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First published online 23 April 2008
doi: 10.1242/dev.015818
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1 The Scripps Research Institute, Department of Cell Biology and Institute of
Childhood and Neglected Disease, La Jolla, CA 92037, USA.
2 University of California and Veterans Administrative Centers, Department of
Orthopaedics and Bioengineering, San Diego, CA 92037, USA.
3 University of Leicester, Department of Biochemistry, Leicester LE1 7RH,
UK.
4 The Scripps Research Institute, Microscopy Core Facility, La Jolla, CA 92037,
USA.
* Author for correspondence (e-mail: umueller{at}scripps.edu)
Accepted 31 March 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7β1 integrin cause myopathy in humans. In mice, β1
integrins control myoblast fusion, the assembly of the muscle fiber
cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The
effector molecules that mediate β1 integrin functions in muscle are not
known. Previous studies have shown that talin 1 controls the force-dependent
assembly of integrin adhesion complexes and regulates the affinity of
integrins for ligands. Here we show that talin 1 is essential in skeletal
muscle for the maintenance of integrin attachment sites at MTJs. Mice with a
skeletal muscle-specific ablation of the talin 1 gene suffer from a
progressive myopathy. Surprisingly, myoblast fusion and the assembly of
integrin-containing adhesion complexes at costameres and MTJs advance normally
in the mutants. However, with progressive ageing, the muscle fiber
cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles
show defects in the ability of talin 1-deficient muscle to generate force.
Collectively, our findings show that talin 1 is essential for providing
mechanical stability to integrin-dependent adhesion complexes at MTJs, which
is crucial for optimal force generation by skeletal muscle.
Key words: Integrin, Talin, Muscular dystrophy, Myopathy, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Durbeej and Campbell,
2002; Durbeej et al.,
1998; Straub et al.,
1997). Far less is known about the function of integrins in
muscle. Integrins are heterodimeric ECM receptors consisting of - and
β-subunits (Hynes, 1992).
Skeletal muscle fibers in vertebrates express many integrin subunits,
including the β1-subunit and its partners 1, 3, 4,
5, 6, 7 and v
(Gullberg et al., 1998).
β1 integrins appear to have partially redundant functions in skeletal
muscle. Accordingly, inactivation of all /β1 integrins by
CRE/lox-mediated ablation of the β1-subunit gene causes defects in
myoblast fusion and sarcomere assembly that are not observed in muscle lacking
individual integrin -subunits
(Schwander et al., 2003). Of
all integrin -subunit gene knockout mice, muscle defects have only been
observed for mice with mutations in the integrin 5- and
7-subunit genes, and the phenotypes manifest later than in
β1-deficient mice. Chimeric mice that lack the integrin 5β1
in muscle develop dystrophic symptoms
(Taverna et al., 1998).
Mutations in the gene for the murine integrin 7-subunit cause defects
in myotendinous junctions (MTJs) (Mayer et
al., 1997) and humans with mutations in the integrin
7-subunit gene suffer from myopathy
(Hayashi et al., 1998).
The cytoplasmic domains of integrins bind to many cytoskeletal and
signaling proteins (Geiger et al.,
2001; Liu et al.,
2000), raising questions about the specific contributions of
individual proteins to integrin function. Talin 1 is a major integrin
effector. It binds to the cytoplasmic domain of several integrin
β-subunits and connects β1 integrins at focal adhesions to the actin
cytoskeleton (Critchley,
2000). Talin 1 binds to focal adhesion components such as vinculin
and focal adhesion kinase (FAK; Ptk2 - Mouse Genome Informatics), two
important regulators of actin dynamics
(Mitra et al., 2005;
Ziegler et al., 2006).
Importantly, the assembly of focal adhesions is dependent on mechanical force,
which regulates recruitment of vinculin to focal adhesions
(Balaban et al., 2001;
Choquet et al., 1997;
Galbraith et al., 2002;
Riveline et al., 2001). Talin
1 is crucial for force-dependent vinculin recruitment, and for the
strengthening of interactions between integrins and cytoskeletal proteins
(Giannone et al., 2003). In
addition, in vitro and in vivo studies have shown that talin 1 modulates the
ligand-binding activity of the extracellular domain of integrins by a
mechanism termed insight-out signaling
(Calderwood, 2004a;
Campbell and Ginsberg, 2004;
Nieswandt et al., 2007;
Petrich et al., 2007).
In skeletal muscle fibers, talin 1 is localized to costameres and MTJs
(Tidball et al., 1986), and
its expression is regulated by mechanical loading
(Frenette and Tidball, 1998).
However, the function of talin 1 in vertebrate muscle is unclear because mice
with a mutation in the talin 1 (Tln1) gene die during gastrulation
(Monkley et al., 2000).
Vertebrates contain a second talin gene (Tln2) encoding talin 2
(McCann and Craig, 1997;
McCann and Craig, 1999;
Monkley et al., 2001), the
function of which in skeletal muscle is likewise unknown. In vitro studies
show that expression of talin 2 is upregulated during myoblast differentiation
into myotubes, whereas talin 1 expression remains unchanged during
differentiation (Senetar et al.,
2007). The genomes of C. elegans and D.
melanogaster contain only one talin gene, which is essential for the
attachment of muscle fibers to surrounding tissue
(Brown et al., 2002;
Cram et al., 2003). The
invertebrate ortholog of the vertebrate integrin β1-subunit gene plays a
similar role, indicating that talin 1 mediates integrin functions in
invertebrate muscle (Brabant et al.,
1996; Brown, 1994;
Brown et al., 2002;
Cram et al., 2003;
Gettner et al., 1995;
Lee et al., 2001;
Leptin et al., 1989;
Volk et al., 1990). However,
the mechanism by which defects in talin lead to the perturbation of adhesion
sites is unclear.
To define the function of talin 1 in skeletal muscle of vertebrates, we have taken a genetic approach and crossed mice carrying a floxed Tln1 allele with mice expressing CRE in developing skeletal muscle. We show here that talin 1 is not essential for the assembly of integrin β1-dependent adhesion complexes at costameres and MTJs. Instead, talin 1 plays an important role in stabilizing adhesion complexes at MTJs, thereby providing resistance against mechanical stress that is exerted during muscle contraction and relaxation. Surprisingly, although talin 2 is abundantly expressed in skeletal muscle, it cannot compensate for loss of talin 1, suggesting that the two talin isoforms are not entirely functionally interchangeable.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Nieswandt et al.,
2007; Petrich et al.,
2007; Schwander et al.,
2003). The mice were on a mixed 129xC57Bl/6 genetic
background. For genotyping, tail DNA was analyzed by PCR using primers for
Tln1 as indicated in Fig.
1: (a) 5'-AAGCAGGAACAAAAGTAGGTCTCC-3'; (b)
5'-GCATCGTCTTCACCACATTCC-3'; and (c)
5'-TAGAGAAGGTTCAGCTGTCAGGG-3'.
Real-time PCR
Transcript levels for talin 1 and talin 2 were determined by quantitative
real-time PCR (RT-PCR). RNA from gastrocnemius muscle from 5-day-old
(n=3) and 6-month-old (n=3) C57Bl/6 mice was prepared using
Trizol LS (Invitrogen). RNA (1.6-2 µg) was reverse-transcribed using the
Superscript III System (Invitrogen). Transcripts were quantitated by RT-PCR
using Chromo4 (MJ Research) and SYBR Green (Applied Biosystems). Two sets of
primers were used for each transcript: Talin 1 set 1,
5'-GGAAATCTGCCGGAGTTTGG-3' and
5'-TTGGCTGTTGGGGTCAGAGA-3'; Talin 1 set 2,
5'-GGGCTGGAGGGAGATGAAGA-3' and
5'-AGAGCCGTGCTCCACTTTCC-3'; Talin 2 set 1,
5'-AAAACCCGAATGAGCCTGTGA-3' and
5'-GAAATCCCTGCCATTGACTCG3'; Talin 2 set 2,
5'-GAAAACCCGAATGAGCCTGTG-3' and
5'-GAAATCCCTGCCATTGACTCG-3'. Primers spanned at least one
exon/intron boundary and were verified in BLAST searches for specificity. To
determine primer efficiency, RNA was diluted serially and linear regression
analysis was applied. The slope of the resulting curve was used as a measure
of PCR efficiency [E=(10-1/slope)-1]. Efficiencies of the primer
pairs were 95-98%. Both set of primers produced comparable results and data
from one set are shown. mRNA levels were determined using the comparative
threshold (Ct) method (Livak and
Schmittgen, 2001) and normalized to glyceraldehyde-3-phosphate
dehydrogenase or β-actin mRNA.
Antibodies, immunohistochemistry and electron microscopy
Rabbit antibodies to talin 1 and talin 2 were produced at Animal Pharm
Services (Healdsburg) using peptides corresponding to amino acids 1830-1850
(mouse talin 1) and 940-957 (mouse talin 2). Immunohistochemistry was carried
out as described (Schwander et al.,
2003) using the following antibodies: mouse monoclonal antibodies
against vinculin (Sigma), dystrophin (clone NCL-DYS2, Novocastra), myosin
heavy chain fast (Sigma) and Ilk (Li et
al., 1999); rabbit polyclonal antibodies against collagen IV
(Chemicon), laminin (Chemicon), talin 1, talin 2 and 7 integrin (kindly
provided by U. Meyer, University of East Anglia, Norwich, UK). Electron
microscopy was carried out as described
(Schwander et al., 2003).
Western blotting
Gastrocnemius muscles were lysed in 1% Triton X-100 in PBS supplemented
with protease inhibitor cocktail (Roche Diagnostics). Equal amounts of
protein, as determined using the Micro BCA Kit (Pierce), were analyzed by
western blotting with the following antibodies: talin 1, vinculin (Sigma),
Erk1/2 total and phosphorylated (Cell Signaling Technology, Danvers, MA), FAK
and FAK-PY397 (UBI). To verify equal loading, membranes were stripped with
Stripping Solution (Chemicon) and probed with antibodies to -tubulin
(Sigma). Signal intensity was determined by densitometry using MetaMorph
Optical Density analysis software (Molecular Devices). Films from three
independent experiments were scanned.
Evans Blue Dye (EBD) uptake and creatine kinase assay
EBD (Sigma) was dissolved in PBS (10 mg/ml) and injected via the tail vein
using 50 µl per 10 g body weight. After 5 hours, mice were sacrificed,
muscles were dissected and visually inspected. Tissues were fixed overnight
with 4% paraformaldehyde (PFA). Sections (12 µm) of gastrocnemius and
diaphragm muscles were analyzed using an Olympus BX50WI epifluorescence
microscope. Measurements of creatine kinase (CK) levels from blood samples of
6- to 7-month-old mice were performed by Antech Diagnostics (Irvine, CA). To
evaluate EBD uptake after exercise, 5-month-old Tln1HSA-CREko and
wild-type mice (4-5 per genotype) were injected with EBD solution and 1 hour
later subjected to exercise. Mice were allowed to warm up by running at 10
meters/minute on a 0° incline for 5 minutes. After a 5 minute rest, mice
were run at 17 meters/minute on a 0° incline for 30 minutes. Animals were
returned to their cages and sacrificed 24 hours after exercise, muscles were
dissected and EBD incorporation evaluated as described above.
Cardiotoxin experiments
50 µl of 10 µM cardiotoxin (Sigma-Aldrich) were injected into the
calf muscles of 8- to 12-week-old mice. The controlateral leg was injected
with 50 µl PBS as a control. Muscles were isolated at 5, 10 and 21 days
following injection, fixed with 4% PFA and embedded in paraffin. Sections were
prepared and stained with Hematoxylin and Eosin and morphology of myofibers
evaluated for regeneration.
Isometric and eccentric contraction cycles
Mice were sacrificed and the fifth EDL (extensor digitorum longus) muscle
was dissected in Ringer's solution (137 mM NaCl, 5 mM KCl, 1 mM
NaH2PO4, 24 mM NaHCO3, 2 mM CaCl2,
1 mM MgSO4, 11 mM glucose, 10 mg/l curare). Using 8-0 silk sutures,
the muscle origin was secured by the tendon at a rigid post and the insertion
was secured by the tendon to the arm of a dual-mode ergometer (model 300B,
Aurora Scientific). Muscle was bathed in Ringer's solution, which was
determined to be 10°C during measurements. Muscle length (ML) was
measured, increased by 10%, and sarcomere length was measured by laser
diffraction at 632.8 nm and adjusted to 3.0 µm. The EDL muscle was
subjected to three passive stretches, one every 3 minutes, each time stretched
10% of ML at the velocity of 0.7 ML/second. Maximum isometric tension was
measured by applying a 400 millisecond train of 0.3 millisecond pulses
delivered at 100 Hz while ML was maintained constant. This measurement was
repeated again after 5 minutes. The stimulation frequency chosen for these
isometric contractions and the other contractions that follow was 100 Hz, as
this frequency produced a fused tetanic contraction, but was low enough to
prevent excessive fatigue. Each muscle underwent ten eccentric contractions
(ECs), one every 3 minutes, each time stretched to 15% of ML at the velocity
of 2 ML/second. Following the EC cycle, the ML was returned to its initial
value and post-eccentric isometric tension was measured by applying a 400
millisecond train of 0.3 millisecond pulses, delivered at 100 Hz while ML was
maintained constant. This measurement was repeated twice, at 5 minute
intervals. Muscle weight was measured to determine the physiological
cross-sectional area (Sam et al.,
2000). Data were acquired using a CA-1000 data board and analyzed
in LabVIEW 7.0 (National Instruments, Austin, TX).
All force and displacement records were stored for offline analysis. Work
was calculated from the force-time and displacement-time records by creating
the force-displacement relationship and integrating this relationship
according to the equation:
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Mice with different recombination events were recovered. In
one line, only the neo cassette was lost, maintaining floxed exons
1-4 (referred to as conditional allele, Tln1flox). In a
second line, the floxed region including the neo cassette and exons
1-4 were removed (referred to as deleted allele, Tln1-)
(Fig. 1A).
Tln1flox/flox mice showed no overt abnormality,
demonstrating that the presence of loxP sites did not affect Tln1
function (data not shown). Tln1-/- embryos died at
gastrulation (6.5-7.5 days post-coitum) (data not shown), thus phenocopying
mice in which the Tln1 allele has been ablated
(Monkley et al., 2000).
To abolish talin 1 expression in skeletal muscle, we took advantage of mice
that express CRE under the control of the human skeletal -actin
(HSA; ACTA1 - Human Gene Nomenclature Database) promoter
(HSA-CRE) (Leu et al.,
2003; Schwander et al.,
2003). We have previously shown that the HSA-CRE
transgene is expressed as early as embryonic day (E) 9.5 in somites, and
throughout all muscle groups by E14.5
(Schwander et al., 2003).
Tln1flox/flox were mated with
Tln1flox/+ mice that also contained a HSA-CRE
transgene (Tln1flox/+ HSA-CRE+/-) to obtain
mutant (HSA-CRE+/- Tln1flox/flox, referred to
as Tln1HSA-CREko mice) and control (HSA-CRE-/-
Tlnflox/flox or HSA-CRE-/-
Tlnflox/+ referred to as wild type because their phenotype was
indistinguishable from C57Bl/6 mice) offspring. To confirm recombination of
the Tln1flox allele in Tln1HSA-CREko mice, we
performed PCR analysis of DNA extracted from skeletal muscle of E14.5 and
postnatal day (P) 2 mice; Tln1flox/flox and
Tln1flox/+ mice that lacked the HSA-CRE transgene
were analyzed in parallel as controls. A 707 bp band corresponding to the
recombined Tln1flox allele was detected in skeletal muscle
of Tln1HSA-CREko mice as early as E14.5
(Fig. 1B), but not in
non-muscle tissue (data not shown) or control mice
(Fig. 1B).
Talin 1 expression is abolished in skeletal muscle
To confirm that we had inactivated Tln1 in Tln1HSA-CREko
mice, we raised in rabbits antibodies that are specific for talin 1 and for
talin 2 (see Materials and methods) and analyzed protein expression in muscles
including gastrocnemius, tibialis and diaphragm. At late embryonic and early
postnatal ages when skeletal muscle fibers are actively forming, talin 1
expression was detected at the sarcolemma of wild-type mice but not in the
mutants (Fig. 2A,B, arrows).
Some cells in the mutants expressed talin 1
(Fig. 2A,B arrowheads), but we
identified them by morphology and co-staining with antibodies to VE-cadherin
(cadherin 5) as endothelial cells (Fig.
2E). Similarly, in muscle from 6-month-old mice, talin 1
expression was not detectable in muscle fibers
(Fig. 2C,D) and only persisted
in blood vessels (Fig. 2D,
arrowhead). Western blot analysis carried out with extracts obtained from
skeletal muscle of P2 and P8 Tln1HSA-CREko mice further confirmed
that talin 1 expression was essentially abolished
(Fig. 2F). Residual expression
was likely to be due to talin 1 expressed in blood vessels. In addition, we
detected talin 2 expression by western blot
(Fig. 2F) and
immunohistochemistry (Fig.
6M,N), but we failed to detect compensatory talin 2 upregulation
upon removal of talin 1 (Fig.
2F, Fig. 6M,N).
The expression levels of the Tln1 and Tln2 transcripts in
gastrocnemius muscle were also determined by real-time PCR. Expression levels
for Tln2 were 4- to 5-fold higher than for Tln1 in both
5-day-old (Tln1, 1.03±0.16; Tln2, 4.70±0.44;
P=0.004) and 6-month-old muscle (Tln1, 1.00±0.06;
Tln2, 4.1±0.31; P=0.002)
(Fig. 2G). This analysis
corroborates earlier findings indicating that Tln2 mRNA is the
predominant isoform expressed in skeletal muscle
(Monkley et al., 2001;
Senetar et al., 2007).
Progressive accumulation of dysmorphic muscle fibers in Tln1HSA-CREko mice
Tln1HSA-CREko mice were born at the expected Mendelian ratio, were
viable and fertile, were indistinguishable in appearance from wild-type
littermates (data not shown), and grew at normal rates
(Fig. 2H). However,
histological examination revealed progressive defects in muscle fiber
morphology. We analyzed several muscle groups including gastrocnemius, soleus
and diaphragm collected from animals between P15 and 6 months. At P15 and P60,
all muscle groups appeared normal (Fig.
3A-H; data not shown); wild-type and Tln1-deficient
muscle fibers were regular in diameter. Morphological defects were obvious in
muscle of Tln1-deficient mice by 6 months of age, and were more
commonly observed in the diaphragm. In contrast to wild-type mice
(Fig. 3I,K), muscle fibers in
mutant animals appeared less regular in diameter and were enlarged
(Fig. 3J,L) and distorted
(Fig. 3M). Dysmorphic fibers
were also evident close to the MTJs of diaphragm muscle
(Fig. 3O,P). Centrally located
nuclei and an accumulation of interstitial cells were occasionally detectable.
However, unlike the situation in mice lacking expression of the integrin
7-subunit (Mayer et al.,
1997), a diffuse centronuclear myopathy was not observed in the
Tln1-deficient mice.
|
). In postnatal muscle, non-proliferating myoblasts are
localized between myofibers and the basal lamina, where they constitute the
satellite cell population. Upon muscle injury, satellite cells proliferate,
fuse and regenerate new myofibers, which are characterized by variable size
and centrally located nuclei (Shi and
Garry, 2006). Immunohistochemical analysis did not provide clear
evidence for talin 1 expression in satellite cells (data not shown). However,
satellite cells may express low levels of talin 1 that were difficult to
detect with our antibodies. Therefore, to evaluate whether the absence of a
centronuclear myopathy was caused by defects in satellite cell function, we
injected gastrocnemius muscles of 2-month-old mice with cardiotoxin and
analyzed muscle fiber morphology 5, 10 and 21 days later. At all time points
Tln1HSA-CREko and wild-type mice presented signs of effective muscle
regeneration. Five days after toxin injection, necrotic fibers and small
fibers with centrally located nuclei, indicative of regenerating fibers, were
evident and immune cell infiltrate was noted (see Fig. S1A-D in the
supplementary material). After 10 days, affected fibers presented with central
located nuclei, but muscle architecture was largely restored (see Fig. S1E,F
in the supplementary material). By 21 days, regeneration was complete (see
Fig. S1G,H in the supplementary material; data not shown). We conclude that
the absence of a centronuclear myopathy in Tln1HSA-CREko mice is not
caused by an impaired capacity of mutant muscle to regenerate.
Talin 1 is not essential to maintain sarcolemmal integrity
An increase in the fragility of the sarcolemma is observed in several forms
of muscular dystrophy (Carpenter and
Karpati, 1979; Schmalbruch,
1975; Straub et al.,
1997; Weller et al.,
1990). To evaluate damage to the sarcolemma, Evans Blue Dye (EBD)
was injected into 6-month-old Tln1HSA-CREko and wild-type mice, and
muscles were dissected after 5 hours to evaluate dye incorporation. Whereas
connective tissue presented a blue coloration, muscles including the diaphragm
(Fig. 3Q,R), gastrocnemius and
soleus (Fig. 3S,T; data not
shown) failed to accumulate EBD. Occasionally, a few EBD-stained fibers were
evident, independent of the genotype of the mice
(Fig. 3U, arrow).
Histologically, these EBD-positive fibers appeared damaged, possibly in
response to normal muscle usage. This, together with the positive coloration
of interstitial tissue, validated successful circulation of the dye.
EBD-positive fibers in damaged muscle appeared fluorescent by light microscopy
(Fig. 3X). In both wild-type
and Tln1-deficient muscle, fluorescence was only observed in tendons
and interstitial tissue, and not in muscle fibers
(Fig. 3V,W).
A more sensitive indication of membrane integrity is gained by evaluating
the efflux of intracellular proteins from damaged muscle fibers
(Rosalki, 1989). In
6-month-old Tln1HSA-CREko mice, serum creatine kinase was elevated
3-fold compared with levels in wild-type mice (1526.20±151.44 U/l
and 593.85±154.23 U/l, respectively)
(Fig. 3Y). This increase is
mild when compared with other mouse models of muscular dystrophy
(Sonnemann et al., 2006).
|
;
Hamer et al., 2002;
McNeil and Khakee, 1992;
Vilquin et al., 1998). EBD was
injected into 5- to 6-month-old mice and, 1 hour after injection, mice were
run on a treadmill for 30 minutes. No major dye incorporation was observed in
diaphragm and gastrocnemius muscle from wild-type (n=4) (see Fig.
S2A,C,F in the supplementary material) or Tln1HSA-CREko
(n=4) (see Fig. S2B,D in the supplementary material) mice. Mild EBD
accumulation was only observed in one gastrocnemius muscle in one mutant mouse
(see Fig. S1E in the supplementary material), and in the paraspinalis muscles
of a second mouse (see Fig. S1G in the supplementary material), but the damage
was mild in comparison with what has been observed in mice carrying mutations
affecting DGC proteins (Sonnemann et al.,
2006).
Collectively, these data suggest that sarcolemmal integrity is mildly
defective when the function of talin 1 is perturbed. Mild damage to the
sarcolemma was also observed in patients and mice with mutations in the gene
for the integrin 7-subunit (Hayashi
et al., 1998; Rooney et al.,
2006).
Talin 1 is not essential for the assembly of integrin complexes at costameres
Talin binds to proteins that are localized to costameres, including β1
integrins, vinculin, actin and FAK
(Critchley, 2000). Since
defects in muscle fiber morphology could result from altered assembly of
costameres, we assessed whether talin 1 ablation impaired costamere assembly
in vivo. We analyzed the distribution of costameric proteins in 6-month-old
mice by immunohistochemistry, but observed no obvious defects. β1
integrins and -actinin were localized normally, forming a regular array
(Fig. 4A-D). Talin 1 directly
binds to vinculin, but vinculin was still localized to costameres in
Tln1-deficient skeletal muscle fibers
(Fig. 4E,F). Consistent with
the lack of extensive sarcolemmal damage
(Fig. 3), localization of
dystrophin was also unaffected (Fig.
4G,H). Morphological defects of muscle fibers could be caused by
defects in the basement membrane around muscle fibers, but no obvious defects
were apparent in the localization of collagen type IV or laminin
(Fig. 4I-L). Sarcomere
integrity was also evaluated by electron microscopy. In both wild-type and
Tln1-deficient skeletal muscle fibers, the sarcomeres were well
organized, with Z-lines evident in both 1-month-old
(Fig. 4M,N) and 6-month-old
(Fig. 4O,P) mice. In
Tln1-deficient muscle fibers, the sarcomeres occasionally appeared
hypercontracted and M-bands were not always evident; the Z-line, which
includes β1-integrin-containing complexes and corresponds to costameres,
was always visible (Fig.
4O,P).
|
|
). Integrin
binding to ECM ligands leads to activation of FAK by autophosphorylation at
Tyr397, creating a binding site for Src, which phosphorylates focal adhesion
components and activates MAPK signaling
(Mitra et al., 2005). In
Tln1HSA-CREko mice, expression levels and phosphorylation of FAK at
Tyr397 were comparable to those of wild-type mice
(Fig. 5A,D). Phosphorylation
levels of Erk1/2 (Mapk3/Mapk1 - Mouse Genome Informatics) were also normal
(Fig. 5B,D). Finally, western
blot analysis with antibodies to vinculin and the integrin β1-subunit
revealed that expression levels of these proteins were unchanged in muscle
from Tln1HSA-CREko mice (Fig.
5C,D). Taken together, these data indicate that loss of talin 1 in
skeletal muscle does not lead to major defects in the assembly of costameres,
or in FAK and MAPK signaling.
Talin 1 is essential for the maintenance of the connection between integrins and myofilaments at MTJs
Alterations in muscle fiber morphology could result from defects at MTJs.
Ultrastructural analysis showed that the structure of the lamina densa at
MTJs, which is absent in mice with genetic ablation of β1 integrins and
altered in 7 integrin mutants
(Miosge et al., 1999;
Schwander et al., 2003), was
still visible in Tln1HSA-CREko mice
(Fig. 6A,B). Immunostaining of
sections from 6-month-old mice demonstrated that the distribution of collagen
IV and laminin was normal at MTJs in Tln1 mutant animals
(Fig. 6C-F). In addition,
7 integrin, vinculin and integrin-linked kinase (Ilk) localized at the
MTJ, suggesting that the integrin complexes at MTJs assemble in the absence of
talin 1 and are largely functional to mediate adhesive interactions
(Fig. 6G-L). We also observed
that talin 2 is localized at the MTJs without any obvious upregulation in the
mutants (Fig. 6M,N). Taken
together, these data provide strong evidence that integrin-dependent adhesion
complexes still form at MTJs in the absence of talin 1.
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Collectively, our findings show that in the absence of talin 1, integrin adhesion complexes still form at MTJs and connect to the muscle fiber cytoskeleton. However, with progressive age, myofilaments detach, suggesting that talin 1 is required for maintaining their interaction with integrin adhesion complexes during mechanical strain.
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;
Lieber et al., 1991). The
difference in isometric stress produced by muscle fibers before and after the
eccentric contraction cycle provides a measure of susceptibility to mechanical
damage (Sam et al., 2000). No
significant difference was observed in the length and mass of isolated
wild-type and Tln1-deficient muscles
(Fig. 8A), and all force values
were normalized against the physiological cross-sectional area
(Fig. 8A), which takes into
account differences in muscle mass, length and in the orientation of muscle
fibers (see Materials and methods). At 2 to 3 months of age, the isometric
stresses produced by Tln1-deficient muscle fibers were comparable to
those of wild-type muscle fibers (P=0.413 pre-eccentric,
P=0.892 post-eccentric) (Fig.
8D). By contrast, force production was markedly impaired in muscle
obtained from 6- to 7-month-old Tln1HSA-CREko mice
(Fig. 8E). In an eccentric
contraction cycle, mutant mice showed a significant reduction in maximum
pre-eccentric (P=0.0276) and post-eccentric (P=0.0358)
isometric stress (Fig. 8F,G).
Additionally, for older Tln1-deficient muscles, the peak stresses
exerted during the eccentric contraction were significantly reduced from
wild-type values in every eccentric contraction except the initial one, which
showed greater variability (see Fig. S3B in the supplementary material).
Similarly, the work performed on the muscle normalized by muscle mass was
decreased in the 6- to 7-month-old Tln1HSA-CREko mice for each
eccentric contraction when compared with the wild type (see Fig. S3D in the
supplementary material). Values of peak stress and work were not significantly
different between the muscles obtained from 2-month-old wild-type and mutant
mice (see Fig. S3A,C in the supplementary material). The forces generated by
wild-type and mutant fibers decreased during the eccentric contraction
protocol (Fig. 8D,F). At both 2
and 7 months, the force drop was comparable between wild-type and
Tln1-deficient muscles (Fig.
8H). However, because 7-month-old mutant muscles exerted
considerably less force during the contraction protocol, reduced damage and
fatigue and consequently a smaller force drop were expected. The greater than
expected force drop in mutant muscle suggests that Tln1-deficient
muscle was susceptible to eccentric-contraction-induced damage. These data are
consistent with a failure in the attachment of skeletal muscle fibers at MTJs.
Overall, we conclude that ablation of talin 1 causes a progressive impairment
of the capacity of muscle to generate and to bear force. |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previous studies have shown that in invertebrates, talin is essential to
mediate interactions between integrins and the muscle fiber cytoskeleton
(Brown et al., 2002;
Cram et al., 2003), but the
mechanism of talin function has remained unclear. By taking advantage of a
vertebrate model system that provides greater access to study the biophysical
properties of muscle, we now provide evidence that talin 1 has an important
biomechanical role in muscle. Studies with cells in culture have shown that
talin 1 localizes to focal adhesions, where it interacts with β1
integrins, FAK, vinculin and actin
(Borowsky and Hynes, 1998;
Critchley, 2000), and with
layilin in membrane ruffles (Borowsky and
Hynes, 1998). The assembly of adhesion complexes at focal
adhesions is controlled by mechanical force. Forces that are applied to
nascent integrin adhesion sites induce a strengthening of the
integrin-cytoskeleton interaction, initiating focal adhesion formation and
promoting maturation (Balaban et al.,
2001; Choquet et al.,
1997; Galbraith et al.,
2002; Riveline et al.,
2001). Mechanical force accelerates the localization of vinculin
to focal adhesions, which depends on talin 1
(Giannone et al., 2003). Our
findings now show an important mechanical function for talin 1 in vertebrate
skeletal muscle in vivo. At MTJs, integrin adhesion complexes containing the
integrin 7- and β1-subunits, Ilk and vinculin assembled in the
absence of talin 1. However, talin 1-deficient MTJs showed greater
susceptibility to mechanical stress-induced damage. MTJs in muscle such as
diaphragm that is under constant workload showed the most prominent defects.
Mechanical measurements on isolated muscle fibers demonstrated that talin
1-deficient fibers generated less force than wild-type muscle fibers. MTJs in
Tln1HSA-CREko mice still contained talin 2 but were unstable,
suggesting that talin 1 and talin 2 are not entirely functionally
interchangeable.
Previous studies have shown that talin 1 is of central importance for
integrin function, regulating interactions of the β1 integrin with the
cytoskeleton and with ECM ligands
(Calderwood, 2004b;
Campbell and Ginsberg, 2004;
Ginsberg et al., 2005;
Nieswandt et al., 2007;
Petrich et al., 2007).
Ablation of the gene encoding the integrin β1-subunit during skeletal
muscle development leads to defects in myoblast fusion and sarcomere assembly
(Schwander et al., 2003). We
were surprised that we did not observe similar defects in
Tln1HSA-CREko mice. The migration and fusion of myoblasts was not
obviously perturbed, and immunohistochemical and ultrastructural analysis
revealed no defects in the assembly of costameres and integrin complexes. How
can these findings be reconciled? Talin 1 might still have essential functions
early in skeletal muscle development, for example in myoblast migration, that
have escaped detection because gene inactivation using HSA-CRE might have been
incomplete at early ages. Furthermore, talin 2 may compensate for some talin 1
functions. In agreement with previous findings
(Monkley et al., 2001;
Senetar and McCann, 2005;
Senetar et al., 2007), we
observed prominent talin 2 expression in skeletal muscle. However, our
findings as well as previous studies suggest that talin 2 and talin 1 are not
entirely interchangeable. First, genes for talin 1 and talin 2 have been
identified in all available vertebrate genomes, indicating that evolutionary
pressure has maintained two talin genes. Second, Tln1-null mice are
embryonically lethal (Monkley et al.,
2000) indicating that Tln2 cannot compensate for
Tln1 in early mouse development. Third, although talin 1 and 2 share
a high degree of identity, amino acid differences in functionally important
domains such as FERM and I/LWEQ (actin-binding) domains have been maintained
throughout evolution (Senetar and McCann,
2005). These differences might confer specific functions to each
protein. Consistent with this interpretation, talin 1 and 2 have different
affinities for F-actin (Senetar et al.,
2004). Fourth, talin 1 and talin 2 have distinct binding partners
in skeletal muscle (Senetar and McCann,
2005). Finally, the two-piconewton slip bond between fibronectin
and the cytoskeleton depends on talin 1. Talin 2 cannot compensate for the
function of talin 1 in this process (Jiang
et al., 2003). The latter finding suggests that talin 1 has a
specialized function in force coupling, which could be especially important in
maintaining MTJs under mechanical duress. Talin 2 is likely to be sufficient
for the assembly of integrin adhesion complexes at MTJs, but in the absence of
talin 1, MTJs progressively fail.
Our data suggest that the functions of talin 1 in the regulation of
integrin activation might be tissue-specific. Unlike the situation in
megakaryocytes and platelets, in which a rapid and discrete modulation of
integrin affinity for the ECM is required for proper function
(Calderwood, 2004a;
Campbell and Ginsberg, 2004;
Nieswandt et al., 2007;
Petrich et al., 2007),
affinity modulation may be less crucial at more stable adhesion complexes at
MTJs. Importantly, integrin activation by talin was evaluated only for the
integrin β1A splice variant, whereas adult skeletal muscle expresses
integrin β1D, which provides a stronger mechanical link to the actin
cytoskeleton.
Finally, our findings have implications for understanding disease
mechanisms. Mutations that affect talin 1 and integrin 7β1 cause
fragility of MTJs, but membrane damage is mild
(Hayashi et al., 1998;
Mayer et al., 1997). By
contrast, MTJs are maintained when the DGC is affected, but plasma membrane
damage is prominent (Straub et al.,
1997). Collectively, these findings suggest that ECM receptors of
the integrin family and their effectors control MTJ stability, whereas the DGC
has a major function in maintaining the integrity of the sarcolemma.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/11/2043/DC1
|
ACKNOWLEDGMENTS |
|---|
7-subunit and Ilk, respectively. We thank Dusko Trajkovic for help with
histology. This research was funded with support from the National Institute
of Health to U.M. (NS046456) and R.L. (AR40050) and from the Wellcome Trust to
D.R.C. (O77532). |
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4; mean ± s.e.). Scale bars:
250 µm in A-P; 200 µm in V-X.
