T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $15.00
The Journal of Cell Biology, Vol. 176, No. 7, March 26, 2007 965–977
Transmission of force from skeletal muscle myofi brils to the
ECM is thought to be mediated largely by intermediate fi la-
ments (IFs). Several IF proteins are expressed in muscle, in-
cluding vimentin, nestin, synemin, syncoilin, lamins, cytokeratins,
and desmin, the major muscle-specifi c IF protein (for review
see Paulin and Li, 2004). The desmin IF network forms a 3D
scaffold surrounding Z-disks, extends from one Z-disk to
the next, and fi nally connects the contractile apparatus to
the plasma membrane at the level of Z-disks but also to organ-
elles such as mitochondria and the nucleus (for review see
Capetanaki, 2002). The dystrophin–glycoprotein complex (DGC)
has been implicated in mediating the IF-ECM link through syn-
coilin and synemin, which interact with desmin and bind to the
DGC protein α-dystrobrevin (Bellin et al., 2001; Newey et al.,
2001; Poon et al., 2002). The DGC is a large protein complex
consisting of integral membrane proteins (α- and β-dystro-
glycan [βDG], α-, β-, γ-, and δ-sarcoglycan, and sarcospan), the
>425-kD large actin-binding protein dystrophin, and dystrophin-
associated proteins such as the syntrophins and α-dystrobrevin.
Components of the DGC are part of the costameric protein
network that, among other proteins, also includes integrins,
vinculin, talin, α-actinin, and caveolin-3. Costameres are sub-
sarcolemmal protein assemblies that circumferentially align in
register with the Z-disks of peripheral myofi brils (for reviews
see Spence et al., 2002; Ervasti, 2003); some authors include
elements located above M-lines and in longitudinal lines in this
term (Bloch et al., 2002).
Muscular dystrophies (MDs) are a group of clinically and
genetically heterogeneous diseases characterized by progres-
sive muscle wasting. Lack of dystrophin leads to the most com-
mon form, Duchenne MD (DMD), but MD can also result from
mutations in genes whose products are not known to associate
with the DGC (Burton and Davies, 2002). Most patients with
plectin defects, who mainly suffer from various subtypes of the
skin blistering disease epidermolysis bullosa (Pfendner et al.,
2005), have also been diagnosed with MD, and muscle phenotypes
have been observed in plectin-defi cient mice (Andrä et al., 1997).
Plectin 1f scaffolding at the sarcolemma
of dystrophic (mdx) muscle fi bers through multiple
interactions with β-dystroglycan
Günther A. Rezniczek,1 Patryk Konieczny,1 Branislav Nikolic,1 Siegfried Reipert,1 Doris Schneller,1
Christina Abrahamsberg,1 Kay E. Davies,2 Steve J. Winder,3 and Gerhard Wiche1
1Max F. Perutz Laboratories, Department of Molecular Cell Biology, University of Vienna, A-1030 Vienna, Austria
2Department of Physiology, Anatomy, and Genetics, Medical Reasearch Council Functional Genetics Unit, Oxford OX1 3QX, England, UK
3Centre for Developmental and Biomedical Genetics, Department of Biomedical Science, University of Sheffi eld, Western Bank, Sheffi eld S10 2TN, England, UK
eight protein isoforms differing only in small N-terminal
sequences (5–180 residues), four of which (plectins 1, 1b,
1d, and 1f) are found at substantial levels in muscle tissue.
Using plectin isoform–specifi c antibodies and isoform ex-
pression constructs, we show the differential regulation of
plectin isoforms during myotube differentiation and their
localization to different compartments of muscle fi bers,
identifying plectins 1 and 1f as sarcolemma-associated
n skeletal muscle, the cytolinker plectin is prominently
expressed at Z-disks and the sarcolemma. Alternative
splicing of plectin transcripts gives rise to more than
isoforms, whereas plectin 1d localizes exclusively to Z-disks.
Coimmunoprecipitation and in vitro binding assays using
recombinant protein fragments revealed the direct bind-
ing of plectin to dystrophin (utrophin) and β-dystroglycan,
the key components of the dystrophin–glycoprotein
complex. We propose a model in which plectin acts as
a universal mediator of desmin intermediate fi lament
anchorage at the sarcolemma and Z-disks. It also explains
the plectin phenotype observed in dystrophic skeletal mus-
cle of mdx mice and Duchenne muscular dystrophy patients.
Correspondence to Gerhard Wiche: firstname.lastname@example.org
Abbreviations used in this paper: ABD, actin-binding domain; βDG, β-dystroglycan;
DGC, dystrophin–glycoprotein complex; DMD, Duchenne MD; EDL, extensor
digitorum longus; IB, immunoblotting; IF, intermediate fi lament; IFM, immuno-
fl uorescence microscopy; IP, immunoprecipitation; MD, muscular dystrophy;
MyHC, myosin heavy chain.
The online version of this article contains supplemental material.
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Supplemental Material can be found at:
JCB • VOLUME 176 • NUMBER 7 • 2007 966
The cytolinker protein plectin is prominently expressed in stri-
ated muscle cells and has been visualized at Z-disks, the sarco-
lemma, and at mitochondria (Wiche et al., 1983; Schröder et al.,
1997; Reipert et al., 1999; Hijikata et al., 2003), but the mole-
cular mechanisms involved in plectin-related muscle disease/
defects are unknown.
Plectin is a large (Mr > 500,000) protein consisting of
N- and C-terminal globular domains separated by an ?200-nm–
long rod. The N-terminal domain contains a multifunctional
actin-binding domain (ABD; Andrä et al., 1998) that is capable of
also interacting with integrin β4 (Rezniczek et al., 1998; Geerts
et al., 1999) and vimentin (Sevcik et al., 2004) and also contains
binding sites for nesprin-3 (Wilhelmsen et al., 2005) and the
nonreceptor tyrosine kinase Fer (Lunter and Wiche, 2002). The
C-terminal domain contains binding sites for IFs (Nikolic et al.,
1996), the γ1 subunit of AMP kinase (Gregor et al., 2006), and
the PKC scaffolding protein RACK1 (Osmanagic-Myers and
Wiche, 2004). Several different plectin isoforms, which are
generated by tissue and cell type–dependent alternative splicing
of transcripts from a single gene with >40 exons, form the basis
for its broad versatility (Fuchs et al., 1999; Rezniczek et al.,
2003). Isoforms with eight alternative N termini have been
identifi ed, and specifi c functions have been linked to distinct
isoforms. Plectin 1a anchors keratin IFs to hemidesmosomes in
basal keratinocytes (Andrä et al., 2003), and a specifi c role in
fi broblast and T cell migration has been demonstrated for plectin 1
(Abrahamsberg et al., 2005).
In skeletal muscle, four isoforms (plectins 1, 1b, 1d, and 1f)
are expressed at considerable levels. In this study, we address
the following issues: Where on the subcellular level are these
plectin isoforms localized in muscle fi bers? What are their
muscle-specifi c (novel) binding partners? Are they differen-
tially regulated during differentiation? What role do they play in
dystrophic muscle, such as that of mdx mice?
Muscle fi ber type–dependent expression
and isoform-specifi c subcellular localization
Plectins 1d, 1f, 1b, and 1, the isoforms most abundantly expressed
in skeletal muscle, show relative mRNA ratios of >10:4:3:1,
respectively (Fuchs et al., 1999). To obtain data about their
expression and localization in skeletal muscle on the protein
level, we isolated the quadriceps, a typical fast-twitch muscle
composed of mainly type 2 fi bers, from 10-wk-old mice and
processed it for immunolabeling. Anti–pan-plectin antiserum
revealed strong subsarcolemmal and moderate sarcoplasmic
staining in cross sections of small diameter fi bers and only faint
sarcoplasmic and sarcolemmal staining in larger diameter fi bers
(Fig. 1 C). On longitudinal sections, Z-disks were stained in
all fi bers, but the signal was much stronger in small diameter
fi bers, where additionally the plasma membrane was stained
(Fig. 1 A). These fi bers, which showed strong autofl uorescence
at 488 nm (Fig. 1, F and H; insets), were positive for myosin
heavy chain (MyHC)–2A (Fig. 1 B; also see E, G, and I), whereas
those with larger diameters were MyHC-2B positive (Fig. 1 K).
Therefore, it appears that in quadriceps, fast 2A fi bers express
plectin at higher levels than type 2B fi bers, as has previously
been reported for type 2 compared with slow type 1 fi bers
(Schröder et al., 1997). Double immunolabeling of plectin 1f
and MyHC-2A on longitudinal sections revealed this plectin iso-
form to be located at Z-disks in 2A fi bers but to be hardly ex-
pressed in 2B fi bers (Fig. 1, D and E; and not depicted). On cross
sections, 2A fi bers showed moderate sarcoplasmic plectin 1f–
specifi c staining as well as irregular and weak staining of the
membrane (Fig. 1, F and G). Staining of longitudinal sections
using a plectin 1–specifi c antiserum revealed this isoform to be
much less abundant, if at all present, at Z-disks. However, a strong
Figure 1. Immunolocalization of plectin isoforms in quadri-
ceps of 10-wk-old mice. Consecutive (except A–C) serial lon-
gitudinal and cross sections were stained with antibodies
(pan-plectin) recognizing all plectin isoforms (A and C) or spe-
cifi cally isoforms 1f (D and F) or 1 (H and J); in addition, anti-
bodies specifi c to MyHC-2A (B, E, G, and I) and -2B (K), all
in combination with Cy5-labeled secondary antibodies, were
used. The boxed area in D (bottom right) is shown enlarged
in the inset. The insets in F and H show autofl uorescence after
excitation at 488 nm recorded in the standard FITC channel.
Bar, 50 μm.
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DIRECT INTERACTION OF PLECTIN WITH β-DYSTROGLYCAN • REZNICZEK ET AL. 967
signal came from sarcolemma-associated structures, primarily
in 2A fi bers (Fig. 1 H). On cross sections, plectin 1–specifi c
signals were detected as irregularly distributed accumulations at
the sarcolemma of 2A but not 2B fi bers (Fig. 1 J).
As we were unsuccessful in generating isoform-specifi c
antibodies directed against plectins 1b and 1d, we ectopically
expressed and visualized GFP fusions of all four full-length
plectin isoforms (1, 1b, 1d, and 1f) in myotubes (Fig. 2, A–D).
Plectin 1 was expressed in a diffuse dotty pattern throughout the
cytosol (Fig. 2 A; see virtual cross sections in insets 1 and 2)
and was concentrated in the vicinity of nuclei. Immunolabeling
with antibodies specifi c for sarcomeric α-actinin, a marker for
Z-disks, revealed that areas positive for α-actinin were com-
pletely devoid of plectin 1 (Fig. 2 A, a and b; see areas marked
by identically positioned arrowheads). Plectin 1b was distrib-
uted throughout the sarcoplasm in a pattern somewhat more
patchy but similar to that observed for plectin 1, also mostly ex-
cluding areas that were positive for α-actinin (Fig. 2 B; a, b, and
cross sections). Plectin 1d was located exclusively at structures
identifi ed as Z-disks (Fig. 2 C; arrowheads in a and b indicate
the same exemplary positions). Contrary to expectations based
on the immunostaining of tissue sections, plectin 1f was found
not to be associated with Z-disks (Fig. 2 D). However, the ob-
served sarcolemma association of this isoform was impressively
confi rmed (Fig. 2 D, virtual cross sections 1–4; a and b show
individual confocal sections as indicated in panel 1). Immuno-
labeling of in vitro–differentiated C2C12 cells with plectin 1– and
1f–specifi c antibodies revealed the same localization of the
native isoforms (unpublished data).
To further investigate the sarcolemma association of plectin,
extensor digitorum longus (EDL) muscle was teased into
sin gle fi bers, which were immunolabeled for plectin and βDG,
a costameric membrane marker (Fig. 2 E). Both proteins co-
localized in costameric structures. Whereas βDG staining
resembled a gridlike pattern (Z-disks and longitudinal lines),
pan-plectin serum revealed prominent Z-disk and perinuclear
localization and only a rare association of plectin with longitu-
dinal lines. Virtual cross and longitudinal sections (Fig. 2 E,
insets 1 and 2) through confocal stacks showed plectin at the
sarcolemma but also extending into the fi bers in regular intervals.
Analysis with isoform-specifi c antibodies showed distinct stain-
ing patterns for plectins 1 and 1f. Whereas plectin 1 was found
in the perinuclear area, at longitudinal lines, and in a dotty
pattern at Z-disks (Fig. 2 F), plectin 1f was strongly expressed at
Figure 2. Expression of full-length plectin isoforms in
differentiated myotubes and immunostaining of teased muscle
fi bers. (A–D) Full-length plectin 1 (A), 1b (B), 1d (C), or 1f (D) ex-
pression plasmids (with C-terminal EGFP; green) were trans-
fected into myoblasts ?12–16 h before differentiation. After
96 h, differentiated myotubes were fi xed with methanol
and processed for immunolabeling using antibodies specifi c
for sarcomeric α-actinin (A–C) or caveolin-3 (D). Secondary
antibodies were Texas red labeled (red), and nuclei were
visualized with Hoechst 33342 (blue). Main panels show
composites of confocal stacks; dotted frames indicate areas
shown in more detail in panels a and b of A–D. Numbered
lines indicate confocal sections shown in correspondingly
labeled insets; virtual cross sections were reconstructed from
confocal stacks using LSM imaging software. Horizontal lines
in insets 1–4 of D indicate the positions of the planes shown in
panels a and b of D. Arrowheads in A and C indicate identi-
cal exemplary positions in panels a and b. (E–H) Teased
fi bers were prepared and processed for IFM as described in
Materials and methods. Primary antibodies used were anti-
serum #123 to plectin (E), anti–plectin 1 (F), anti–plectin 1f
(G and H), monoclonal anti-βDG (E), anti–α-actinin (F and G),
and antidystrophin (H). Plectin-specifi c antibodies were de-
tected with Cy3-conjugated secondary antibodies (green),
and all others were detected with Cy5-conjugated secondary
antibodies (red). Panels a and b show individual colors of the
merged images. The virtual sections shown in insets 1 and 2
in E are from a different fi ber. The line in H indicates the plane
of section shown in inset 1. Bars in A and E apply to A–D and
E–H, respectively; bars in panel b of C and E apply to panels
a and b of A–D and E–H, respectively. Bars, 20 μm.
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JCB • VOLUME 176 • NUMBER 7 • 2007 968
Z-disks and tightly encircled nuclei (Fig. 2 G). Intriguingly, when
the staining patterns for both isoforms are merged, they match
that observed with pan-specifi c plectin antibodies, suggesting
that plectins 1 and 1f are the major sarcolemma-associated
isoforms. Costaining of plectin 1f with dystrophin revealed a
partial colocalization of both proteins (Fig. 2 H). At the sarco-
lemma, plectin 1f was concentrated at Z-disks and extended
into the fi ber, whereas dystrophin was limited to the sarcolemma
but was also found between Z-disks (Fig. 2 H, inset 1).
Coexpression of plectin 1f and dystrophin
during myoblast differentiation
To defi ne the role of plectin in the process of differentiation
from myoblasts to myotubes, we profi led the expression of
plectin and other skeletal muscle proteins (Fig. 3). After 96 h of
differentiation, myoblast cultures had formed myotubes that
started to twitch (unpublished data). During differentiation,
plectin isoform 1 expression peaked at 8–16 h, which is similar
to that of utrophin. Plectin 1f was expressed only later, starting
between 24 and 48 h, and reached plateau levels after 72 h.
Interestingly, dystrophin showed a very similar expression pro-
fi le, whereas βDG was detectable earlier (16 h), and caveolin-3
was not detected before 48 h. Integrin α7B was already ex-
pressed in myoblasts and showed peak levels after ?48 h
Plectin interacts with the EF-ZZ domains
of dystrophin and utrophin
The spatially and temporally coordinated expression of plec-
tin 1f and dystrophin suggested a possible direct interaction of
both proteins. In immunoprecipitation (IP) experiments using
IP lysates (see Materials and methods) from wild-type muscle
tissue, plectin coprecipitated with dystrophin and vice versa
(Fig. 4 A, lanes 5 and 7). From mdx IP lysates, which were
used as negative controls, plectin was not precipitated using
dystrophin antibodies (Fig. 4 A, lanes 6 and 8). Interestingly,
although plectin was expressed at higher levels in mdx com-
pared with wild-type muscles (Fig. 4 A, lanes 1 and 2), less of
it was immunoprecipitated from mdx (Fig. 4 A, lanes 5 and 6;
also see Fig. 6, A and C), indicating a shift of plectin into an
insoluble pool. Utrophin and plectin were coprecipitated from
rat fibroblast lysates (unpublished data). To identify inter-
acting subdomains of the proteins, we immobilized His-tagged
fragments of utrophin, including its N-terminal ABD, the C ter-
minus, and the entire WW-ZZ domain as well as its three sub-
domains WW, EF, and ZZ on nitrocellulose membranes and
overlaid them with various plectin samples (Fig. S1 A, avail-
able at http://www.jcb.org/cgi/content/full/jcb.200604179/DC1;
summarized in Fig. 4 B). Using either purified full-length
plectin or plectin-rich cell lysates, we found plectin bound
to the ABD and WW-ZZ domain of utrophin but not to its
C-terminal part. A recombinant plectin ABD showed similar
specifi city, although its binding to the utrophin ABD was very
weak compared with that of full-length plectin. When over-
laid onto WW-ZZ subdomains, positive signals were obtained
for the EF and ZZ domains, whereas a fragment encoded by
exons 9–12 of plectin did not show binding to any of the utro-
phin fragments used. Confi rming these results, a Eu3+-labeled
version of the plectin ABD specifi cally bound to WW-ZZ
domains of utrophin and dystrophin when overlaid onto micro-
titer plate–immobilized proteins (Fig. S1 B). Furthermore, the
WW-ZZ domain of dystrophin competed with that of utrophin
for plectin ABD binding as did actin (with considerably higher
effi ciency; Fig. S1 C). This suggested that simultaneous binding
of actin and WW-ZZ domains to the plectin ABD was unlikely
Up-regulation of sarcolemmal plectin
in mdx muscle
To defi ne the relation of plectin with costameric membrane com-
plexes in muscles lacking dystrophin, we characterized plectin
localization in skeletal muscle fi bers at different stages of MD
in mdx mice by immunolabeling cross sections of quadriceps
Figure 3. Protein expression profi ling during the differentiation of myo-
blasts in vitro. Myoblasts isolated from plectin (+/+)/p53 (−/−) mice were
differentiated in vitro for up to 96 h. Immediately before the start of differ-
entiation and after 4, 8, 16, 24, 48, 72, and 96 h, cells were lysed in
sample buffer, and proteins were separated by SDS-PAGE on 5% (plectin,
dystrophin, and utrophin) or 15% (βDG, caveolin-3, integrin α7B, and
tubulin) gels and analyzed by IB. Bands were scanned and evaluated den-
sit ometrically. Signals were normalized to tubulin and represent the means
of at least triplicate experiments. 100% corresponds to the highest expres-
sion of each protein during the course of differentiation. Error bars (SD;
rarely >10%) have been omitted for clarity.
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DIRECT INTERACTION OF PLECTIN WITH β-DYSTROGLYCAN • REZNICZEK ET AL. 969
from 2-, 4-, and 14-wk-old animals with plectin-specifi c anti-
bodies (Fig. 5, A–K). Compared with normal muscle, no differ-
ences were observed at the (prenecrotic) age of 2 wk (not
depicted) and in unaffected areas of quadriceps from 4-wk-old
(peak necrotic) mdx mice (Fig. 5, A and B and D and E). Plectin
1f was found in the sarcoplasm and irregularly at the sarco-
lemma, whereas plectin 1 was localized only in subsarcolemmal
accumulations. Dystrophic areas were clearly distinguished by
the presence of high numbers of small-diameter fi bers with cen-
tralized nuclei and loose connective tissue, expressing very high
levels of plectins 1 and 1f (Fig. 5, C and F). After 14 wk, most
fi bers had already passed through one round of degeneration/
regeneration, and, as was expected from fi ndings in DMD
muscles (Schröder et al., 1997), we observed increased plectin
staining at the sarcolemma of regenerated fi bers when using
a pan-plectin antibody (unpublished data). Isoform-specifi c anti-
bodies revealed that this increased sarcolemmal staining was
caused by the up-regulation of plectin 1f (Fig. 5, H and I vs. G).
This was especially evident in 2B fi bers, which are identifi ed as
large-diameter fi bers lacking autofl uorescence (Fig. 5 I; insets
in G–I show autofl uorescence). For plectin 1, on the other hand,
we could not detect notable differences between mdx and con-
trol samples (Fig. 5, J and K). Thus, during the regeneration
of mdx muscles, plectins 1 and 1f were both up-regulated in
regenerating myotubes, but only plectin 1f associated with the
sarcolemma and stayed there at high levels after regeneration
To obtain quantitative estimates of plectin up-regulation
in mdx mice compared with other sarcolemma-associated
proteins, we prepared KCl-washed microsomes from skeletal
muscle of 8–10-wk-old mdx and control mice (Ohlendieck and
Campbell, 1991; Cluchague et al., 2004). Compared with total
muscle lysates (Fig. 6 A), microsome fractions from control
muscle were enriched in DGC components (dystrophin, utro-
phin, and βDG) and the membrane markers caveolin-3 and
integrin α7B; in addition, actin and plectins 1 and 1f but not
tubulin were detected in microsomes (Fig. 6 B). Comparing
corresponding control and mdx samples, we found increased
levels of plectin (?170%) and utrophin (?140%) in total mus-
cle lysates, whereas those of actin were similar (Fig. 6 C). Total
plectin was two- to threefold more abundant in mdx versus con-
trol microsome fractions, with relative levels of plectin 1 and
plectin 1f of ?300% and ?150%, respectively. Interestingly,
the levels of utrophin in the sarcolemmal fraction were only
?50% of those found in the wild type, suggesting a weaker
membrane association of utrophin in mdx muscle. With our
lysis protocol (see Materials and methods), the levels of βDG
were found at ?50% compared with the wild type, although
mdx βDG levels as high as ?100% of wild type have been
reported when samples were treated with cholate detergent
(Cluchague et al., 2004). Caveolin-3 appeared slightly increased,
and no notable difference was observed in the case of actin.
Interestingly, the mdx levels of integrin α7B were approxi-
mately fourfold increased (Fig. 6 D). Thus, these biochemical
data were in agreement with the observed up-regulation of plec-
tin in mdx muscle and the sarcolemma association of isoform 1f
observed in the immunolabeling of tissue sections.
Utrophin is likely not the preferred binding
partner of plectin at the sarcolemma
of mature mdx muscle fi bers
To assess whether utrophin was substituting for dystrophin as a
linker protein between βDG and plectin in dystrophin-defi cient
muscle, we stained cross sections of mdx gastrocnemius for
βDG and utrophin (Fig. 7, A–L). The antiserum to βDG gave
a strong signal in control samples of all ages (2, 4, and 14 wk;
Figure 4. Co-IP of plectin with dystrophin and direct
binding of plectin to the utrophin/dystrophin EF-ZZ
domain via its ABD. (A) Total and IP lysates of wild-
type (wt) and mdx muscle tissues were prepared as
described in Materials and methods. IP lysates were
incubated without (IP: Control) or with antibodies spe-
cifi c for plectin (IP: Ple) or dystrophin (IP: Dys). After
separation and transfer to nitrocellulose, total lysates
and precipitated samples were probed with anti-
bodies specifi c for plectin (top) or dystrophin (bottom).
(B) Schematic drawing of plectin and utrophin show-
ing locations of binding interfaces, fragments used in
blot overlay assays (bars above and below drawings),
and a summary of binding data. Actual data are
shown in Fig. S1 (available at http://www.jcb.org/
cgi/content/full/jcb.200604179/DC1). N and C
termini of proteins are indicated. ABD, actin-binding
domain. Overlaid plectin was either purifi ed from cell
cultures (C6) or contained in cell lysates (804G). ++,
strong binding; +/−, weak binding; −, no binding.
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JCB • VOLUME 176 • NUMBER 7 • 2007 970
Fig. 7, A, E, and I) and reduced but still clearly positive sig-
nals in the corresponding mdx samples (Fig. 7, C, G, and K).
Actively regenerating mdx fi bers, which are marked by small
diameters and centralized nuclei, showed a much stronger staining
at the age of 4 wk that was almost similar in intensity to the sig-
nal in control fi bers (Fig. 7 G). Utrophin was strongly expressed
at the sarcolemmas of 2-wk-old control animals, whereas only
limited staining was observed in corresponding mdx muscle
samples (Fig. 7, compare B with D). At later developmental
stages of normal muscle, utrophin was detectable at myotendi-
nous (Fig. 7 F, arrowheads) and neuromuscular junctions (not
depicted), but no general sarcolemmal staining was observed
(Fig. 7, F and J). In the case of 4- and 14-wk-old mdx muscle,
utrophin was present exclusively in regenerating small-diameter
fi bers (Fig. 7, H and L; asterisks) and at myotendinous junctions
(Fig. 7 H, arrowheads). Faint sarcolemmal utrophin-specifi c
staining that was not visible in wild-type muscle was observed
in mdx muscle (Fig. 7, H and L). The low levels of utrophin and
the positive identifi cation of βDG at the sarcolemma of mdx
muscle prompted us to costain teased wild-type and mdx muscle
fi bers for plectin and βDG (Fig. 7, M–R). Both mAbs as well as
an antiserum to βDG revealed that the gridlike staining pattern
typical for costameres was lost in mdx fi bers, and, instead, βDG
was found exclusively above Z-disks together with plectin 1f
(Fig. 7, compare the insets of N and P, which are magnifi ed in Q
and R). We also examined microsome fractions and sections
prepared from muscles of mdx/utr−/− mice and found a similar
situation as in mdx muscles (Fig. S2, available at http://www
Direct interaction of plectin with 훃DG
via multiple binding sites
The redistribution of βDG to sites above Z-disks where plectin
1f was concentrated suggested that plectin could directly inter-
act with the cytoplasmic domain of βDG. Co-IP of both pro-
teins from lysates of C2C12 myoblasts, mouse keratinocytes,
and the human colon adenocarcinoma cell line CaCo-2 using
anti-βDG antibodies was successful (Fig. 8 A). Using no or
irrelevant antibodies, neither plectin nor βDG was detectable in
the corresponding precipitates (unpublished data). Plectin and
βDG could also be coprecipitated from lysates of skeletal mus-
cle from mdx mice (Fig. 8 B). Immuno-EM confi rmed the close
association of both proteins at the sarcolemma of muscle fi bers
(Fig. 8 C). When the plasma membrane was lost as a result of
Triton X-100 extraction, βDG remained anchored to subsarco-
lemmal fi lamentous structures (Fig. 8, white arrowheads), which
were also positive for plectin (Fig. 8 D). βDG and plectin
la beling was most prominent at subsarcolemmal regions over-
lying Z-disks. Additionally, the plectin label was concentrated
at the periphery of Z-disks (Fig. 8 E).
To map the βDG-binding sites on plectin, a panel of
His-tagged plectin fragments representing different structural
domains (Fig. 9, A and B) were blotted onto nitrocellulose and
overlaid with the cytoplasmic domain of βDG. The WW-ZZ
domain of dystrophin, which binds to the C terminus of βDG
(Ilsley et al., 2002), and the utrophin ABD or BSA were used as
positive and negative controls, respectively. Using βDG- specifi c
antibodies for detection, we found an interaction of βDG with
two nonoverlapping plectin fragments (Fig. 9 C). One was en-
coded by plectin exons 12–24, representing part of the plakin
domain located C terminally of the ABD within the plectin
N-terminal globular domain, and the other corresponded to the
C terminus of plectin, starting within repeat domain 4. No other
protein tested showed binding except for the dystrophin WW-
ZZ domain. To narrow down the region of βDG involved in
binding to plectin, a fragment (βDGcyt∆DBS) corresponding
to roughly 70% of the βDG cytoplasmic domain (lacking the
C-terminal region containing the dystrophin/utrophin-binding
motif) was overlaid onto the same panel of recombinant pro-
teins (Fig. 9 D). It bound to both plectin fragments identifi ed
before but bound much weaker to the C-terminal plectin frag-
ment (Ple R4-C), suggesting that additional C-terminal βDG
sequences were needed for effi cient binding to this fragment.
As expected, the truncated βDG fragment failed to bind to the
dystrophin WW-ZZ domain.
Binding of βDG to both plectin fragments was effi ciently
blocked by the dystrophin WW-ZZ domain (Fig. 9 E). When
dystrophin was added to the overlay solutions at an equimolar
ratio, βDG–plectin binding was only slightly reduced, but
Figure 5. Distribution of plectin isoforms 1 and 1f in muscle during the
course of MD in mdx mice. Cross sections of quadriceps from 4- and
14-wk-old mdx and normal control mice were immunolabeled with plectin
1f– (A–C and G–I) and plectin 1–specifi c antibodies (D–F, J, and K). Secondary
antibodies used were Cy5 labeled. Areas of unaffected (B and E), actively
regenerating (C and F), and regenerated (H, I, and K) mdx muscles are
shown. Insets in G–I show autofl uorescence after excitation at 488 nm,
with autofl uorescent fi bers corresponding to type 2A fi bers. n, sectioned
nuclei. Bar, 20 μm.
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DIRECT INTERACTION OF PLECTIN WITH β-DYSTROGLYCAN • REZNICZEK ET AL. 971
increasing the molar ratios to 1:5 or 1:10 in favor of dystrophin
led to strongly reduced binding and no binding, respectively.
Unexpectedly, in this competition experiment, positive signals
were observed in two additional lanes (Ple E1–12 and Utr ABD;
Fig. 9 E), suggesting that the dystrophin WW-ZZ domain me-
diated the indirect binding of βDG to the ABDs of utrophin
Figure 6. Comparative (semiquantitative) IB analysis of total
cell lysates and microsome fractions from control and mdx
skeletal muscles. (A and B) Scans of typical bands obtained
by IB. (C and D) Bands were evaluated as described in Fig. 3.
Signals were normalized to tubulin (total lysates) or total pro-
tein content (microsome fractions) and are shown relative to
protein amounts in control (wild type) samples (100%). Values
represent means ± SD (error bars) of at least three gel runs
using samples from two independent preparations.
Figure 7. Expression of 훃DG and utrophin during the course
of MD in mdx mice and localization of 훃DG in teased muscle
fi bers. (A–L) Cross sections of gastrocnemius from 2-, 4-, and
14-wk-old normal control (A, B, E, F, I, and J) and mdx (C, D,
G, H, K, and L) mice were immunolabeled using antibodies
specifi c for βDG (A, C, E, G, I, and K) and utrophin (B, D, F,
H, J, and L). Note the expression (albeit reduced at ages 2
and 14 wk) of βDG in mdx muscle fi bers. Utrophin is present
at the sarcolemma of muscle fi bers from young (<4 wk old)
animals (normal and mdx) as well as in regenerating fi bers
from adolescent and adult mdx animals; at these stages,
utrophin localization in nonregenerating fi bers is virtually con-
fi ned to myotendinous junctions both in normal and mdx mice
(arrowheads in F and H). Asterisks in H and L denote areas of
active regeneration in mdx muscle. (M–R) Teased fi bers from
normal and mdx muscles from 8-wk-old mice were stained with
pan-plectin mouse antiserum #123 (pan), rabbit antiserum to
plectin 1f (insets) and rabbit antiserum #1710 (AS), or mouse
mAbs (insets) to βDG. Control and mdx samples were stained
in parallel, and images were recorded using the same settings.
Q and R show magnifi cations of the areas labeled Q and R in
the insets in N and P. Bars (A–L), 50 μm; (M–P) 20 μm.
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JCB • VOLUME 176 • NUMBER 7 • 2007 972
and plectin. To confi rm the simultaneous binding of WW-ZZ
domains to plectin and βDG, we performed an overlay assay
in which the utrophin WW-ZZ domain was immobilized on
microtiter plates and incubated with constant amounts of Eu3+-
labeled βDG in the presence of increasing concentrations of
labeled or unlabeled versions of the plectin ABD (Fig. 9 F). In
the fi rst case, the amounts of labeled protein bound remained
unchanged (Fig. 9 F, gray bars), whereas signals were additive
(Fig. 9 F, black bars) in the latter, clearly demonstrating that
plectin and βDG bound to independent binding sites within the
Distinct regulation and localization
of plectin isoforms in skeletal muscle
Muscle formation is an incremental process in which differenti-
ating myoblasts fuse and form primary and fi nally secondary
myotubes. As this process involves massive rearrangements of
the cytoskeleton, it was not unexpected to fi nd that plectin iso-
forms were differentially expressed during its course. Of special
interest was the striking similarity of the temporal expression
patterns of plectin 1f and dystrophin, which suggested a role for
this particular plectin isoform in the formation and maturation
of costameres. This is supported by the observed exclusive
membrane association of a recombinant version of this isoform
in differentiated myotubes but not at the myoblast stage, where
dystrophin is absent. A similar role of plectin had been sug-
gested previously by Schröder et al. (2000, 2002), who con-
cluded from their myoblast differentiation experiments that the
association of plectin with Z-disks is a prerequisite for formation
of the intermyofi brillar desmin cytoskeleton and, furthermore,
that plectin is a component of primary longitudinal adhe-
sion structures, which are precursors of costameres that form
mature costameres only after being subjected to contractile
forces. This would also explain the apparent discrepancy in
plectin 1f localization in the tissue and in teased fi bers (sarco-
lemma and Z-disks) versus transfected myotubes (sarcolemma
only), as the latter represent a less mature stage. Interestingly,
using mAb 121 to plectin, Schröder et al. (2002) identifi ed a
membrane-associated plectin variant that is up-regulated during
human myotube differentiation. Our results would suggest that
this variant is plectin 1f. However, it is unexplainable how a
mAb with an epitope in plectin’s rod domain could specifi cally
detect one rod-containing isoform (1f) over others.
Immunostaining of muscle tissue revealed that plectin ex-
pression levels in individual fi bers varied and were dependent
on the fi ber type. In cross sections of normal striated human
muscle, a moderate to intense cytoplasmic and sarcolemmal
staining of plectin has been reported in type 1 (slow twitch)
fi bers, whereas only faint staining of the sarcolemma was ob-
served in type 2 (fast twitch) fi bers (Schröder et al., 1997). In
the present study, we show using an antiserum to plectin not
discriminating among isoforms that in quadriceps (a typical fast
muscle composed of mainly type 2 fi bers), plectin clearly was
localized at Z-disks in both type 2A and 2B fi bers, with a
stronger signal in 2A fi bers. This corresponds well with the in-
tense staining obtained with plectin 1f–specifi c antibodies in
this fi ber type. Neither with anti–plectin 1f nor anti–plectin 1
antibodies did we detect substantial Z-disk staining in 2B
fi bers. Based on this observation, one other plectin isoform ex-
pressed in skeletal muscle, plectin 1b or 1d, must be associated
Figure 8. Co-IP and ultrastructural colocalization of plectin
and 훃DG. (A) Cell lysates from C2C12 myoblasts (M), mouse
keratinocytes (K), and CaCo-2 (C) cells were subjected to IP
using anti-βDG antiserum; antibodies used for detection are
indicated. (B) mdx muscle lysates were immunoprecipitated
with the indicated antibodies and probed for βDG. (C–E)
Preembedding immunogold labeling of teased muscle fi bers
extracted with Triton X-100. Gold particles labeling plectin
(5 nm) and βDG (10 nm) were silver enhanced. In C, note the
intense labeling of both proteins at the plasma membrane lo-
cally separated from the muscle fi ber. The exterior of the fi ber (e)
contains extracellular material, whereas the blistered interior (i)
is empty. In D, a sarcomere positioned beneath the sarcolemma
is shown. Although the plasma membrane is lost by Triton
X-100 extraction, βDG as well as plectin remain anchored to
fi lamentous structures (arrowheads) in the subsarcolemmal
region. Also note plectin labeling in the interior of the fi ber at
Z-disks. In E, details of a sarcomeric region proximal to a Z-disk
are shown. Labeling of βDG is restricted to the subsarcolemmal
region with incomplete detachment of the sarcolemma. While
most of the βDG is located at fi lamentous structures (white
arrowheads), it can also be observed (arrow) in association
with the sarcolemma (black arrowheads). Bars, 500 nm.
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DIRECT INTERACTION OF PLECTIN WITH β-DYSTROGLYCAN • REZNICZEK ET AL. 973
with Z-disks in type 2B fi bers. From our overexpression experi-
ments, we conclude that this isoform is plectin 1d, as it was
found localized exclusively at Z-disks. At this time, the molecular
mechanism of this targeting is unknown.
Plectin is associated with the DGC
via multiple interfaces
Using co-IP and in vitro binding assays, we demonstrate direct
interactions of plectin via multiple interfaces with components
of the DGC, including (1) direct binding of its plakin domain
to the cytoplasmic domain of βDG; (2) direct binding of its
C-terminal portion to βDG; (3) binding of its ABD to the WW-ZZ
domains of dystrophin and utrophin; and (4) binding of its ABD
to the ABD of utrophin (Figs. 4 and 9, schematics). Whether the
ABD of plectin would also interact with that of dystrophin re-
mains an open question considering that the functionalities of
the ABDs of dystrophin and utrophin differ (Rybakova et al.,
2006), whereas their WW-ZZ domains are highly conserved
(Hnia et al., 2007). Plectin and dystrophin had previously been
coimmunoprecipitated from muscle lysates, but their interaction
was assumed to be indirect via actin (Hijikata et al., 2003). In
dystrophin-lacking mdx mice, one may thus expect to fi nd the
reduced sarcolemma association of plectin, but our immuno-
fl uorescence and tissue fractionation experiments revealed that
plectin was instead enriched at the sarcolemma of mdx muscle.
It was widely believed that dystrophin defi ciency in skeletal mus-
cles of mdx mice and DMD patients leads to the reduced expression
and sarcolemmal association of dystrophin-associated proteins,
including a strong reduction or even absence of βDG immuno-
reactivity (Ohlendieck and Campbell, 1991) despite its mRNA
levels being similar to those in normal samples (Ibraghimov-
Beskrovnaya et al., 1992; Rouger et al., 2002). However, in
a recent study, Cluchague et al. (2004) challenged this view
when they demonstrated that in mdx muscle samples treated
with 2% cholate, βDG was detectable at levels comparable with
those of wild-type samples. The authors proposed that βDG
was targeted to the plasma membrane normally in dystrophin-
defi cient mdx muscles but remained inaccessible to antibodies
and, when tissues were lysed, became part of an SDS-insoluble
pool. Using our protocols, we also found considerable levels of
βDG in mdx skeletal muscle microsome fractions (?50% of
wild type even without cholate treatment), and we were able to
immunodetect βDG with variable intensities throughout mdx
muscle regeneration. Thus, our results support the hypothesis of
Cluchague et al. (2004), and the direct interaction of plectin
with βDG provides an explanation for the observed increase in
sarcolemmal plectin in mdx muscle (Fig. 10). In the absence of
dystrophin, more plectin can bind to βDG, causing at the same
time the redistribution and accumulation of βDG above Z-disks,
where plectin is normally localized. Matching our βDG immuno-
staining results, Yurchenco et al. (2004) have observed a cor-
responding redistribution of αDG in teased mdx fi bers.
It has been suggested that utrophin could substitute for
dystrophin in dystrophic muscles, but its intimate association
with βDG may be limited to the time of regeneration only (Tinsley
et al., 1998). The ?50% reduction of utrophin observed in
Figure 9. Direct binding of 훃DG to N- and C-terminal
domains of plectin. (A) Schematic representation of plectin,
βDG, dystrophin, and protein fragments used for in vitro inter-
action assays. Binding interfaces between plectin, dystrophin,
and βDG are indicated by brackets connected by lines. ABD,
actin-binding domain; IFBD, IF-binding domain; TM, trans-
membrane domain; DBS, dystrophin-binding site. N and C
termini of proteins are indicated. (B–E) Blot overlay assay. A
panel of protein fragments recombinantly expressed in bacteria
(B; Coomassie) was immobilized on nitrocellulose membranes
and overlaid with βDGcyt (C) or βDGcyt∆DBS, a shorter
fragment comprising amino acids 765–857 and lacking
the dystrophin-binding site (D). Bound proteins were detected
using mAbs to βDG (C) or via the S tag contained in
βDGcyt∆DBS (D). In E, membranes were overlaid with
βDGcyt in the presence of Dys WW-ZZ, a fragment corre-
sponding to the WW-ZZ domain of dystrophin. Molar ratios
(βDGcyt/Dys WW-ZZ) were 1:1, 1:5, and 1:10 in the large,
small top, and small bottom panels, respectively. (F) Microtiter
plate competition binding assay. Recombinantly expressed
and purifi ed WW-ZZ domains of utrophin (immobilized) were
overlaid with constant amounts of a Eu3+-labeled version of
the full-length cytoplasmic domain of βDG (βDGcyt; white
bars; 100% indicated by the dotted line) and increasing
amounts of unlabeled (gray bars) or Eu3+-labeled (black bars)
recombinant plectin ABD. Data represent mean ± SD (error
bars) of a typical experiment performed with triplicate wells.
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JCB • VOLUME 176 • NUMBER 7 • 2007 974
microsome preparations from muscle of ?10-wk-old mdx mice
despite the overall higher expression of utrophin (?150% of
wild-type levels) would support this notion. Furthermore, this
observation also suggests that plectin binds to βDG with a higher
affi nity than utrophin. Remaining sarcolemmal utrophin staining
that was observed in transgenic mdx mice overexpressing Dp71,
a short splice variant of dystrophin lacking rod and N-terminal
domains (Cox et al., 1994; Greenberg et al., 1994), was explained
by Cox et al. (1994) to possibly be caused by the binding of utro-
phin to subsarcolemmal actin via its ABD. Our fi nding that βDG
immunostaining signals observed in mdx tissue from 4-wk-old
animals was almost as strong as in normal muscle would also fi t
the proposed model (Fig. 10), as the higher expression of utro-
phin during the phase of peak necrosis/regeneration may dis-
place plectin from βDG and, thus, restore accessibility of the
epitopes masked by plectin. Similarly, the overexpression of
Dp71 (or other dystrophin or utrophin deletion variants) in mdx
restored normal DGC components, whereas a full phenotypic
rescue was achieved only by proteins with functional ABDs and
intact C-terminal βDG-binding domains (Tinsley et al., 1998).
Plectin acts as a universal mediator
of IF anchorage
The DGC has been considered to be responsible for connecting
the subsarcolemmal actin cytoskeleton to the ECM, and disrup-
tion of this link causes a dystrophic phenotype. However, in re-
cent years, it has been established that the contractile actions of
a muscle fi ber are mechanically integrated by desmin IFs, which
are responsible for linking individual myofi brils laterally with
each other and to the sarcolemma at the level of the Z-disks.
Previous studies have implicated the DGC as the transmem-
brane complex linking the IF network with the ECM (for
r eviews see Blake and Martin-Rendon, 2002; Capetanaki, 2002;
Paulin and Li, 2004). The necessary link would be created by
an α-dystrobrevin–synemin/syncoilin–desmin bridge. Synemin
may also directly interact with vinculin, providing an alternative
anchorage of desmin IFs to costameres (Bellin et al., 2001).
Plectin directly interacts with desmin via its C-terminal IF-
binding domain (Reipert et al., 1999) and also with multiple
components of the DGC, including its transmembrane core pro-
tein βDG. Thus, we propose that plectin acts as a direct linker
between the DGC and the desmin IF network. There is prece-
dence for such a function of plectin in basal keratinocytes, where
the protein directly links the keratin IF network to the cyto-
plasmic domain of the β subunit of the laminin receptor integrin
α6β4 (Rezniczek et al., 1998). It could be that synemin plays
the essential role in establishing the direct linkages between
heteropolymeric IFs and the myofi brillar Z-disk and costameric
regions, and plectin might only provide additional structural
support at these sites. However, this would be in confl ict with
observations in differentiating human skeletal muscle cultures,
where plectin was already localized in a cross-striated pattern,
whereas desmin was still found in longitudinal fi laments (Schröder
et al., 2000).
Recently, it was shown that besides type III and IV IFs,
the cytokeratins K8 and K19 are also expressed in striated mus-
cle and localize to Z-disks and M-lines and that K19 directly
interacts with the dystrophin ABD (Stone et al., 2005). Whereas
plectin has been shown to directly bind to keratins 5, 14, and 18
(Geerts et al., 1999; Steinböck et al., 2000), it is unknown
whether it can also interact with the muscle-specifi c keratins
and possibly plays a role in their anchorage as well.
Based on our observations, we propose the following
model for plectin’s association with the DGC (Fig. 10). Because
(1) binding of plectin to βDG via its C-terminal binding site
was abolished in the absence of the C-terminal part of βDG’s
cytoplasmic domain (harboring the PPxY motif required for
interaction with the WW domain of dystrophin and utrophin;
see Ilsley et al., 2002 for a discussion of the WW domain and its
interactions) and (2) binding of plectin to βDG was effi ciently
blocked by dystrophin, only a portion of the βDG cytoplasmic
tails would normally be available for binding to plectin when
dystrophin is present. However, plectin could remain associated
with the DGC via binding of its ABD to dystrophin (utrophin).
Figure 10. Model of DGC-cytoskeleton linkage via plectin in normal and
mdx/DMD muscle fi bers. Under normal conditions (left), plectin molecules
are associated with the DGC by binding to βDG via the binding site in its
plakin domain and/or by binding to dystrophin via its ABD, leaving the
IF-binding domain available for binding to the desmin IF network. Binding
of dystrophin and plectin to βDG may occur simultaneously at costameric
structures above Z-disks but not M-lines (smaller-scale structures). When
dystrophin is absent (right), plectin can additionally bind with its C-terminal
domain to βDG using a binding site on βDG normally occupied by
dystrophin. This leads to increased levels of plectin at the sarcolemma and
to a redistribution of βDG (and DGCs) from their normal gridlike localiza-
tion (top left) to areas overlying Z-disks only (top right). Furthermore, the
tight interaction with multiple plectin molecules causes the complex to
become SDS insoluble (see Discussion). Binding to βDG via the C-terminal
domain could leave the plectin ABD available for binding to fi lamentous
actin, potentially fulfi lling to some degree dystrophin’s function of linking
the DGC to the actin cytoskeleton. However, the ABD of plectin and potentially
also signaling molecules scaffolded on plectin may have adverse regula-
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DIRECT INTERACTION OF PLECTIN WITH β-DYSTROGLYCAN • REZNICZEK ET AL.975
In such a constellation, the plectin C-terminal domain, sepa-
rated from the N terminus by the ?200-nm–long rod domain,
would be exposed and available for binding to muscle IFs (des-
min and potentially also cytokeratins 8/19). In the absence of
dystrophin, however, the available binding sites on βDG are oc-
cupied by plectin, leading to the increased sarcolemmal plectin
1f signal observed in mdx, mdx/utr−/−, and DMD muscle fi bers
and consequently to an increased insolubility of βDG and mask-
ing of its epitopes. Binding via the C terminus would also leave
the plectin ABD available for interaction with fi lamentous actin,
possibly taking over some of the responsibilities of dystrophin.
Finally, based on the cellular targeting of overexpressed recom-
binant full-length versions of plectin isoforms, we propose plectin
isoform 1d to be involved in the anchorage of desmin IFs to
Z-disks (Fig. 10, bottom).
DGC signaling: plectin as a scaffold?
Recently, several proteins involved in signaling such as nonre-
ceptor tyrosine kinase Fer, the PKC scaffolding protein RACK1,
and the key enzyme involved in energy homeostasis, AMP ki-
nase, have been identifi ed as novel interaction partners of plec-
tin and led to the proposal that plectin acts as a scaffolding
platform for signaling proteins in addition to serving as a cyto-
skeletal linker. Having established plectin as a component of the
DGC with multiple binding interfaces to its key components,
it will now be a challenge to defi ne its role in DGC-mediated
signaling. An important consequence implied by our model
could be that misguided signaling mediated by the accumula-
tion of plectin scaffolds at the sarcolemma of mdx and DMD
dystrophic muscle contributes to the disease phenotype.
Materials and methods
Full-length (mouse) plectin isoform cDNA constructs (including respective
5′ untranslated regions) encoding proteins with C-terminal GFP have been
described previously (Rezniczek et al., 2003). For bacterial expression of
plectin fragments, the corresponding cDNAs were PCR amplifi ed by using
primers with EcoRI tails and were cloned into pJD1, a modifi ed pET-15b
(Novagen) that was obtained by replacing the EcoRI–BamHI fragment of
pBN120 with that from pAD29 (Nikolic et al., 1996); expressed proteins
contained an N-terminal His tag and a C-terminal c-myc tag. The following
plectin fragments were used: Ple E1–12 (M1–R654), Ple E9–12 (E419–V541),
Ple E12–24 (M546–E1128), Ple Rod (E2235–Q2577), Ple R1–R3 (A2762–K3852),
and Ple R4–C (L3850–A4687), which were all from a rat (X59601), as well as
Ple ABD (D181–N418) from a mouse (NM_201389). To express N-terminally
His-tagged versions of human utrophin (X69086) fragments, Utr ABD (S19–
D261), Utr WW-ZZ (A2798–M3113), Utr WW (A2798–K2868), Utr EF (I2869–
S3014), Utr ZZ (N3015–M3113), and Utr C-terminal (M3204–M3433) EcoRI-fl anked
cDNAs were cloned into pBN120 (Nikolic et al., 1996). The WW-ZZ
domain (A3041–M3356) of human dystrophin (X14298) was also expressed
from pBN120. Fragments βDGcyt (L765–P895) and βDGcyt∆DBD (L765–D857)
of human βDG (NM_004393) were expressed from pLJ1 (a pET32a
[Novagen]-derived plasmid in which the sequence between the NcoI and
XhoI restriction sites has been replaced by 5′-A A T T C C T G G T G C C A C G C G G-
T T C T -3′) as proteins with N-terminal Trx-His-S tags and C-terminal His tags.
The cDNA fragments encoding Ple ABD and Dys WW-ZZ were also in-
serted into the EcoRI site of pMal-c2 (New England Biolabs, Inc.) to generate
fusion proteins with N-terminal maltose-binding protein.
For immunoblotting (IB), IP, immunofl uorescence microscopy (IFM), and
immuno-EM, the following antibodies were used: mAbs 5B3 (IB) and 7A8
(EM; Rezniczek et al., 2004) to plectin; antisera #9 (IB), #46 (IFM), and
#123 (IFM) to plectin (Andrä et al., 2003); anti–plectin isoform 1 antiserum
(IB and IFM; Abrahamsberg et al., 2005); anti–plectin isoform 1f anti-
serum (IB and IFM), which was prepared and affi nity purifi ed as described
previously (Abrahamsberg et al., 2005) using amino acids M1–K28 of
plectin 1f (NM_212539) as immunogen; mAb EA-53 (Sigma-Aldrich) to
sarcomeric α-actinin (IFM); mAb AC-40 (Sigma-Aldrich) to actin (IB); mAb
B-5-1-2 (Sigma-Aldrich) to tubulin (IB); mAb 43DAG1/8D5 (IB, IFM, and
IP; Novocastra) and rabbit antisera #1709 and #1710 (Tyr 895-P; IB,
IFM, IP, and EM; Ilsley et al., 2001) to βDG; anti-utrophin antiserum RAB5
(IB, IP, and IFM; James et al., 2000); mAb DY4/6D3 (Novocastra) to dys-
trophin (IB); mAb (clone 26) to caveolin-3 (IB and IFM; BD Biosciences);
anti–integrin α7 antiserum (IB; provided by U. Mayer, University of East
Anglia, Norwich, UK; Cohn et al., 1999); mAbs to MyHC-2A (SC-71) and
-2B (BF-F3; IFM; hybridomas were obtained from the German Resource
Center for Biological Material; Schiaffi no et al., 1989); and mAb to myc
epitope tag (1-9E10.2; IB; American Type Culture Collection). As secondary
antibodies, we used goat anti–rabbit IgG AlexaFluor488 (Invitrogen), goat
anti–mouse IgG Texas red (Jackson ImmunoResearch Laboratories), and
donkey anti–rabbit Cy5 (Jackson ImmunoResearch Laboratories) for IFM
and used goat anti–rabbit and goat anti–mouse IgGs conjugated to AP or
HRP (Jackson ImmunoResearch Laboratories) for IB.
Thin sections (3–5 μm for longitudinal and 8–10 μm for cross sections)
were prepared from skeletal muscle (quadriceps and gastrocnemius) dis-
sected from C57BL/10 control and mdx mice (Institut für Labortierkunde,
Medical University of Vienna) and frozen in liquid nitrogen–cooled isopentane.
Sections were placed on slides, fi xed with acetone for 10 min, and
in cubated for 1 h in 5% goat serum in PBS to block nonspecifi c binding of
antibodies. Samples were incubated with primary and secondary anti-
bodies diluted in PBS for 1 h each. Signal specifi city was controlled by the
omission of primary antibodies or by using normal mouse or rabbit serum
in their place. To prepare teased fi bers, mice were anesthetized with isofl u-
rane and perfused with 2% PFA in PBS. EDL was dissected and incubated
with the same fi xing solution for 10 min. Using fi ne forceps, the muscle was
teased into single fi bers, which were then adhered onto chrome-alaun/
gelatin-coated slides. Slides were blocked with PBS containing 0.1% BSA
and 0.1% Triton X-100 for 1 h, incubated for 2 h with primary antibodies
(diluted in blocking solution), washed with PBS for 30 min, incubated with
secondary antibodies (diluted in PBS) for 1.5 h, and washed again with PBS.
Finally, samples were briefl y rinsed with water and mounted in Mowiol.
Myoblast transfection and differentiation
Immortalized (p53 negative) mouse myoblasts (Gregor et al., 2006) were
cultivated on collagen-coated (5 mg/ml in PBS overnight; Sigma-Aldrich)
tissue culture dishes in F-10 (Invitrogen) medium containing 20% FCS, 2.5
ng/ml human basic FGF (Promega), 100 U/ml penicillin, and 100 μg/ml
streptomycin. Myoblasts were transfected using FuGENE6 (Roche), and
differentiation was initiated after 12–16 h by switching the medium to
DME containing 5% horse serum, 100 U/ml penicillin, and 100 μg/ml
streptomycin. After 4–6 d, myotubes were fi xed with chilled (−20°C) meth-
anol and processed for immunolabeling and subsequent laser-scanning
confocal microscopy. For IB analysis of protein expression profi les during
differentiation, myoblast cultures were differentiated, and cells were lysed
directly in reducing SDS sample buffer at different time points.
Fluorescence microscopy and imaging
Immunolabeled tissue and cell samples mounted in Mowiol were viewed in
a fl uorescence microscope (Axiophot; Carl Zeiss MicroImaging, Inc.) using
plan Neofl uar 40× NA 1.2 (for tissue sections; Carl Zeiss MicroImaging,
Inc.) and plan Apochromat 63× NA 1.3 (for cells and teased fi bers; Carl
Zeiss MicroImaging, Inc.) objectives. Confocal images were recorded using
the LSM510 module (Carl Zeiss MicroImaging, Inc.) and the LSM510 soft-
ware package (version 3.2 SP2; Carl Zeiss MicroImaging, Inc.). Images
were processed using LSM Image Browser (generation of projections of
confocal stacks; gamma/contrast adjustments; version 3.2; Carl Zeiss
MicroImaging, Inc.) and Photoshop CS2 (cropping and splitting of color
channels; Adobe) and were mounted/labeled using Illustrator CS2 (Adobe).
For preembedding immuno-EM, perfusion-fi xed (2% PFA in PBS) adult
rat EDL was dissected and teased into small fi ber bundles before shock
freezing in liquid nitrogen–cooled isopentane. Samples were thawed in PBS,
treated with 0.2% Triton X-100 in PBS for 1 h, and blocked for 1 h in 0.1%
Triton X-100, 0.1% BSA, and 1:25 normal goat serum in PBS (blocking
solution). For primary immunolabeling, teased fi bers were incubated
on November 21, 2007
JCB • VOLUME 176 • NUMBER 7 • 2007 976
overnight at 4°C in a mixture of mAb 7A8 to plectin and antiserum
#1710 to βDG (diluted in blocking solution without serum). After washing
for 1 h (0.2% BSA in PBS), samples were incubated overnight at 4°C
in a mixture of gold-conjugated goat anti–mouse (5 nm) and goat anti–
rabbit (10 nm) secondary antibodies (British Biocell International). Gold-
labeled fi bers were washed in PBS, postfi xed in 2.5% glutaraldehyde for
30 min, and washed in double-distilled water before silver enhancement
for 1 h (R-GENT SE-EM kit; Aurion). Samples were immersed in 0.5%
OsO4 in PBS for 15 min, dehydrated, and embedded in epoxy resin
(agar 100; Agar Scientifi c Ltd.). Thin sections were cut with an ultra-
microtome (Ultracut S; Leica), mounted on copper grids, counterstained
with uranyl acetate and lead citrate, and examined at 80 kV in an elec-
tron microscope (JEM-1210; JEOL). Digital images were acquired and
processed using a camera (Morada; Olympus) and the analySIS soft-
ware package (Olympus).
Preparation of total muscle lysates and microsome fractions
Hind leg muscles were dissected from C57BL/10 control and mdx mice,
snap frozen in liquid nitrogen, and ground in a mortar. Muscles were
homogenized in solution A (20 mM Na4P2O7, 20 mM Na-PO4, pH 7.4,
0.303 M sucrose, 0.5 mM EDTA, 1 mM MgCl2, 2 mM PMSF, and Com-
plete mini protease inhibitor cocktail [Roche]) using a Dounce homogenizer
(?10 strokes). Part of the crude homogenate (total muscle lysate) was
mixed with an equal volume of SDS sample buffer (0.4 M Tris, pH 6.8,
0.5 M DTT, 10% SDS, 50% glycerol, and 0.1% bromophenol blue) for
further analysis; the rest was centrifuged for 15 min at 20,000 g, and the
pellet was rehomogenized. Combined supernatants were fi ltered through
six layers of cheesecloth and centrifuged for 15 min at 25,000 g. The
pellet was discarded. To the supernatant, solid KCl was added to a fi nal
concentration of 0.6 M. After centrifugation for 35 min at 200,000 g, the
pellet was resuspended in solution B (20 mM Tris-maleate, pH 7.4, 0.303 M
sucrose, 0.6 M KCl, and the same protease inhibitors as in solution A).
After incubating for 1 h, KCl-washed microsomes were pelleted for 35 min
at 200,000 g and resuspended in solution B without KCl. 5 g of muscle
yielded 0.5 ml of microsome suspension. All steps were performed at 4°C
on ice. For subsequent IB analysis, microsome suspensions were mixed
with 5 vol SDS sample buffer.
Gel electrophoresis and IB
Proteins were separated using standard 5 or 15% SDS-PAGE. Note the
considerably higher concentration of SDS (?60 mg/ml) in our samples
compared with standard conditions (?20 mg/ml). Under these conditions,
immunoblot analysis of microsome fractions generally gave much better
results, which are likely caused by the enhanced solubilization of membrane-
associated protein complexes in these lipid-rich fractions. For IB, proteins
were transferred to nitrocellulose membranes, and membranes were
blocked with 5% nonfat dried milk in PBS–0.05% Tween 20 and incubated
with primary and AP-conjugated secondary antibodies. For quantitation,
stained membranes were scanned, and bands were evaluated using the
ImageQuant 5.1 software package (Molecular Dynamics). Normalization
factors based on total protein content or tubulin signals were applied to the
Mouse myoblasts (Gregor et al., 2006), mouse keratinocytes (Andrä et al.,
2003), and CaCo-2 (HTB-37; American Type Culture Collection) were cul-
tured as recommended or described previously. IP with antisera to plectin
and βDG was performed essentially as described previously (James et al.,
2000). In brief, clarifi ed cell extracts in RIPA buffer were incubated for 2 h
at 4°C with antibodies or, for control experiments, without antibodies or
with host sera. Immunocomplexes were collected by centrifugation after a
further 1-h incubation with protein A– or G–Sepharose beads (GE Health-
care) and extensive washing with RIPA buffer and were subsequently ana-
lyzed by IB. For IP from muscle tissue, total muscle lysates were prepared
as for microsome preparations with the addition of 0.5% Triton X-100 to
solution A. Part of the lysates was mixed with SDS sample buffer (total
lysates), and the rest was incubated for 3 h with protein A–Sepharose beads
and centrifuged (IP lysates) before incubation with antibodies overnight.
Immunocomplexes were captured by protein A–Sepharose beads and
eluted with SDS sample buffer.
Expression and purifi cation of protein fragments
Recombinant protein fragments were expressed in Escherichia coli
BL21(DE3) and purifi ed as described previously (Rezniczek et al., 2004).
Blot overlay assay
Protein fragments were transferred to nitrocellulose membranes after 10%
SDS-PAGE. Membranes were blocked with 5% BSA in TBS containing
0.5% Tween 20 and overlaid with 10 μg/ml of proteins in 20 mM Hepes,
pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, and 5% BSA and incu-
bated overnight at 4°C with agitation. Bound proteins were detected by IB
using protein- or epitope tag–specifi c primary and HRP-conjugated secondary
antibodies or (in the case of His- and S-tagged fragments) by using the India
HIS detection system (Pierce Chemical Co.) and HRP-conjugated S protein
Microtiter plate–binding assay
The experimental details of this binding assay have been described previ-
ously (Rezniczek et al., 2004). In brief, proteins immobilized on microtiter
plates were overlaid with Eu3+-labeled proteins in solution at different
concentrations. After washing, the amounts of proteins bound were deter-
mined by measuring Eu3+ fl uorescence in comparison with a standard.
Online supplemental material
Fig. S1 shows the binding data (blot overlay assays) summarized in the
table in Fig. 4 B as well as additional microtiter plate–binding data.
Fig. S2 shows immunofl uorescence images of tissue sections from wild-
type and mdx/utr−/− mice coimmunolabeled with antibodies to plectin
and βDG as well as immunoblots of wild-type and mdx/utr−/− muscle
lysates using primary antibodies to plectin, dystrophin, utrophin, and
βDG. Online supplemental material is available at http://www.jcb.org/
We thank U. Mayer for providing antibodies to integrin α7B.
This work was supported by the Austrian Science Research Fund grant
P17862-B09 (to G. Wiche) and the Wellcome Trust Research Career Develop-
ment award 042180 (to S.J. Winder).
Submitted: 28 April 2006
Accepted: 16 February 2007
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on November 21, 2007