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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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The disruption of the Tmod1–tropomyosin interaction in permeabilized and extracted cardiac myocytes results in the loss of actin filaments. (A) Cardiac myocytes permeabilized with saponin and extracted in 0.5 M KCl were stained for myosin (a), tropomyosin (b), or Tmod1 (c): all three sarcomeric components were extracted, whereas the actin filaments were unaffected (c and d, costained). Biotinylated tropomyosin (e) and recombinant Tmod1 (f) were reconstituted onto the ghost myofibrils (g). (B) Cardiac myocytes extracted and reconstituted with biotinylated tropomyosin followed by a mixture of Tmod1 with MOPC-21 (h–k) showed no disruption of the striated actin filaments (i), tropomyosin (j), and Tmod1 (k) upon addition of MOPC-21 (h and i; j and k, both costained). In contrast, the addition of mAb17 (l–o) or mAb8 (p–s) resulted in the loss of actin filaments (m and q), tropomyosin (n and r), and Tmod1 (o and s) (n and o; r and s, both costained). The addition of mAb8 (t) in the absence of recombinant Tmod1 had no effect on the actin filaments (u) or on tropomyosin (v). Bars, 10 μm.
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fig5: The disruption of the Tmod1–tropomyosin interaction in permeabilized and extracted cardiac myocytes results in the loss of actin filaments. (A) Cardiac myocytes permeabilized with saponin and extracted in 0.5 M KCl were stained for myosin (a), tropomyosin (b), or Tmod1 (c): all three sarcomeric components were extracted, whereas the actin filaments were unaffected (c and d, costained). Biotinylated tropomyosin (e) and recombinant Tmod1 (f) were reconstituted onto the ghost myofibrils (g). (B) Cardiac myocytes extracted and reconstituted with biotinylated tropomyosin followed by a mixture of Tmod1 with MOPC-21 (h–k) showed no disruption of the striated actin filaments (i), tropomyosin (j), and Tmod1 (k) upon addition of MOPC-21 (h and i; j and k, both costained). In contrast, the addition of mAb17 (l–o) or mAb8 (p–s) resulted in the loss of actin filaments (m and q), tropomyosin (n and r), and Tmod1 (o and s) (n and o; r and s, both costained). The addition of mAb8 (t) in the absence of recombinant Tmod1 had no effect on the actin filaments (u) or on tropomyosin (v). Bars, 10 μm.

Mentions: To gain additional insights into the mechanism of the thin filament disruption phenotype, we used a thin filament reconstitution assay. This assay allows us to selectively study the assembly properties of individual thin filament components (in the absence of thick filament components), as well as interfere with specific thin filament protein interactions. Using this assay, we previously demonstrated that binding of Tmod1 to the pointed ends of actin filaments required the prior reconstitution of tropomyosin along the actin filaments in “ghost myofibrils” (Gregorio and Fowler, 1995). In brief, ghost myofibrils were prepared by permeabilizing cardiac myocytes with saponin and extracting them in 0.5 M KCl. Under these conditions, many sarcomeric components including the thick filaments (Fig. 5 a) and the thin filament components, tropomyosin (Fig. 5 b) and Tmod1 (Fig. 5 c), were removed (Gregorio and Fowler, 1995), as shown by a lack of immunofluorescent detection. Actin filaments (Fig. 5, d and g), as well as the major Z-line component α-actinin, and titin filaments remained intact in the extracted cells (unpublished data). Purified tropomyosin (Fig. 5 e) and recombinant Tmod1 (Fig. 5 f) were reconstituted on the existing actin filaments (Fig. 5 g) and assembled in their typical sarcomeric distributions. In fact, actin filaments alone, or those with tropomyosin plus Tmod alone, were stable for >60 min at room temperature (unpublished data). Remarkably, the addition of exogenous tropomyosin followed by a mixture of recombinant Tmod1 with mAb17 (Fig. 5, l–o), or Tmod1 with mAb8 (Fig. 5, p–s), resulted in a dramatic loss of actin filaments (Fig. 5, m and q, respectively): a phenotype that appeared identical to that observed in intact cells. In contrast, reconstitution of tropomyosin followed by the addition of a mixture of Tmod1 with MOPC-21 (Fig. 5, h–k), the addition of mAb8 in the absence of Tmod1 (Fig. 5, t–v), or the addition of mAb8 with Tmod1 in the absence of reconstituted tropomyosin (unpublished data) had no effect on the actin filaments. These data indicate that once the actin–tropomyosin filaments are capped by Tmod1, the interaction between Tmod1 and tropomyosin must be intact to prevent depolymerization of the thin filaments (see Discussion).


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)

The disruption of the Tmod1–tropomyosin interaction in permeabilized and extracted cardiac myocytes results in the loss of actin filaments. (A) Cardiac myocytes permeabilized with saponin and extracted in 0.5 M KCl were stained for myosin (a), tropomyosin (b), or Tmod1 (c): all three sarcomeric components were extracted, whereas the actin filaments were unaffected (c and d, costained). Biotinylated tropomyosin (e) and recombinant Tmod1 (f) were reconstituted onto the ghost myofibrils (g). (B) Cardiac myocytes extracted and reconstituted with biotinylated tropomyosin followed by a mixture of Tmod1 with MOPC-21 (h–k) showed no disruption of the striated actin filaments (i), tropomyosin (j), and Tmod1 (k) upon addition of MOPC-21 (h and i; j and k, both costained). In contrast, the addition of mAb17 (l–o) or mAb8 (p–s) resulted in the loss of actin filaments (m and q), tropomyosin (n and r), and Tmod1 (o and s) (n and o; r and s, both costained). The addition of mAb8 (t) in the absence of recombinant Tmod1 had no effect on the actin filaments (u) or on tropomyosin (v). Bars, 10 μm.
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Related In: Results  -  Collection

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fig5: The disruption of the Tmod1–tropomyosin interaction in permeabilized and extracted cardiac myocytes results in the loss of actin filaments. (A) Cardiac myocytes permeabilized with saponin and extracted in 0.5 M KCl were stained for myosin (a), tropomyosin (b), or Tmod1 (c): all three sarcomeric components were extracted, whereas the actin filaments were unaffected (c and d, costained). Biotinylated tropomyosin (e) and recombinant Tmod1 (f) were reconstituted onto the ghost myofibrils (g). (B) Cardiac myocytes extracted and reconstituted with biotinylated tropomyosin followed by a mixture of Tmod1 with MOPC-21 (h–k) showed no disruption of the striated actin filaments (i), tropomyosin (j), and Tmod1 (k) upon addition of MOPC-21 (h and i; j and k, both costained). In contrast, the addition of mAb17 (l–o) or mAb8 (p–s) resulted in the loss of actin filaments (m and q), tropomyosin (n and r), and Tmod1 (o and s) (n and o; r and s, both costained). The addition of mAb8 (t) in the absence of recombinant Tmod1 had no effect on the actin filaments (u) or on tropomyosin (v). Bars, 10 μm.
Mentions: To gain additional insights into the mechanism of the thin filament disruption phenotype, we used a thin filament reconstitution assay. This assay allows us to selectively study the assembly properties of individual thin filament components (in the absence of thick filament components), as well as interfere with specific thin filament protein interactions. Using this assay, we previously demonstrated that binding of Tmod1 to the pointed ends of actin filaments required the prior reconstitution of tropomyosin along the actin filaments in “ghost myofibrils” (Gregorio and Fowler, 1995). In brief, ghost myofibrils were prepared by permeabilizing cardiac myocytes with saponin and extracting them in 0.5 M KCl. Under these conditions, many sarcomeric components including the thick filaments (Fig. 5 a) and the thin filament components, tropomyosin (Fig. 5 b) and Tmod1 (Fig. 5 c), were removed (Gregorio and Fowler, 1995), as shown by a lack of immunofluorescent detection. Actin filaments (Fig. 5, d and g), as well as the major Z-line component α-actinin, and titin filaments remained intact in the extracted cells (unpublished data). Purified tropomyosin (Fig. 5 e) and recombinant Tmod1 (Fig. 5 f) were reconstituted on the existing actin filaments (Fig. 5 g) and assembled in their typical sarcomeric distributions. In fact, actin filaments alone, or those with tropomyosin plus Tmod alone, were stable for >60 min at room temperature (unpublished data). Remarkably, the addition of exogenous tropomyosin followed by a mixture of recombinant Tmod1 with mAb17 (Fig. 5, l–o), or Tmod1 with mAb8 (Fig. 5, p–s), resulted in a dramatic loss of actin filaments (Fig. 5, m and q, respectively): a phenotype that appeared identical to that observed in intact cells. In contrast, reconstitution of tropomyosin followed by the addition of a mixture of Tmod1 with MOPC-21 (Fig. 5, h–k), the addition of mAb8 in the absence of Tmod1 (Fig. 5, t–v), or the addition of mAb8 with Tmod1 in the absence of reconstituted tropomyosin (unpublished data) had no effect on the actin filaments. These data indicate that once the actin–tropomyosin filaments are capped by Tmod1, the interaction between Tmod1 and tropomyosin must be intact to prevent depolymerization of the thin filaments (see Discussion).

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