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MICROSCOPY RESEARCH AND TECHNIQUE 48:131–141 (2000)
Plasma Membrane Cytoskeleton of Muscle:
A Fine Structural Analysis
SIMON C. WATKINS,1* MICHAEL J. CULLEN,2 ERIC P. HOFFMAN,3 AND LYNN BILLINGTON1
1Department
of Cell Biology and Physiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261
of Neuroscience, University of Newcastle upon Tyne, Newcastle upon Tyne, Tyne and Weir, England NE1 7RU
3Department of Human Genetics, National Children’s Medical Center, Washington, DC 20010
2Department
KEY WORDS
dystrophin; electron microscopy; light microscopy; dystrophin cytoskeleton
ABSTRACT
The discovery of dystrophin and its definition as the causative molecule in
Duchenne Muscular Dystrophy has led to a renewed interest in the molecular structure of the
muscle fiber plasma membrane and its association with the extracellular basal lamina. The original
identification of dystrophin gave credence to the possibility that the plasma membrane of the muscle
fiber may be highly organized and involved in maintaining appropriate homeostasis in this actively
contracting cellular system. In this review, we examine the currently known members of the muscle
fiber plasma membrane cytoskeleton and the interactions that occur between the different members
of this complex using histological, electron microscopic, and confocal methods. From our studies and
others cited in this review, it is clear that the dystrophin cytoskeletal complex is not completely
understood and component molecules continue to be discovered. Perhaps equally importantly,
currently defined molecules (such as alpha-actinin or neuronal nitric oxide synthase) are being
recognized as being specifically associated with the complex. What is striking from all of the studies,
to date, is that while we are able to identify members of the dystrophin cytoskeletal complex and
while we are able to associate mutations of individual molecules with disease(s), we are still unable
to truly define the roles of each of the molecules in maintaining the normal physiology of the muscle
fiber. Microsc. Res. Tech. 48:131–141, 2000. r 2000 Wiley-Liss, Inc.
INTRODUCTION
Since the discovery of dystrophin in 1987 and the
elucidation of its fundamental role in the pathogenesis
of Duchenne muscular dystrophy (DMD) (Hoffman et
al., 1987), public interest and clinical research have
focused largely on attempts to correct the effects of
mutations in the dystrophin gene with drug therapy
(e.g., Barton-Davis et al., 1999; DeSilva et al., 1987;
Haye and Williams, 1994, 1998; Sansome et al., 1993),
with surgical intervention (e.g., Bach and McKeon,
1991; Forst and Forst, 1995, 1999; Smith et al., 1993)
and with molecular and cellular therapeutics (e.g.,
Floyd et al, 1998; Howell et al., 1998; Karpati, 1989;
Karpati and Petrof, 1998; Law et al., 1993, 1994; Miller
et al., 1987; Tremblay et al., 1993). However, the
plasmalemmal cytoskeleton of the skeletal muscle fiber
is now recognized to be an extremely complex multimolecular network, and mutations of these so-called dystrophin-associated proteins will give rise to diverse and
commonly debilitating myopathies. The first discovery
of a dystrophin associated protein was by Campbell and
Kahl (1989) who reported the association of dystrophin
with an integral membrane glycoprotein soon after the
discovery of dystrophin. Since this time, there has been
a continuous series of discoveries that demonstrate the
complexity of the dystrophin cytoskeleton. In addition
to dystrophin, there are at least fourteen proteins that
link the cytoplasmic myofibrillar contractile elements
to the signal transducing molecules of the extracellular
matrix and provide structural support to the sarcolemma (Fig. 1). These proteins include an extracellular
r 2000 WILEY-LISS, INC.
protein (␣-dystroglycan), seven integral membrane proteins (␤-dystroglycan, sarcospan, and ␣-, ␤-, ␦, ␥-, and
⑀-sarcoglycan, four cytoplasmic proteins (␣-actinin-2
and syntrophin ␣1, ␤1, and ␤2) and two isoforms of a
subsarcolemmal protein (␣-dystrobrevin-1, -2). It is
certain that together with dystrophin these proteins
form a critical membrane cytoskeleton at the muscle
fiber plasma membrane, functionally responsible for
the structural and possibly signal transduction pathway between the contractile elements of a muscle fiber
and its extracellular matrix (Ervasti and Campbell,
1993). Description of the physical inter-relationship(s)
between these proteins and with dystrophin, and the
possible role(s) of these proteins in the DMD and other
myopathies is the focus of this review.
DYSTROPHIN-ASSOCIATED PROTEINS
Dystroglycan Complex
The dystroglycan complex (DGC) was initially purified from digitonin-solubilized rabbit skeletal muscle
membranes (Ervasti et al., 1990). Two of the components, ␣- and ␤-dystroglycan (156 and 43 DAG, respectively) are post-translationally spliced products of a
single gene and are termed dystroglycans because of
their demonstrated, direct interaction with dystrophin
(dystro-) and the heavily glycosylated (-glycan) nature
of the ␣-form (Ibraghimova-Beskrovnaya et al., 1992).
*Correspondence to: Simon C. Watkins Ph.D., BSTS 225, 3500 Terrace St.,
University of Pittsburgh, Pittsburgh PA 15261. E-mail: [email protected]
Received 22 September 1999; accepted in revised form 10 October 1999
132
S.C. WATKINS ET AL.
Fig. 1.
Schematic representation of dystrophin and the dystrophin-associated proteins.
Sequence analysis of rabbit skeletal muscle cDNAs
reveals a reading frame of 2,685 bases encoding a
precursor polypeptide of 895 amino acids with a predicted molecular weight of 97 kDa (IbraghimovBeskrovnaya et al., 1992). The N-terminal portion of
the precursor polypeptide encodes the 56 kDa core
protein of ␣-dystroglycan while the C-terminal portion
is processed into the mature ␤-dystroglycan. Carbohydrate moieties constitute up to two thirds of the molecular mass of ␣-dystroglycan. Beta-dystroglycan has a
large extracellular domain with four potential N-glycosylation sites, a transmembrane domain, and a 120 amino
acid cytoplasmic tail.
Expression of the dystroglycans is ubiquitous. An
early report showed ␤-dystroglycan to be present in
brain and lung (Ibraghimova-Beskrovnaya et al., 1992)
More recently, a comprehensive immunoblot analysis of
monkey tissues showed the molecule to be present in
every tissue examined including lung, liver, uterus,
aorta, cerebrum, cerebellum, spinal cord, and peripheral nerve (Mizuno et al., 1994). Alpha-dystroglycan
has also been found in brain tissue (Gee et al., 1993;
Montanaro et al., 1995; Tian et al., 1996) and peripheral nerve fibers (Yamada et al., 1994). Importantly, in
these locales, the proteins are generally functioning as
laminin receptors.
Studies of dystrophin binding properties using protein binding assays show that its linkage to the integral
membrane components of the DGC is via ␤-dystroglycan (Suzuki et al., 1994). The dystroglycan binding site
on dystrophin is located in a widely extended region
encompassing the cysteine-rich domain and the first half
of the C-terminal domain. ␤-dystroglycan also binds
with utrophin or dystrophin related protein, a homolog
of dystrophin via an SH3 domain-binding region in its
C-terminus (Jung et al., 1995). Further in vitro localization shows that dystrophin from skeletal muscle and
brain binds to the cytoplasmic domain of ␤-dystrogly-
can (Jung et al., 1995) and that the binding site is on
the second half of hinge 4 and the cysteine-rich domain
(amino-acids 3054–3271) of dystrophin. The dystrophin
binding site is in the proline-rich section at the extreme
end of the C-terminus of ␤-dystroglycan (amino acids
880–895).
Alpha-dystroglycan is a heavily glycosylated, mucinlike protein that links the transmembrane ␤-dystroglycan to the extracellular matrix protein, laminin (Smalheiser and Kim, 1995). Laminin, a major component of
the basal lamina, binds to ␣-dystroglycan via the G
repeats of its ␣2 chain (Talts et al., 1999). Alphadystroglycan is a receptor for agrin (Bowe et al., 1994;
Gee et al., 1994; Peng et al., 1998, 1999; Sugiyama et
al., 1994), and, thus, may contribute to the clustering of
acetylcholine receptors at the neuromuscular junction
(Peng et al., 1998). Mycobacterium leprae, the bacterium responsible for leprosy, complexed with laminin
utilizes ␣-dystroglycan as a target for entry into peripheral nerve Schwann cells (Rambukkana et al., 1998).
The morphological relationships of the dystroglycans
and the proteins that they bind to have, until recently,
been based almost entirely on (very thorough) biochemical information. Immunofluorescence studies have
shown that the dystroglycans, like dystrophin, are localized at the sarcolemma (Durbeej et al., 1998; James et
al., 1996; Kawajiri et al., 1996, 1998; Peng et al., 1998),
but for precise information on how they are positioned
relative to dystrophin, ultrastructural immunolabeling
is required. Immunogold labeling of ␣-dystroglycan
shows that the gold conjugate is localized to the outer
face of the sarcolemma, usually on structures projecting from the surface (Fig. 2a) and when ␣-dystroglycan
and dystrophin are double labeled on the same section,
dystrophin is found on the proximal side of the membrane frequently opposite dystroglycan labeling sites
(Fig. 2a).
MICROSCOPY OF THE DYSTROPHIN CYTOSKELETON
133
Fig. 2. a. Immunogold labeling of ␣-dystroglycan (10nm particles).
Alpha-dystroglycan localizes to the outer surface of the sarcolemma
usually on structures projecting from the surface. b. Double immuno-
gold labeling of ␣-dystroglycan (15nm particles) and dystrophin.
Dystrophin (10nm particles) localizes to the inner surface of the
sarcolemma. (Scale bar ⫽ 100nm).
Immunogold labeling of ␤-dystroglycan shows that
the gold conjugate lies on the membrane or extremely
close to it, on either side of the membrane (Fig. 3a)
(Cullen et al., 1998). The primary antibody that was
used was raised against 15 of the last 16 amino acids at
the C-terminus of ␤-dystroglycan, i.e., at the cytoplasmic end of the molecule. The observation that the gold
conjugate is sometimes positioned on the extracellular
side of the membrane can probably be attributed to the
fact that the immunogold probe can lie up to the length
of two antibody molecules distant, i.e., ⬃20 nm, from
the epitope that it is labeling. Because the antibody was
to the C-terminus and because the C-terminus is involved in binding to dystrophin, it could be predicted
that double labeling of ␤-dystroglycan and dystrophin
would show close colocalization. Double labeling experiments using antibodies to the C-termini of both molecules show that this is indeed the case (Fig. 3b) and
134
S.C. WATKINS ET AL.
Fig. 3. a. Immunogold labeling of ␤-dystroglycan. The gold conjugate lies on or very near to both sides
of the sarcolemma. b. Double immunogold labeling of the C-termini of ␤-dystroglycan (10nm particles)
and dystrophin (5nm particles). (Scale bar ⫽ 100nm).
careful measurement of the distance and orientation of
one gold probe with respect to the other shows that the
two epitopes must be separated by ⬍15 nm (Cullen et
al., 1998).
While much new information can be gained by performing immunolabeling on cryosections, there is a
restriction in what the technique can achieve because
one is viewing a membrane in what is essentially just
one linear dimension. If the investigator wishes to view
the arrangement of a protein at a membrane face on, in
two dimensions, a different technique needs to be
employed. The technique of choice is freeze-fracture in
which membranes are split down the center of the
phospholipid bilayer to expose membrane-associated
proteins. There are histological artifacts associated
with combining this technique with immunolabeling
because of the thawing required for the antibody incubation stages (Abeysekera and Robards, 1995) but
preliminary studies examining the relationship between ␤-dystroglycan and dystrophin are encouraging.
Figure 4a shows a platinum-carbon replica of an
extracellular face of a muscle plasma membrane that
was immunogold labeled for ␤-dystroglycan. The 10-nm
gold particles are distributed over the membrane in a
pattern that can be shown statistically to be nonrandom. When the nearest-neighbor distances between
labeling sites from a number of such electronmicrographs are measured, the resulting histogram of repeating ␤-dystroglycan molecules has a clear mode at
approximately 140 nm, i.e., at value close to that
obtained from cryosections (Fig. 4b). Figure 4c. shows a
fracture face that has been double labeled for ␤-dystro-
MICROSCOPY OF THE DYSTROPHIN CYTOSKELETON
135
glycan (10 nm gold) and dystrophin C-terminus (5 nm
gold). As was shown on the cryosections, these two
epitopes colocalize extremely closely. Interestingly, when
this procedure was repeated using an antibody to the
N-terminus of dystrophin, the colocalization is not seen
(S. Stevenson, personal communication).
It is instructive to examine to what extent the
localization studies carried out are in accord with the
models derived from biochemical studies. Examination
of a number of the published models shows that ␣-dystroglycan lies in an extracellular position binding to
laminin in the extracellular matrix (Matsumura and
Campbell, 1994; Ohlendieck, 1996; Ozawa et al., 1995;
Winder et al., 1995). The ultrastructural results are
entirely in accord with this, both when ␣-dystroglycan
is labeled by itself or with another protein. The immunogold labeling of ␤-dystroglycan indicates that it is a
transmembrane protein and, moreover, the close proximity to the C-terminus of dystrophin when double
labeling is carried out, is in agreement with dystrophin
binding at the end of the C-terminus of ␤-dystroglycan.
The resolution of freeze fracture experiments is high,
but labeling sensitivity is still relatively poor and limits
any major conclusions of direct molecular interactions.
However, the distribution of nearest-neighbor distances suggest that the dystroglycans, along with other
components of the DGC, may be separated by dystrophin molecules. Analogous with spectrin, dystrophin is
usually depicted as an anti-parallel dimer with apposing N- and C-termini in close proximity to each other.
However, it has been alternatively suggested, on the
basis of amino acid sequence analysis, that there is
little evidence for dimerization (Chan and Kunkel,
1997; Rybakova and Ervasti, 1997; Winder et al., 1995,
1996) and thus the N-terminus may be positioned at
some distance from the C-terminus. The doublelabeling experiments in which the N-terminus of dystrophin and the C-terminus of ␤-dystroglycan were not
seen to colocalize, lends support to this interpretation.
While the absence of dystrophin is central to the
pathogenesis of DMD, the exact effect of this loss on the
functional physiology of the muscle cell is still not
understood. As the members of the DGC are tightly
interlinked in an interdependent fashion, it is possible
that the pathological sequelae of mutations in the
dystrophin gene may have as much to do with the
cellular functions of the associated proteins as with
dystrophin itself.
It is highly likely the dystroglycans are essential to
normal homeostasis (particularly given their ubiquitous distribution) and that any mutations causing
complete dystroglycan loss are lethal (Jung et al.,
1995). Two studies support this assertion. In one,
myotubes derived from cells transfected with an antisense dystroglycan construct show a preferential loss of
␣-dystroglycan and undergo apoptosis in vitro (Mon-
Fig. 4. a. Platinum-carbon replica of an extracellular face of a
sarcolemma immunogold labeled for ␤-dystroglycan. (Scale bar ⫽
100nm). b. Nearest-neighbor distances between labeling sites of
␤-dystroglycan have a mode of approximately 140nm. c. Freezefracture immunolabeling of dystrophin (5nm particles with open
arrows) and ␤-dystroglycan (10nm particles and closed arrows). (Scale
bar ⫽ 100nm).
136
S.C. WATKINS ET AL.
tanaro et al., 1999). In another, homozygous mouse
embryos with a null allele of the dystroglycan gene
exhibit disrupted patterns of laminin and collagen and
undergo early death in embryonic development due to
the disruption of the Reichert’s membrane (Williamson
et al., 1997). Other than a single report that mutations
in ␤-dystroglycan cause a limb-girdle muscular dystrophy (LGMD) as demonstrated by immunostaining and
immunoblot analysis (Salih et al., 1996), there have
been no other demonstrated causative associations
between mutations in the dystroglycans and non-lethal
myopathies.
Sarcoglycan Complex
The sarcoglycan complex is comprised of ␣-sarcoglycan (previously termed adhalin and 50 DAG), ␤-sarcoglycan (A3b), ␦-sarcoglycan, and ⑀-sarcoglycan. Alphasarcoglycan and ␥-sarcoglycan have been found to be
present only in skeletal and cardiac muscle by immunoblotting (Yamamoto et al., 1994) whereas ␤-, ␦- and
⑀-sarcoglycan are ubiquitously distributed (Ettinger et
al., 1997; McNally et al., 1998).
The sarcoglycan complex is considered to be associated with the dystrophin axis via a lateral association
with the dystroglycan complex, but the exact binding
sites between the sarcoglycans and dystroglycans have
yet to be identified (Ozawa et al., 1995; Suzuki et al.,
1994). The cDNA of ␣-sarcoglycan was cloned and
shown to include a region coding for a transmembrane
domain (McNally et al., 1994; Roberds et al., 1993) and
⑀-sarcoglycan shares a high structural homology to
␣-sarcoglycan (McNally et al., 1998). Madhaven and
Jarrett (1995) found that ␣-sarcoglycan was bound by
the cysteine-rich sequences of dystrophin using overlay
binding experiments to probe gel blots of purified rabbit
muscle DGC.
The first immunocytochemical reports of the localization of ␣- and ␥-sarcoglycan appeared quickly after
their discovery (Ohlendieck and Campbell, 1991) and
have been followed by a large number of papers incorporating immunofluorescence studies, many comparing
diseased and normal muscle and some reporting developmental changes (e.g., Di Blasi et al., 1996; Matsumura et al., 1992a,b, 1993a,b; Noguchi et al., 1995;
Vater et al., 1995; Yamamoto et al., 1994). Without
exception, these papers show that the sarcoglycans are
normally present at the sarcolemma of the myofibers.
Using an antibody that recognizes an epitope in the
extracellular portion of ␣-sarcoglycan, we carried out
immunogold labeling on control and DMD biopsy
samples and detected the gold probe close to the
sarcolemma and mostly on its peripheral side (Cullen et
al., 1994). A more recent study, using triple immunogold
labeling shows that ␣-sarcoglycan localizes to the outer
surface of the sarcolemma and that ␤- and ␦-sarcoglycan localize to the inner surface (Wakayama et al.,
1999).
A deficiency of ␣-sarcoglycan has been associated
with severe childhood autosomal recessive muscular
dystrophy (SCARMD), a disease whose symptoms and
pathology are very similar to DMD (Ben Hamida et al.,
1983). SCARMD was first described in North African
(Ben Hamida et al., 1983) and Middle Eastern countries (Salih et al., 1993) but has since been described in
European (Fardeau et al., 1993), American (Passos-
Bueno et al., 1993a,b, 1999; Vainzof et al., 1999a), Asian
(Hayashi et al., 1995; Higuchi et al., 1994, 1997) and
Australian families (Jones et al., 1998). Since its first
association with ␣-sarcoglycan deficiency (Matsumura
et al., 1992a), it has become clear that SCARMD is not a
single homogeneous genetic disease, but a group of
muscular dystrophies with similar phenotypes caused
by damage to several different genes (Carrie et al.,
1997; Duclos et al., 1998, Holt and Campbell, 1998;
Mizuno et al., 1995; Passos-Bueno et al., 1996; Romero
et al., 1995).
Linkage analysis reveals that some cases of sarcoglycanopathy (now termed limb girdle muscular dystrophy
type 2C) are linked to chromosome 13q12 and display a
deficiency of ␥-sarcoglycan; some SCARMD phenotypes
are independent of any mechanism involving the primary gene defect (Ozawa et al., 1995). Human patients
with mutations in the ␥-sarcoglycan present lower
levels of dystrophin, ␣- and ␤-sarcoglycan, and disrupted laminin patterns, thus showing that abnormalities of dystrophin may, in some cases, be a secondary
phenomenon (Jones et al., 1998; Vainzof et al., 1999b).
Ultrastructural studies in a case of ␥-sarcoglycanopathy (LGMD2C) suggest that loss or reduction of the
sarcoglycans results in an instability in the plasma
membrane, which becomes abnormally indented and
convoluted (Hassoni and Cullen, 1999). A murine model
of ␥-sarcoglycan deficiency produces a dystrophic phenotype that does not display a secondary loss of dystrophin or of laminin and dystroglycan (Hack et al., 1998).
The mice do show a deficiency of ␤- and ␦-sarcoglycan,
thus providing additional evidence that muscular dystrophy can occur in the absence of alterations in
dystrophin levels.
A form of autosomal recessive muscular dystrophy in
the BIO 14.6 hamster, another animal model of sarcoglycanopathy, has been reported to be associated with a
selective loss of ␦-sarcoglycan and has now been shown
to lack the entire sarcoglycan complex (Mizuno et al.,
1995). The primary defect is due to a large deletion in
the 5’ region of the gene (Sakamoto et al., 1997). Using
intravenous injection of Evans blue dye as an in vivo
tracer assay, Straub et al. (1998) found that extensive
fiber damage takes place in the skeletal and cardiac
muscle of the BIO 14.6 hamster due to the disruption of
the sarcolemma. Delta-sarcoglycan gene transfer has
recently been shown to restore the sarcoglycan complex
and sarcolemmal integrity in hamsters (Crosbie et al.,
1999; Greelish et al., 1999; Holt et al., 1998; Li et al.,
1999).
Loss of ␤-sarcoglycan or ␦-sarcoglycan results in the
disruption of the sarcoglycan-sarcospan complex in
knockout mice. Beta-sarcoglycan-deficiency leads to
progressive muscular dystrophy with extensive degeneration and regeneration (Araishi et al., 1999). Deltasarcoglycan deficiency affects both skeletal and smooth
muscle, the latter of which leads to the perturbation of
vascular function and cardiomyopathy (Coral-Vazquez
et al., 1999)
Syntrophin Complex
The syntrophins constitute a heterogeneous group of
58-kDa intracellular dystrophin-binding and membrane-associated proteins. A 58-kDa peripheral membrane protein was first identified in the Torpedo electric
MICROSCOPY OF THE DYSTROPHIN CYTOSKELETON
organ (Froehner et al., 1987) and this has since been
placed in the syntrophin family. Syntrophin isolated
from rabbit skeletal muscle appears as a triplet by
one-dimensional SDS electrophoresis and, when separated by two-dimensional gel electrophoresis, appears
as two clusters of 58-kDa proteins with different isoelectric points (pI). One, ␣-syntrophin, is slightly acidic
(pI ⫽ 6.4) and the other, ␤-syntrophin, is quite basic
(pI ⫽ 9.0) (Yamamoto et al., 1993). From predicted
amino acid sequences and calculated isoelectric points,
␤-syntrophin has been shown to have two isoforms: ␤1
(p1 ⫽ 9.0) and ␤2 (pI ⫽ 9.4) (Ahn et al., 1996).
Alpha1-syntrophin is located on the inner surface of
the sarcolemma of skeletal and cardiac muscle and
interacts with the carboxy-terminal region of dystrophin (Ahn et al., 1996; Castello et al., 1996; Suzuki et
al., 1995; Yang et al., 1995;). Beta1- and ␤2-syntrophin
are found in a variety of tissues including muscle (Ahn
et al., 1996). As all three syntrophins bind to dystrophin
in vitro, it is likely that their differential localization is
regulated by interactions with other proteins.
Little is known about the normal function of the
syntrophins and their association with neuromuscular
disease. The syntrophins are down-regulated in the
absence of normal dystrophin and their absence may
contribute to the dystrophic phenotype (Peters et al.,
1994; Tachi et al., 1997). As shown by immunogoldlabeling electron microscopy, ␣1-syntrophin colocalizes
with nitric-oxide synthase (nNOS) at the sarcolemma
and may bind nNOS (Wakayama et al., 1997). Until
recently, the absence of sarcolemmal nNOS in DMD
patients and mdx mice was thought to contribute to
myofiber necrosis (Brenman et al., 1995; Chang et al.,
1996; Haycock et al., 1996). In nNOS-dystrophin null
mdx mice, typical dystrophic pathogenesis takes place
in the absence of nitric oxide synthesis (Crosbie et al.,
1998). However, in ␣1-syntrophin knockout mice, nNOS
is absent at the sarcolemma but the structural and
functional properties of the muscles remain relatively
normal (Kameya et al., 1999), thus making the role of
the syntrophins even more obscure.
Sarcospan
Sarcospan, formerly known as A5 (Yoshida and
Ozawa, 1990), is a 25-kDa multisubunit sarcolemmal
complex. Northern blot analysis shows ubiquitous expression of a 4.5-kb transcript of sarcospan but a 6.5-kb
transcript is found exclusively in skeletal and cardiac
muscle. In skeletal muscle, sarcospan is enriched at
neuromuscular junctions and myotendinous junctions
(Crosbie et al., 1999). Topology algorithms predict that
sarcospan has four transmembrane spanning helices
with both N- and C-terminal domains located intracellularly, which render sarcospan structurally and functionally analogous to the tetraspan superfamily of
proteins (Crosbie et al., 1997). Tetraspans are thought
to facilitate control of cell growth, migration, and
adhesion through interactions between transmembrane proteins (Laguaudriere-Gesbert et al, 1997; Maecker et al., 1997; Wice and Gordon, 1995; Wright and
Tomlinson, 1994). Whether or not sarcospan provides
the same functions is not known.
Sarcospan precipitates with the dystrophin-glycoprotein complex in a wheat germ agluttinin-Sepharose ion
exchange column (Crosbie et al, 1997) and is dependent
137
upon the presence of the sarcoglycan complex for its
expression. In ␦-sarcoglycan deficient BIO14.6 hamsters and in ␣-sarcoglycan null mice, both the sarcoglycan complex and sarcospan are missing from the sarcolemma. Injection of adenovirus encoding ␦-sarcoglycan
into BIO 14.6 muscles restores sarcospan as well as the
sarcoglycan complex mentioned previously (Crosbie et
al., 1999).
The role of sarcospan in muscle disease has yet to be
established. It is known that the presence of sarcospan
is greatly reduced in DMD muscle compared with
normal muscle (Crosbie et al., 1997). This reduction
may contribute to the dystrophic phenotype in DMD. In
addition, Crosbie et al. (1997) propose that mutations
in the sarcospan gene cause congenital fibrosis of the
extraocular muscles (CFEOM) as the gene encoding
sarcospan maps to the same narrow region of chromosome 12 as the disease locus for CFEOM.
Alpha-Dystrobrevin
Alpha-dystrobrevin is a dystrophin homologue that
coprecipitates with the both the acetylcholine receptor
complex and the dystrophin glycoprotein complex (Blake
et al., 1996; Sadoulet-Puccio et al., 1997). In human
skeletal muscle, alternative splicing of the dystrobrevin
locus on chromosome 18q12.1–12.2 (Ambrose et al.,
1997; Sadoulet-Puccio et al., 1996) produces five isoforms of dystrobrevin, two of which are found in skeletal muscle: full-length ␣-dystrobrevin-1 (84 kDa) and a
C-terminus truncated ␣-dystrobrevin-2 (65 kDa). During development, ␣-dystrobrevin-1 is found on the cell
surface and acetylcholine-rich receptor areas of the
myotube. Following agrin-induced acetylcholine receptor clustering, ␣-dystrobrevin-1 becomes relocalized
beneath the sarcolemma and restricted to the synaptic
region of the myofibers (Nawrotzki et al., 1998). Alphadystrobrevin-2 is concentrated at the synapse but is
also present on the extrasynaptic sarcolemma in mature muscle (Peters et al., 1998). The association of
␣-dystrobrevin with the synaptic region suggests that it
may have a role in synapse formation and/or stability
(Sadoulet-Puccio et al., 1997).
The amount of ␣-dystrobrevin is severely reduced in
DMD patients and in patients with LGMD arising from
loss of one or all of the sarcoglycans (Metzinger et al.,
1997; Puca et al., 1998) as well as in mdx mice
(Nawrotzki et al., 1998) and thus may contribute to the
dystrophic phenotype. Other than this possibility, no
disease has been associated with the loss or mutation of
␣-dystrobrevin.
Alpha-Actinin-2
The most recently named component of the dystrophin-glycoprotein complex is ␣-actinin-2 (Hance et al.,
1999). The C-terminal region of dystrophin interacts
with skeletal muscle ␣-actinin-2 and actin in the yeast
two-hybrid system. Alpha-actinin-2 colocalizes with the
sarcolemma of tissue culture cells and purified sarcolemma vesicles. Alpha-actinin-2 links overlapping actin
filaments every 38 nm at the vertebrate muscle Z-lines
(Yamaguchi et al., 1985) Alpha-actinin-2 also binds to
the ␤1 subunit of integrin (Otey et al., 1990) thus
providing critical links between F-actin, dystrophin,
and the transmembrane protein, integrin (Pavalko et
138
S.C. WATKINS ET AL.
al., 1990). There are yet no known diseases directly
associated with the mutations in the ␣-actinin-2 gene.
Conclusions
Over the last 12 years, our understanding of the
muscle fiber plasma membrane cytoskeleton has grown
enormously, from the first identification of the dystrophin molecule, to the recognition that the plasma
membrane of an actively and constantly contracting
cell is supported by an elaborate complex of cytoskeletal
molecules. It is certain, given the selective and debilitating diseases that are associated with each of the
molecules, that the functionality of each component of
the membrane cytoskeleton is complex. However, it is
somewhat surprising that while we have been able to
identify many (though probably not all) of the components of the muscle fiber cytoskeleton, we have little
idea of the true function of each member of the complex.
Since Rowland’s first suggestion of the ‘‘membrane
hypothesis’’ of DMD (1981) it was apparent that a
failure of maintenance of integrity of the muscle fiber
plasma membrane may be central to the pathology of
DMD. The recognition that dystrophin was specifically
associated with the plasma membrane, gave convenient support to the notion that its function was to
protect the muscle fiber from damage during repeated
cycles of contraction and relaxation. However, it is clear
that the role(s) of the members of the plasma membrane cytoskeleton may go far beyond this simple
function. Perhaps the most intriguing feature of mutations of the complex, which suggest functionalities far
beyond simply protecting the cell membrane from damage, is the diversity of disease phenotypes caused by
mutations of the complex. Selective mutations give rise
to DMD, congenital muscular dystrophy, limb girdle
muscular dystrophy, or oculo-pharangeal muscular dystrophy. Why should selective mutations cause diverse
and selected dystrophies of different muscles? Unfortunately, the solution to this question must await resolution of the actual molecular function of each member of
the growing family of proteins that make up the
dystrophin cytoskeleton.
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