Limits...
Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy.

Kee AJ, Schevzov G, Nair-Shalliker V, Robinson CS, Vrhovski B, Ghoddusi M, Qiu MR, Lin JJ, Weinberger R, Gunning PW, Hardeman EC - J. Cell Biol. (2004)

Bottom Line: Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals.These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure.Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

View Article: PubMed Central - PubMed

Affiliation: Muscle Development Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia.

ABSTRACT
Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

Show MeSH

Related in: MedlinePlus

Expression of NS γ-TM products and nonmuscle actins in skeletal muscles. Shown are representative Western blots of protein (10 μg) from a range of mouse skeletal muscles and brain using antibodies that detect NS Tms from the γ-TM gene (A and B), γ-actins (C), and β-actin (D). The CG3 antibody (A) specifically recognizes the γ-TM 1b exon and detects all known NS products from this gene, whereas the γ9d antibody (B) recognizes a subset of these NS Tms, those that contain exon 9d (Fig. 1 C). A novel Tm isoform was detected at high levels in a subset of the muscles examined (EOM, soleus, and diaphragm), a 34-kD protein from γ-TM containing exon 1b (A, CG3 antibody). This novel isoform has not been detected in nonmuscle tissues. Both γ- and β-actin were detected in all muscles (C and D, respectively), but the levels were far greater for the former versus later isoforms. (Note that exposure to film was 45 s for γ-actin vs. 20 min for β-actin.) The γ-actin antibody also detects γ-smooth actin and so some of the signal with this antibody may be this isoform. (E) A representative Coomassie-stained gel used for the Westerns showing equal protein loading for the muscle extracts. Muscle abbreviations: ECU, extensor carpi ulnaris; EDL, extensor digitorum longus; EOM, extraocular muscles; FDP, flexor digitorum profundus; gastroc, gastrocnemius; quad, quadriceps; diaph, diaphragm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172434&req=5

fig2: Expression of NS γ-TM products and nonmuscle actins in skeletal muscles. Shown are representative Western blots of protein (10 μg) from a range of mouse skeletal muscles and brain using antibodies that detect NS Tms from the γ-TM gene (A and B), γ-actins (C), and β-actin (D). The CG3 antibody (A) specifically recognizes the γ-TM 1b exon and detects all known NS products from this gene, whereas the γ9d antibody (B) recognizes a subset of these NS Tms, those that contain exon 9d (Fig. 1 C). A novel Tm isoform was detected at high levels in a subset of the muscles examined (EOM, soleus, and diaphragm), a 34-kD protein from γ-TM containing exon 1b (A, CG3 antibody). This novel isoform has not been detected in nonmuscle tissues. Both γ- and β-actin were detected in all muscles (C and D, respectively), but the levels were far greater for the former versus later isoforms. (Note that exposure to film was 45 s for γ-actin vs. 20 min for β-actin.) The γ-actin antibody also detects γ-smooth actin and so some of the signal with this antibody may be this isoform. (E) A representative Coomassie-stained gel used for the Westerns showing equal protein loading for the muscle extracts. Muscle abbreviations: ECU, extensor carpi ulnaris; EDL, extensor digitorum longus; EOM, extraocular muscles; FDP, flexor digitorum profundus; gastroc, gastrocnemius; quad, quadriceps; diaph, diaphragm.

Mentions: Western blots reveal that adult skeletal muscles express two NS Tms at significant levels (Fig. 2, A and B). The expression of NS Tms varies widely between different muscles, with the extraocular muscle (EOM), soleus, and diaphragm muscles having particularly high levels of NS Tms. A novel 34-kD muscle-specific isoform was detected by the CG3 antibody in these muscles (very low amounts in flexor digitorum profundus muscle also; Fig. 2 A). All known products recognized by CG3 migrate at 30 kD (e.g., Tm5NM1), therefore this is likely to be a larger protein with a novel carboxy terminus because it is not recognized by the γ9d antibody (Fig. 2 B; no 34-kD band in the Western blots) or antibodies against the other carboxy-terminal exons from this gene (not depicted). In contrast to the muscle-specific expression of this novel isoform, γ9d detected a band that was present in all skeletal muscles examined (Fig. 2 B). This band (30 kD) is likely to be Tm5NM1, as a band of this size was not detected with an antibody (WS5/9d; Percival et al., 2004) that preferentially recognizes Tm5NM2, the only other exon 9d-containing product from the γ-TM gene (Dufour et al., 1998a). We have also examined the expression of nonmuscle actins that are known to be present in skeletal muscle, γ-actin, and β-actin (Fig. 2, C and D, respectively). γ-Actin and β-actin were expressed in all muscles examined, but the level of expression of γ-actin was much greater than β-actin (Fig. 2, C and D, respectively). Because the γ-actin antibody also detects γ-smooth actin (not depicted) we cannot exclude the possibility that at least some of the γ-actin signal is due to this isoform.


Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy.

Kee AJ, Schevzov G, Nair-Shalliker V, Robinson CS, Vrhovski B, Ghoddusi M, Qiu MR, Lin JJ, Weinberger R, Gunning PW, Hardeman EC - J. Cell Biol. (2004)

Expression of NS γ-TM products and nonmuscle actins in skeletal muscles. Shown are representative Western blots of protein (10 μg) from a range of mouse skeletal muscles and brain using antibodies that detect NS Tms from the γ-TM gene (A and B), γ-actins (C), and β-actin (D). The CG3 antibody (A) specifically recognizes the γ-TM 1b exon and detects all known NS products from this gene, whereas the γ9d antibody (B) recognizes a subset of these NS Tms, those that contain exon 9d (Fig. 1 C). A novel Tm isoform was detected at high levels in a subset of the muscles examined (EOM, soleus, and diaphragm), a 34-kD protein from γ-TM containing exon 1b (A, CG3 antibody). This novel isoform has not been detected in nonmuscle tissues. Both γ- and β-actin were detected in all muscles (C and D, respectively), but the levels were far greater for the former versus later isoforms. (Note that exposure to film was 45 s for γ-actin vs. 20 min for β-actin.) The γ-actin antibody also detects γ-smooth actin and so some of the signal with this antibody may be this isoform. (E) A representative Coomassie-stained gel used for the Westerns showing equal protein loading for the muscle extracts. Muscle abbreviations: ECU, extensor carpi ulnaris; EDL, extensor digitorum longus; EOM, extraocular muscles; FDP, flexor digitorum profundus; gastroc, gastrocnemius; quad, quadriceps; diaph, diaphragm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172434&req=5

fig2: Expression of NS γ-TM products and nonmuscle actins in skeletal muscles. Shown are representative Western blots of protein (10 μg) from a range of mouse skeletal muscles and brain using antibodies that detect NS Tms from the γ-TM gene (A and B), γ-actins (C), and β-actin (D). The CG3 antibody (A) specifically recognizes the γ-TM 1b exon and detects all known NS products from this gene, whereas the γ9d antibody (B) recognizes a subset of these NS Tms, those that contain exon 9d (Fig. 1 C). A novel Tm isoform was detected at high levels in a subset of the muscles examined (EOM, soleus, and diaphragm), a 34-kD protein from γ-TM containing exon 1b (A, CG3 antibody). This novel isoform has not been detected in nonmuscle tissues. Both γ- and β-actin were detected in all muscles (C and D, respectively), but the levels were far greater for the former versus later isoforms. (Note that exposure to film was 45 s for γ-actin vs. 20 min for β-actin.) The γ-actin antibody also detects γ-smooth actin and so some of the signal with this antibody may be this isoform. (E) A representative Coomassie-stained gel used for the Westerns showing equal protein loading for the muscle extracts. Muscle abbreviations: ECU, extensor carpi ulnaris; EDL, extensor digitorum longus; EOM, extraocular muscles; FDP, flexor digitorum profundus; gastroc, gastrocnemius; quad, quadriceps; diaph, diaphragm.
Mentions: Western blots reveal that adult skeletal muscles express two NS Tms at significant levels (Fig. 2, A and B). The expression of NS Tms varies widely between different muscles, with the extraocular muscle (EOM), soleus, and diaphragm muscles having particularly high levels of NS Tms. A novel 34-kD muscle-specific isoform was detected by the CG3 antibody in these muscles (very low amounts in flexor digitorum profundus muscle also; Fig. 2 A). All known products recognized by CG3 migrate at 30 kD (e.g., Tm5NM1), therefore this is likely to be a larger protein with a novel carboxy terminus because it is not recognized by the γ9d antibody (Fig. 2 B; no 34-kD band in the Western blots) or antibodies against the other carboxy-terminal exons from this gene (not depicted). In contrast to the muscle-specific expression of this novel isoform, γ9d detected a band that was present in all skeletal muscles examined (Fig. 2 B). This band (30 kD) is likely to be Tm5NM1, as a band of this size was not detected with an antibody (WS5/9d; Percival et al., 2004) that preferentially recognizes Tm5NM2, the only other exon 9d-containing product from the γ-TM gene (Dufour et al., 1998a). We have also examined the expression of nonmuscle actins that are known to be present in skeletal muscle, γ-actin, and β-actin (Fig. 2, C and D, respectively). γ-Actin and β-actin were expressed in all muscles examined, but the level of expression of γ-actin was much greater than β-actin (Fig. 2, C and D, respectively). Because the γ-actin antibody also detects γ-smooth actin (not depicted) we cannot exclude the possibility that at least some of the γ-actin signal is due to this isoform.

Bottom Line: Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals.These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure.Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

View Article: PubMed Central - PubMed

Affiliation: Muscle Development Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia.

ABSTRACT
Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

Show MeSH
Related in: MedlinePlus