Limits...
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.

Show MeSH
Stabilization of thin filaments by phalloidin before addition of mAb17 or mAb8 prevents thin filament disruption. Cardiac myocytes were extracted and actin filaments were stabilized with AlexaFluor 488 phalloidin before the addition of tropomyosin and Tmod1. MOPC-21 was added to the myocytes (a–d) and had no apparent effect on actin filaments (b), tropomyosin (c), and Tmod1 (d) (a and b; c and d, both costained). Notably, the addition of mAb17 (e–h) or mAb8 (i–l) along with the purified Tmod1 had no effect on actin filaments (f and j), tropomyosin (g and k), and Tmod1 (h and l), which were stabilized by phalloidin (e,f; i,j; g,h; k,l, all costained). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172850&req=5

fig6: Stabilization of thin filaments by phalloidin before addition of mAb17 or mAb8 prevents thin filament disruption. Cardiac myocytes were extracted and actin filaments were stabilized with AlexaFluor 488 phalloidin before the addition of tropomyosin and Tmod1. MOPC-21 was added to the myocytes (a–d) and had no apparent effect on actin filaments (b), tropomyosin (c), and Tmod1 (d) (a and b; c and d, both costained). Notably, the addition of mAb17 (e–h) or mAb8 (i–l) along with the purified Tmod1 had no effect on actin filaments (f and j), tropomyosin (g and k), and Tmod1 (h and l), which were stabilized by phalloidin (e,f; i,j; g,h; k,l, all costained). Bar, 10 μm.

Mentions: To determine if stabilizing the actin filaments before addition of mAb17 or mAb8 with Tmod1 would affect the loss of thin filaments, actin filaments were first stabilized with jasplakinolide or phalloidin, before the reconstitution of tropomyosin and Tmod1 (Fig. 6, data shown for phalloidin treatment). Strikingly, both tropomyosin (Fig. 6, c, g, and k) and Tmod1 (Fig. 6, d, h, and l) remained striated along the stabilized myofibrils after the addition of the Tmod1–mAb17 mixture (Fig. 6 e) or Tmod1–mAb8 mixture (Fig. 6 i). Thus, stabilization of the actin filaments via phalloidin or jasplakinolide, before disruption of the interaction of Tmod1 and tropomyosin, was sufficient to prevent thin filament depolymerization. These data from the reconstitution assay also suggest that the thin filament disassembly phenotype observed is a direct effect of disrupting the interaction of Tmod1 with tropomyosin and is likely not influenced by other soluble, regulatory, or sarcomeric components.


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)

Stabilization of thin filaments by phalloidin before addition of mAb17 or mAb8 prevents thin filament disruption. Cardiac myocytes were extracted and actin filaments were stabilized with AlexaFluor 488 phalloidin before the addition of tropomyosin and Tmod1. MOPC-21 was added to the myocytes (a–d) and had no apparent effect on actin filaments (b), tropomyosin (c), and Tmod1 (d) (a and b; c and d, both costained). Notably, the addition of mAb17 (e–h) or mAb8 (i–l) along with the purified Tmod1 had no effect on actin filaments (f and j), tropomyosin (g and k), and Tmod1 (h and l), which were stabilized by phalloidin (e,f; i,j; g,h; k,l, all costained). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172850&req=5

fig6: Stabilization of thin filaments by phalloidin before addition of mAb17 or mAb8 prevents thin filament disruption. Cardiac myocytes were extracted and actin filaments were stabilized with AlexaFluor 488 phalloidin before the addition of tropomyosin and Tmod1. MOPC-21 was added to the myocytes (a–d) and had no apparent effect on actin filaments (b), tropomyosin (c), and Tmod1 (d) (a and b; c and d, both costained). Notably, the addition of mAb17 (e–h) or mAb8 (i–l) along with the purified Tmod1 had no effect on actin filaments (f and j), tropomyosin (g and k), and Tmod1 (h and l), which were stabilized by phalloidin (e,f; i,j; g,h; k,l, all costained). Bar, 10 μm.
Mentions: To determine if stabilizing the actin filaments before addition of mAb17 or mAb8 with Tmod1 would affect the loss of thin filaments, actin filaments were first stabilized with jasplakinolide or phalloidin, before the reconstitution of tropomyosin and Tmod1 (Fig. 6, data shown for phalloidin treatment). Strikingly, both tropomyosin (Fig. 6, c, g, and k) and Tmod1 (Fig. 6, d, h, and l) remained striated along the stabilized myofibrils after the addition of the Tmod1–mAb17 mixture (Fig. 6 e) or Tmod1–mAb8 mixture (Fig. 6 i). Thus, stabilization of the actin filaments via phalloidin or jasplakinolide, before disruption of the interaction of Tmod1 and tropomyosin, was sufficient to prevent thin filament depolymerization. These data from the reconstitution assay also suggest that the thin filament disassembly phenotype observed is a direct effect of disrupting the interaction of Tmod1 with tropomyosin and is likely not influenced by other soluble, regulatory, or sarcomeric components.

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