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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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Model of two identified functional domains of Tmod1. (I and IV) Stable thin filaments are comprised of actin (gray), tropomyosin (red), and Tmod1 (blue cap). (II) Disrupting the interaction of Tmod1 and tropomyosin (red) with mAb17 or mAb8 (black) appears to alter the functional properties of Tmod1 (blue square), which imparts instability at the pointed ends of the thin filaments. Note, the shape change is meant to indicate that Tmod1 acquires a new functionality, but the exact alteration is unknown. (III) The conferred instability results in a loss of thin filaments via depolymerization from the pointed ends. (V) In contrast, disrupting Tmod1's actin capping activity with mAb9 results in an elongation of actin filaments at the pointed ends (VI). These results suggest at least two functional roles for Tmod1: stabilizing/preventing depolymerization of thin filaments through its interaction with tropomyosin, and inhibiting elongation/maintenance of actin filaments lengths through its actin capping activity.
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fig7: Model of two identified functional domains of Tmod1. (I and IV) Stable thin filaments are comprised of actin (gray), tropomyosin (red), and Tmod1 (blue cap). (II) Disrupting the interaction of Tmod1 and tropomyosin (red) with mAb17 or mAb8 (black) appears to alter the functional properties of Tmod1 (blue square), which imparts instability at the pointed ends of the thin filaments. Note, the shape change is meant to indicate that Tmod1 acquires a new functionality, but the exact alteration is unknown. (III) The conferred instability results in a loss of thin filaments via depolymerization from the pointed ends. (V) In contrast, disrupting Tmod1's actin capping activity with mAb9 results in an elongation of actin filaments at the pointed ends (VI). These results suggest at least two functional roles for Tmod1: stabilizing/preventing depolymerization of thin filaments through its interaction with tropomyosin, and inhibiting elongation/maintenance of actin filaments lengths through its actin capping activity.

Mentions: In this work, we specifically blocked the interaction of Tmod1 with one of its three known binding partners, tropomyosin, in live chick cardiac myocytes. We disrupted this interaction by microinjecting function-blocking antibodies against the NH2-terminal region of Tmod1 and by microinjecting a recombinant NH2-terminal Tmod1 fragment containing the tropomyosin binding site. These approaches resulted in a striking phenotype: a loss of thin filaments and cessation of contractile activity. To further investigate the mechanism of this observed phenotype, we transfected myocytes with GFP–α-tropomyosin, which assembled along the lengths of the thin filaments, and then microinjected these cells with mAb17. This live cell imaging strategy allowed us to visualize the loss of thin filaments in real time, revealing that the thin filaments depolymerized progressively from their pointed ends. Results from a cell permeabilization assay further revealed that the loss of thin filaments, upon perturbation of the interaction of Tmod1 with tropomyosin, is prevented by the prior stabilization of the actin filaments with phalloidin or jasplakinolide. These observations reveal exciting, novel functions for both tropomyosin and Tmod1. For tropomyosin, our study provides direct support for its proposed role as a thin filament stabilizing component in vertebrate muscle. For Tmod1, we found that its two distinct structural and ligand-binding regions, the NH2-terminal tropomyosin-binding domain and COOH-terminal actin capping domain, have unique roles at the pointed ends of the thin filaments that can be distinguished in the context of live myocytes. The interaction of Tmod1 with tropomyosin at the pointed ends is critical for maintaining the integrity of thin filaments, by preventing their depolymerization in live cardiac myocytes (Fig. 7, I–III). The phenotype we observed is strikingly different from the phenotype obtained from inhibiting the interaction of Tmod1 with actin, which revealed that Tmod1's capping activity is required to prevent abnormal elongation of the thin filaments from their pointed ends (Gregorio et al., 1995) (Fig. 7, IV–VI). Together, we conclude that Tmod1 is a multifunctional protein in cardiac muscle, involved in maintaining the lengths and stability of the thin filaments.


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)

Model of two identified functional domains of Tmod1. (I and IV) Stable thin filaments are comprised of actin (gray), tropomyosin (red), and Tmod1 (blue cap). (II) Disrupting the interaction of Tmod1 and tropomyosin (red) with mAb17 or mAb8 (black) appears to alter the functional properties of Tmod1 (blue square), which imparts instability at the pointed ends of the thin filaments. Note, the shape change is meant to indicate that Tmod1 acquires a new functionality, but the exact alteration is unknown. (III) The conferred instability results in a loss of thin filaments via depolymerization from the pointed ends. (V) In contrast, disrupting Tmod1's actin capping activity with mAb9 results in an elongation of actin filaments at the pointed ends (VI). These results suggest at least two functional roles for Tmod1: stabilizing/preventing depolymerization of thin filaments through its interaction with tropomyosin, and inhibiting elongation/maintenance of actin filaments lengths through its actin capping activity.
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Related In: Results  -  Collection

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fig7: Model of two identified functional domains of Tmod1. (I and IV) Stable thin filaments are comprised of actin (gray), tropomyosin (red), and Tmod1 (blue cap). (II) Disrupting the interaction of Tmod1 and tropomyosin (red) with mAb17 or mAb8 (black) appears to alter the functional properties of Tmod1 (blue square), which imparts instability at the pointed ends of the thin filaments. Note, the shape change is meant to indicate that Tmod1 acquires a new functionality, but the exact alteration is unknown. (III) The conferred instability results in a loss of thin filaments via depolymerization from the pointed ends. (V) In contrast, disrupting Tmod1's actin capping activity with mAb9 results in an elongation of actin filaments at the pointed ends (VI). These results suggest at least two functional roles for Tmod1: stabilizing/preventing depolymerization of thin filaments through its interaction with tropomyosin, and inhibiting elongation/maintenance of actin filaments lengths through its actin capping activity.
Mentions: In this work, we specifically blocked the interaction of Tmod1 with one of its three known binding partners, tropomyosin, in live chick cardiac myocytes. We disrupted this interaction by microinjecting function-blocking antibodies against the NH2-terminal region of Tmod1 and by microinjecting a recombinant NH2-terminal Tmod1 fragment containing the tropomyosin binding site. These approaches resulted in a striking phenotype: a loss of thin filaments and cessation of contractile activity. To further investigate the mechanism of this observed phenotype, we transfected myocytes with GFP–α-tropomyosin, which assembled along the lengths of the thin filaments, and then microinjected these cells with mAb17. This live cell imaging strategy allowed us to visualize the loss of thin filaments in real time, revealing that the thin filaments depolymerized progressively from their pointed ends. Results from a cell permeabilization assay further revealed that the loss of thin filaments, upon perturbation of the interaction of Tmod1 with tropomyosin, is prevented by the prior stabilization of the actin filaments with phalloidin or jasplakinolide. These observations reveal exciting, novel functions for both tropomyosin and Tmod1. For tropomyosin, our study provides direct support for its proposed role as a thin filament stabilizing component in vertebrate muscle. For Tmod1, we found that its two distinct structural and ligand-binding regions, the NH2-terminal tropomyosin-binding domain and COOH-terminal actin capping domain, have unique roles at the pointed ends of the thin filaments that can be distinguished in the context of live myocytes. The interaction of Tmod1 with tropomyosin at the pointed ends is critical for maintaining the integrity of thin filaments, by preventing their depolymerization in live cardiac myocytes (Fig. 7, I–III). The phenotype we observed is strikingly different from the phenotype obtained from inhibiting the interaction of Tmod1 with actin, which revealed that Tmod1's capping activity is required to prevent abnormal elongation of the thin filaments from their pointed ends (Gregorio et al., 1995) (Fig. 7, IV–VI). Together, we conclude that Tmod1 is a multifunctional protein in cardiac muscle, involved in maintaining the lengths and stability of the thin filaments.

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