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The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes.

Mudry RE, Perry CN, Richards M, Fowler VM, Gregorio CC - J. Cell Biol. (2003)

Bottom Line: In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments.These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability.These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724, USA.

ABSTRACT
Actin (thin) filament length regulation and stability are essential for striated muscle function. To determine the role of the actin filament pointed end capping protein, tropomodulin1 (Tmod1), with tropomyosin, we generated monoclonal antibodies (mAb17 and mAb8) against Tmod1 that specifically disrupted its interaction with tropomyosin in vitro. Microinjection of mAb17 or mAb8 into chick cardiac myocytes caused a dramatic loss of the thin filaments, as revealed by immunofluorescence deconvolution microscopy. Real-time imaging of live myocytes expressing green fluorescent protein-alpha-tropomyosin and microinjected with mAb17 revealed that the thin filaments depolymerized from their pointed ends. In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments. These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability. These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.

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Disrupting the interaction between Tmod and tropomyosin in live cardiac myocytes results in a perturbation of other sarcomeric components. Cardiac myocytes were microinjected with MOPC-21 Fabs (a, c, e, g, i, and k) or mAb17 Fabs (b, d, f, h, j, and l), incubated for 1 h, fixed, and stained for tropomyosin (a and b, red), α-actinin (c, d, k, and l, red), myosin (e and f, red), I-band titin (g and h, red) or M-line titin (i and j, red; k and l, blue). Myocytes injected with mAb17 Fabs showed a loss of tropomyosin consistent with the loss of actin filaments (b). Myocytes injected with mAb17 or mAb17 Fabs displayed a perturbed staining pattern for α-actinin (d), myosin (f), and titin (h and j). Strikingly, myocytes injected with mAb17 Fabs displayed alternating M-line titin (l, blue) and α-actinin (l, red) staining in the absence of detectable actin (l, green). (inset) Enlarged view of l. (arrowheads) Closely spaced remnants of striated staining patterns. Bar, 10 μm.
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fig4: Disrupting the interaction between Tmod and tropomyosin in live cardiac myocytes results in a perturbation of other sarcomeric components. Cardiac myocytes were microinjected with MOPC-21 Fabs (a, c, e, g, i, and k) or mAb17 Fabs (b, d, f, h, j, and l), incubated for 1 h, fixed, and stained for tropomyosin (a and b, red), α-actinin (c, d, k, and l, red), myosin (e and f, red), I-band titin (g and h, red) or M-line titin (i and j, red; k and l, blue). Myocytes injected with mAb17 Fabs showed a loss of tropomyosin consistent with the loss of actin filaments (b). Myocytes injected with mAb17 or mAb17 Fabs displayed a perturbed staining pattern for α-actinin (d), myosin (f), and titin (h and j). Strikingly, myocytes injected with mAb17 Fabs displayed alternating M-line titin (l, blue) and α-actinin (l, red) staining in the absence of detectable actin (l, green). (inset) Enlarged view of l. (arrowheads) Closely spaced remnants of striated staining patterns. Bar, 10 μm.

Mentions: Next, to determine whether mAb17-induced Tmod1 and actin disassembly was accompanied by disassembly of tropomyosin, cells microinjected with mAb17 Fabs were stained for tropomyosin. A loss of tropomyosin staining was clearly observed, which is consistent with the loss of actin filaments (see Fig. 4 b), indicating that the entire thin filament had disassembled. This result suggested that the interaction of Tmod1 with tropomyosin is necessary to stabilize the association of tropomyosin with the actin filaments in cardiac myocyte sarcomeres.


The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes.

Mudry RE, Perry CN, Richards M, Fowler VM, Gregorio CC - J. Cell Biol. (2003)

Disrupting the interaction between Tmod and tropomyosin in live cardiac myocytes results in a perturbation of other sarcomeric components. Cardiac myocytes were microinjected with MOPC-21 Fabs (a, c, e, g, i, and k) or mAb17 Fabs (b, d, f, h, j, and l), incubated for 1 h, fixed, and stained for tropomyosin (a and b, red), α-actinin (c, d, k, and l, red), myosin (e and f, red), I-band titin (g and h, red) or M-line titin (i and j, red; k and l, blue). Myocytes injected with mAb17 Fabs showed a loss of tropomyosin consistent with the loss of actin filaments (b). Myocytes injected with mAb17 or mAb17 Fabs displayed a perturbed staining pattern for α-actinin (d), myosin (f), and titin (h and j). Strikingly, myocytes injected with mAb17 Fabs displayed alternating M-line titin (l, blue) and α-actinin (l, red) staining in the absence of detectable actin (l, green). (inset) Enlarged view of l. (arrowheads) Closely spaced remnants of striated staining patterns. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172850&req=5

fig4: Disrupting the interaction between Tmod and tropomyosin in live cardiac myocytes results in a perturbation of other sarcomeric components. Cardiac myocytes were microinjected with MOPC-21 Fabs (a, c, e, g, i, and k) or mAb17 Fabs (b, d, f, h, j, and l), incubated for 1 h, fixed, and stained for tropomyosin (a and b, red), α-actinin (c, d, k, and l, red), myosin (e and f, red), I-band titin (g and h, red) or M-line titin (i and j, red; k and l, blue). Myocytes injected with mAb17 Fabs showed a loss of tropomyosin consistent with the loss of actin filaments (b). Myocytes injected with mAb17 or mAb17 Fabs displayed a perturbed staining pattern for α-actinin (d), myosin (f), and titin (h and j). Strikingly, myocytes injected with mAb17 Fabs displayed alternating M-line titin (l, blue) and α-actinin (l, red) staining in the absence of detectable actin (l, green). (inset) Enlarged view of l. (arrowheads) Closely spaced remnants of striated staining patterns. Bar, 10 μm.
Mentions: Next, to determine whether mAb17-induced Tmod1 and actin disassembly was accompanied by disassembly of tropomyosin, cells microinjected with mAb17 Fabs were stained for tropomyosin. A loss of tropomyosin staining was clearly observed, which is consistent with the loss of actin filaments (see Fig. 4 b), indicating that the entire thin filament had disassembled. This result suggested that the interaction of Tmod1 with tropomyosin is necessary to stabilize the association of tropomyosin with the actin filaments in cardiac myocyte sarcomeres.

Bottom Line: In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments.These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability.These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724, USA.

ABSTRACT
Actin (thin) filament length regulation and stability are essential for striated muscle function. To determine the role of the actin filament pointed end capping protein, tropomodulin1 (Tmod1), with tropomyosin, we generated monoclonal antibodies (mAb17 and mAb8) against Tmod1 that specifically disrupted its interaction with tropomyosin in vitro. Microinjection of mAb17 or mAb8 into chick cardiac myocytes caused a dramatic loss of the thin filaments, as revealed by immunofluorescence deconvolution microscopy. Real-time imaging of live myocytes expressing green fluorescent protein-alpha-tropomyosin and microinjected with mAb17 revealed that the thin filaments depolymerized from their pointed ends. In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments. These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability. These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.

Show MeSH
Related in: MedlinePlus