIH3T3 fibroblasts expressing CCD causing myc-tagged mutants α-act D1Y or α-act E334A. (b) Graphs of counts of fibroblasts which have myc-actin

b. % cells with incorporation of stress fibers

<figure><img src="image_10.png" /></figure>

c. myoblast

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

incorporation in stress fibers, and fibroblasts which show diffuse cytoplasmic myc staining, *n* = 20 cells, error bars show SEM between two independent counts of the same experiment by different persons. (c) Myoblast expressing WT myc-α-actin, and myc-tagged act L221P, D292V, and P332S. In (a) and (c) Myc is in red and phalloidin in green on all merged pictures. Scale bars are 20 µm.

fuse cytoplasmic cytochrome C, endo G, or AIF staining, which indicates that mitochondria are not desintegrating nor losing their inner membrane potential.

Most CCD patients have mutations in the RyR1 resulting in some cases to a modification of the Ca²⁺ release complex arrangement with depletion of RyR1 from the cores [Herasse et al., 2007]. Since in photoreceptor cells RyR1 channels seem to be positioned on specific sites of the endoplasmic reticulum via interaction with the F-actin system [Baumann, 2001; Wang

et al., 2002] we investigated if the expressed CCD causing α-actin mutants D1Y and E334A possibly induce a mislocalization of this calcium channel on the sarcoplasmic reticulum.

Therefore we localized the RYRs in myotubes expressing the CCD causing α-actin mutants by immunofluorescence staining of the Ryanodine Receptor (Fig. 6). We observed no differences in staining pattern between untransfected, WT, and mutant α-actin expressing myotubes. RyR1 staining appears as intracytoplasmic.
<table><caption>TABLE II. Summary of the Cellular Phenotypes we Observed Upon Expression of Seven Myopathy Causing α-Skeletal Muscle Actin Mutants</caption><thead><tr><th>Mutant</th><th>Phenotype</th><th>Biochemical phenotype</th><th>Cell line</th><th>Cellular phenotype</th></tr></thead><tbody><tr><td rowspan="3">WT</td><td rowspan="3">–</td><td rowspan="3">Normal</td><td>Fibroblast</td><td>Normal</td></tr><tr><td>Myocyte</td><td>Normal</td></tr><tr><td>Myotube</td><td>Normal</td></tr><tr><td rowspan="3">D1Y</td><td rowspan="3">CCD</td><td rowspan="3">Normal</td><td>Fibroblast</td><td>Normal</td></tr><tr><td>Myocyte</td><td>Normal</td></tr><tr><td>Myotube</td><td>No incorporation in nascent sarcomeres</td></tr><tr><td rowspan="3">E334A</td><td rowspan="3">CCD</td><td rowspan="3">Normal</td><td>Fibroblast</td><td>Normal</td></tr><tr><td>Myocyte</td><td>Normal</td></tr><tr><td>Myotube</td><td>No incorporation in nascent sarcomeres</td></tr><tr><td rowspan="6">L221P</td><td rowspan="6">CFTD<sup>†</sup></td><td rowspan="6">Normal</td><td>Fibroblast</td><td>Phalloidin negative nemaline rods and aggregates in the cytoplasm diffuse cytoplasmic myc-actin staining</td></tr><tr><td>Myocyte</td><td>Normal</td></tr><tr><td>Myotube</td><td>Thick curved actin fibers</td></tr><tr><td>Fibroblast</td><td>Phalloidin negative nemaline rods and aggregates in the cytoplasm</td></tr><tr><td>Myocyte</td><td>Normal</td></tr><tr><td>Myotube</td><td>Thick curved actin fibers</td></tr><tr><td rowspan="3">D292V</td><td rowspan="3">CFTD<sup>†</sup></td><td rowspan="3">Normal</td><td>Fibroblast</td><td>Diffuse cytoplasmic myc-actin staining</td></tr><tr><td>Myocyte</td><td>Normal</td></tr><tr><td>Myotube</td><td>No incorporation in nascent sarcomeres</td></tr><tr><td rowspan="3">P332S</td><td rowspan="3">CFTD</td><td rowspan="3">Normal</td><td>Fibroblast</td><td>Normal</td></tr><tr><td>Myocyte</td><td>Normal</td></tr><tr><td>Myotube</td><td>No incorporation in nascent sarcomeres</td></tr></tbody></table>

mic dots, sometimes aligned along the longitudinal axe (Figs. 6a–6d). This distribution of RyR clusters is classical in developing myotubes and is consistent with the location of the sarcoplasmic reticulum along the myofibrils [Castellani et al., 1996; Flucher et al., 1993, 1994].

Variations in intracellular free $Ca^{2+}$ concentration were measured in myotubes loaded with the fluorescent calcium probe Fura-2. Myotubes were stimulated with caffeine in order to directly activate the RyR and to analyze calcium release responses in cells expressing α-actin mutants. The expression of both D1Y and E334A α-actin mutants did not prevent the calcium response to caffeine stimulation, and the calcium signals were not changed compared to control cells (Figs. 6e and 6f). This showed that calcium release properties and RyR1 functions are not altered by the presence of the α-actin mutants causing CCD.

## DISCUSSION

### CFTD and CCD Causing α-Actin Mutants are Correctly Folded and Stable

In this study we investigated two α-actin mutants causing CCD and three α-actin mutants implicated in CFTD. Using our in vitro folding and band-shift assay, we showed that all five mutants fold correctly, are stable and bind to the ABPs DNAseI and VDBP. For the CFTD mutants, our biochemical data complement the results reported by Clarke et al. [Clarke et al., 2007]. These authors showed, using immunoprecipitation, that mutants α-actin L221P, D292V, P332S incorporate in filaments and that α-actin D292V and α-actin P332S

function in an in vitro motility assay [Clarke et al., 2007]. This is consistent with our observation that the mutants are folded to a functional conformation, given they interact with the ABPs tested. However for α-actin D292V the interaction with tropomyosin seems to be particularly disrupted [Clarke et al., 2007].

### CCD Causing α-Actin Mutants Incorporate Normally in Actin Structures and do not Affect the Distribution of Mitochondria or Ryanodine Receptors in Cultured Myotubes

The two α-actin mutants associated with CCD incorporate normally in nonmuscle specific actin structures in cultured fibroblasts and undifferentiated Sol8 myoblasts. On the other hand, we observed no or little incorporation of these mutants between the nascent z-lines in differentiating myotubes, suggesting these mutants are not included in muscle specific structures, i.e. those forming sarcomeres. So the structurally defects of the cytoskeleton are clearly muscle specific. A summary of the cellular phenotypes we observed for the mutants causing CCD is given in Table II.

The molecular mechanism behind core myopathies has not yet been unraveled. In patients, the CCD causing α-actin mutants induce core lesions, which differ in size, number and appearance. The muscle biopsies of the family affected with the D1Y mutation show broad, unstructured, central cores that do not run along the entire long axis of the muscle, which are typical CCD cores, whereas those of the family expressing the E334A α-actin mutant show minicores, and a single muscle fiber
<figure><img src="image_13.png" /><figcaption>Fig. 3. CFTD associated α-actin mutant D292V induces nemaline rods (upper and middle panel) and displays aggregate formation (lower panel) in NIH 3T3 fibroblasts. Arrows indicate rods.</figcaption></figure>

contains multiple cores [Kaindl et al., 2004]. However, by performing cytochrome c, endonuclease G, or AIF stainings, we could not detect defects in the distribution of mitochondria in early myotubes expressing the CCD causing α-actin mutants. The stainings further indicate that mitochondria are not disintegrating, nor losing their inner membrane potential at this stage of differentiation. We cannot rule out that formation of cores can only be observed in more mature muscle cells, as this may be a long term physiopathologic process or an adaptation to more general metabolic alterations.

In 90% of the patients with CCD, mutations in RyR1 occur. This is a sarcoplasmic reticulum channel,

which mediates Ca²⁺ release during excitation-contraction coupling. Two models exist to explain the consequences of these mutations. The first model predicts that the RyR1 mutations lead to a sustained calcium leak. The second model suggests a loss of functional connection between RyR1 and the dihydropyridine receptor DHPR-α1 in the T-tubuli, leading to excitation-contraction uncoupling [Herasse et al., 2007]. Our immunostainings on RyR1 showed no specific phenotypes for the CCD causing α-actin mutants in myotubes after 5 days of differentiation. The localization of RyRs clusters along the longitudinal axis of the cell is consistent with the classical observed distribution in differentiating myo-

<caption>Fig. 4. In myotubes, CCD causing mutants do not incorporate between nascent Z-lines in differentiating myotubes, CFTD causing mutants induce curved thickened actin fibers. (a) myc-staining and troponin-T staining or α-actinin staining of Sol8 myotubes expressing WT α-actin, (b–d) α-actinin staining and myc-staining of Sol8 myotubes expressing α-actin D1Y and E334A, (e) α-actin L221P, D292V and P332S, and (f) α-actin D154N. Scale bars are 20 µm. Underneath the pictures of α-actin WT, D1Y, L221P, and D292V expressing cells are zoomed-in pictures of the yellow square regions.</caption>
<figure><img src="image_14.png" /><figcaption>Figure 4.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>Fig. 5. Localization of mitochondria appears normal in myotubes expressing CCD causing α-actin mutants. (a–c) Myc-staining of Sol8 myotubes expressing WT α-actin (a), α-actin D1Y (b), and α-actin E334A (c). (a'–c') Corresponding stainings of mitochondria with an anti-cytochrome C antibody. (a''–c'') Stainings of mitochondria of myc-a-actin expressing myotubes with an anti-endonuclease G antibody (c''') and a corresponding myc-actin staining of (c'''). Scale bars are 20 µm.</figcaption></figure>

tubes and is not markedly different between untrans-
fected, WT or mutant expressing myotubes [Yuan et al.,
1991]. The expression of α-actin mutants causing CCD
did also not alter the calcium release properties from
RyR1 in myotubes suggesting that there are no direct
relations between physiopathologic pathways due to
RyR1 mutations and alterations due to α-actin mutants.

In Cultured Cells Some CFTD Causing α-Actin
Mutants Display Partly Similar Phenotypes as NM
Causing Mutants Suggesting Molecular
Mechanisms of CFTD and NM may be
Partly Related

Expression of the N-terminally myc-tagged CFTD
causing α-actin mutants in cultured fibroblasts and undif-
ferentiated Sol8 myoblasts reveals that all the mutants
are able to incorporate in nonmuscle specific actin fila-

mentous structures. However, respectively, 90 and 85%
of fibroblasts expressing α-actin L221P and P332S show
additionally a diffuse cytoplasmic localization of the
myc-tagged actin, suggesting these mutants do not fully
incorporate in actin filaments.

A phenotype which is similar to our previous
observations expressing NM causing actin mutants, is
that mutants α-actin D292V and, to a lesser extent, α-
actin L221P induce aggregate and/or rod formation
when expressed in fibroblasts. A small amount of the
phalloidin negative rods in α-actin D292V expressing
fibroblasts appears intranuclear or perinuclear, but the
majority of the rods is seen in long cell extensions of the
spread cells. These rods in the cell extensions are spe-
cific for this mutant and, to our knowledge, not earlier
described for NM causing α-actin mutants. The lack of
phalloidin staining of the rods does not indicate they
<figure><img src="image_16.png" /><figcaption>Fig. 6. Localization of RyR1 and calcium release properties were normal in myotubes expressing CCD causing α-actin mutants. (a–d) Myc stainings and corresponding RyR1 stainings of untransfected Sol8 myotubes (a) or Sol8 myotubes expressing WT α-actin (b), α-actin D1Y (c), and α-actin E334A (d). Scale bars are 20 µm. (e) Representative examples of intracellular calcium transients in Sol8 cells induced by 10 mM caffeine in control myotubes or in myotubes trans-</figcaption></figure>

fected with the α-actin mutant D1Y or E334A. Calcium transients were recorded with the fluorescent probe Fura-2/AM in F + 4 myotubes. Bold lines correspond to the superfusion of caffeine. (f) Histogram of calcium transient parameters induced by 10 mM caffeine. Histograms correspond to variations in amplitude of Fura-2 fluorescence ratio. The bar graphs correspond to the mean ± sem. *n* corresponds to the number of experiments.
contain monomeric actin, but it may mean that the filaments in the rods are decorated with an actin binding protein that occludes the phalloidin binding sites. For instance it is well known that cofilin decorated filaments are phalloidin negative [Minamide et al., 2000]. A summary of the cellular phenotypes we observed for the CFTD causing α-actin mutants is given in Table II.

The fact that CFTD causing α-actin mutants L221P and D292V induce the main characteristics of NM in fibroblasts, i.e. actin aggregates, and in the case of α-actin D292V rods, is perhaps surprising. However, Clarke et al. made a similar observation for C-terminally GFP-tagged α-actin D292V, in C2C12 myoblasts [Clarke et al., 2007], but not for α-actin L221P. This difference with our observations can be due to the use of different tags as we previously described that the type and position of the tags can influence observed phenotypes [Rommelaere et al., 2003]. Alternatively the difference may be due to the used cell lines, since we recently observed that, for certain NM causing α-actin mutants, rod formation is more abundant in fibroblasts [Costa et al., 2004] than in muscle cells (Rommelaere et al., in preparation). Additionally, in Sol8 myotubes, some of these mutants induce the formation of thickened actin fibers, which are sometimes curved. These cells also have no striated α-actinin pattern suggesting sarcomere formation is also affected. Incidentally such fibers also appear in some differentiated Sol8 myotubes expressing NM causing α-actin mutants, e.g. G15R [Bathe et al., 2007] and D154N (Fig. 4d). Nevertheless the observation that some CFTD causing mutants display similar phenotypes as NM causing α-actin mutants suggests that in some cases the underlying molecular mechanisms of CFTD and NM may be (partly) common.

## A Relation Between NM, CCD and CFTD?

Our observation of similar phenotypes of CFTD and NM associated mutants in cultured cells is in line with the fact that some α-actin mutations associated with NM affect size and distribution of muscle fiber types, which is characteristic for CFTD [Goebel et al., 1997; Schroder et al., 2004; Hutchinson et al., 2006]. On the other hand the coexistence of rods and core lesions in patients suffering from NM caused by α-actin mutations has been described [Jungbluth et al., 2001]. This suggests that CCD and NM may also share partly common dysfunctions of the actin cytoskeleton. Mutations in the *cofilin-2* gene also induce a myopathy where both, nemaline bodies and minicores, are observed [Agrawal et al., 2004]. Additionally patients suffering from CCD caused by mutations in the RyR1 can present nemaline rods in their muscle biopsies [Monnier et al., 2000; Scacheri et al., 2000]. It is also striking that there is one case where CFTD and minicores have been found in one and

the same family. This suggests a common, or similar, mechanism behind CCD and CFTD pathogenesis [Jongpiputvanich et al., 1995]. Taken together, it seems that NM, CFTD, and CCD may have related defects. This leads us to the question: what could be the underlying molecular mechanism of NM, CFTD, and CCD associated with α-actin mutations? For the α-act D292V CFTD causing mutant, Clarke et al. suggested a defect in tropomyosin binding [Clarke et al., 2007]. This links to Ca²⁺ sensitivity as the tropomyosin function is under control of this ion via the troponin complex [Gordon et al., 2000]. Although the possibility of altered Ca²⁺ regulation has not been studied in NM, it has been earlier suggested that muscle weakness can be caused by a reduced Ca²⁺ sensitivity of the contractile apparatus in this disease [Michele et al., 1999; Gommans et al., 2003] and the idea is reinforced by the fact that NM can also be caused by mutations in tropomyosin and troponin [Clarkson et al., 2004]. CCD can of course be considered as a disease where Ca²⁺ homeostasis is affected, given that the majority of CCD cases are caused by mutations in RyR1. Although expression of both D1Y and E334A α-actin mutants did not alter the calcium response of cells, we cannot rule out a reduced Ca²⁺ sensitivity of the contractile apparatus here as well.

## ACKNOWLEDGMENTS

We thank Davina Tondeleir for the fibroblast counts.

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<figure><img src="image_17.png" /></figure>

Author Proof