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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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mAb17 and mAb8 specifically bind to the NH2-terminal Tmod1 residues 6–57, recognize Tmod1 in embryonic chick heart lysates, and disrupt its interaction with tropomyosin. (a) Coomassie blue–stained gel of full-length Tmod1 (lane 1,1–359) and Tmod1 fragments (lanes 2–6, residues 6–184, 35–359, 95–359, 6–94, and 6–5, respectively). (b) Western blots of samples in panel a were probed with mAb17 that recognized full-length (lane 1) and NH2-terminal Tmod1 (lanes 2, 5, and 6), but not COOH-terminal Tmod1 (lanes 3 and 4). (c) Western blots of embryonic heart extracts were probed with secondary antibodies alone (2° alone), MOPC-21, mAb17, or mAb8. mAb8 and mAb17 detected a band at ∼40 kD, corresponding to Tmod1. (d) Tmod1 nitrocellulose dots were preincubated with mAb8, mAb17, or mAb9, followed by incubation with 125I-tropomyosin (125I-TM). Preincubating the Tmod1 dots with increasing concentrations of mAb17 (closed squares) or mAb8 (open circles) resulted in a dramatic decrease in the percentage of tropomyosin that bound to Tmod1, as compared with mAb9 which did not disrupt the interaction (closed triangle). (e) Tmod1 dots were incubated with 125I-tropomyosin (125I-TM), followed by incubation with mAb8 (closed squares), or MOPC21 (open circles) for varying periods of time. Addition of mAb8 resulted in a significant dissociation of tropomyosin from Tmod1.
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fig1: mAb17 and mAb8 specifically bind to the NH2-terminal Tmod1 residues 6–57, recognize Tmod1 in embryonic chick heart lysates, and disrupt its interaction with tropomyosin. (a) Coomassie blue–stained gel of full-length Tmod1 (lane 1,1–359) and Tmod1 fragments (lanes 2–6, residues 6–184, 35–359, 95–359, 6–94, and 6–5, respectively). (b) Western blots of samples in panel a were probed with mAb17 that recognized full-length (lane 1) and NH2-terminal Tmod1 (lanes 2, 5, and 6), but not COOH-terminal Tmod1 (lanes 3 and 4). (c) Western blots of embryonic heart extracts were probed with secondary antibodies alone (2° alone), MOPC-21, mAb17, or mAb8. mAb8 and mAb17 detected a band at ∼40 kD, corresponding to Tmod1. (d) Tmod1 nitrocellulose dots were preincubated with mAb8, mAb17, or mAb9, followed by incubation with 125I-tropomyosin (125I-TM). Preincubating the Tmod1 dots with increasing concentrations of mAb17 (closed squares) or mAb8 (open circles) resulted in a dramatic decrease in the percentage of tropomyosin that bound to Tmod1, as compared with mAb9 which did not disrupt the interaction (closed triangle). (e) Tmod1 dots were incubated with 125I-tropomyosin (125I-TM), followed by incubation with mAb8 (closed squares), or MOPC21 (open circles) for varying periods of time. Addition of mAb8 resulted in a significant dissociation of tropomyosin from Tmod1.

Mentions: To study the functional significance of the interaction of Tmod1 with tropomyosin, we first sought to identify monoclonal anti-Tmod1 antibodies that disrupted the interaction of the molecules in vitro. Previous work showed that the extreme NH2-terminal end of Tmod1 (amino acids 6–34) is essential for binding to skeletal muscle tropomyosin (Babcock and Fowler, 1994). We generated two monoclonal antibodies to chicken Tmod1 (mAb8 and mAb17) that specifically recognized the NH2-terminal end of Tmod1 by Western blot analysis (Fig. 1 b). Both antibodies recognized full-length Tmod1 (Fig. 1 b, lane 1), and recombinant Tmod1 fragments including residues 6–184, 6–94 and 6–57, but failed to recognize fragments including residues 35–359 and 95–359 (data shown for mAb17). These results demonstrated that mAb17 and mAb8 recognize epitopes within the NH2-terminal residues 6–34 of Tmod1, which contain the skeletal muscle tropomyosin binding site. To demonstrate the specificity of these antibodies, Western blot analyses of embryonic chick cardiac myocyte extracts were performed, revealing that mAb17 and mAb8 specifically recognized a single 40-kD polypeptide, corresponding to the molecular mass of Tmod1 (Fig. 1 c).


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)

mAb17 and mAb8 specifically bind to the NH2-terminal Tmod1 residues 6–57, recognize Tmod1 in embryonic chick heart lysates, and disrupt its interaction with tropomyosin. (a) Coomassie blue–stained gel of full-length Tmod1 (lane 1,1–359) and Tmod1 fragments (lanes 2–6, residues 6–184, 35–359, 95–359, 6–94, and 6–5, respectively). (b) Western blots of samples in panel a were probed with mAb17 that recognized full-length (lane 1) and NH2-terminal Tmod1 (lanes 2, 5, and 6), but not COOH-terminal Tmod1 (lanes 3 and 4). (c) Western blots of embryonic heart extracts were probed with secondary antibodies alone (2° alone), MOPC-21, mAb17, or mAb8. mAb8 and mAb17 detected a band at ∼40 kD, corresponding to Tmod1. (d) Tmod1 nitrocellulose dots were preincubated with mAb8, mAb17, or mAb9, followed by incubation with 125I-tropomyosin (125I-TM). Preincubating the Tmod1 dots with increasing concentrations of mAb17 (closed squares) or mAb8 (open circles) resulted in a dramatic decrease in the percentage of tropomyosin that bound to Tmod1, as compared with mAb9 which did not disrupt the interaction (closed triangle). (e) Tmod1 dots were incubated with 125I-tropomyosin (125I-TM), followed by incubation with mAb8 (closed squares), or MOPC21 (open circles) for varying periods of time. Addition of mAb8 resulted in a significant dissociation of tropomyosin from Tmod1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172850&req=5

fig1: mAb17 and mAb8 specifically bind to the NH2-terminal Tmod1 residues 6–57, recognize Tmod1 in embryonic chick heart lysates, and disrupt its interaction with tropomyosin. (a) Coomassie blue–stained gel of full-length Tmod1 (lane 1,1–359) and Tmod1 fragments (lanes 2–6, residues 6–184, 35–359, 95–359, 6–94, and 6–5, respectively). (b) Western blots of samples in panel a were probed with mAb17 that recognized full-length (lane 1) and NH2-terminal Tmod1 (lanes 2, 5, and 6), but not COOH-terminal Tmod1 (lanes 3 and 4). (c) Western blots of embryonic heart extracts were probed with secondary antibodies alone (2° alone), MOPC-21, mAb17, or mAb8. mAb8 and mAb17 detected a band at ∼40 kD, corresponding to Tmod1. (d) Tmod1 nitrocellulose dots were preincubated with mAb8, mAb17, or mAb9, followed by incubation with 125I-tropomyosin (125I-TM). Preincubating the Tmod1 dots with increasing concentrations of mAb17 (closed squares) or mAb8 (open circles) resulted in a dramatic decrease in the percentage of tropomyosin that bound to Tmod1, as compared with mAb9 which did not disrupt the interaction (closed triangle). (e) Tmod1 dots were incubated with 125I-tropomyosin (125I-TM), followed by incubation with mAb8 (closed squares), or MOPC21 (open circles) for varying periods of time. Addition of mAb8 resulted in a significant dissociation of tropomyosin from Tmod1.
Mentions: To study the functional significance of the interaction of Tmod1 with tropomyosin, we first sought to identify monoclonal anti-Tmod1 antibodies that disrupted the interaction of the molecules in vitro. Previous work showed that the extreme NH2-terminal end of Tmod1 (amino acids 6–34) is essential for binding to skeletal muscle tropomyosin (Babcock and Fowler, 1994). We generated two monoclonal antibodies to chicken Tmod1 (mAb8 and mAb17) that specifically recognized the NH2-terminal end of Tmod1 by Western blot analysis (Fig. 1 b). Both antibodies recognized full-length Tmod1 (Fig. 1 b, lane 1), and recombinant Tmod1 fragments including residues 6–184, 6–94 and 6–57, but failed to recognize fragments including residues 35–359 and 95–359 (data shown for mAb17). These results demonstrated that mAb17 and mAb8 recognize epitopes within the NH2-terminal residues 6–34 of Tmod1, which contain the skeletal muscle tropomyosin binding site. To demonstrate the specificity of these antibodies, Western blot analyses of embryonic chick cardiac myocyte extracts were performed, revealing that mAb17 and mAb8 specifically recognized a single 40-kD polypeptide, corresponding to the molecular mass of Tmod1 (Fig. 1 c).

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