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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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Disruption of the interaction between Tmod1 and tropomyosin in live cardiac myocytes results in a loss of actin (thin) filaments. Cardiac myocytes were microinjected with MOPC-21 (a and b), mAb17 (c and d), mAb17 Fab fragments (i and j), mAb8 (e and f), or mAb95 (g and h). Injected cells were observed using an AlexaFluor 594–conjugated anti–mouse IgG to detect the injected antibody (a, c, and e). Microinjection of mAb17 or mAb8 resulted in a loss of actin filaments as detected by AlexaFluor 488 phalloidin (d and f, arrowheads) or using anti–α-actin antibodies (j, arrowheads). Normal actin filaments were seen in myocytes injected with MOPC-21 (b, arrows) or with mAb95 (h). Fibroblasts were also microinjected with mAb17 (k and l); no perturbation of actin filaments was seen (l). To test for recovery, injected cells were incubated for 48 h before staining. Actin filaments were easily visualized at this time point (m, arrows). A recombinant Tmod1 fragment containing residues 1–130, mixed with MOPC-21 (n, to identify injected cells) was microinjected into cardiac myocytes and incubated for 1 h. Microinjection of the Tmod1 fragment containing the tropomyosin binding site resulted in a loss of actin filaments (o). Bars, 10 μm.
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fig2: Disruption of the interaction between Tmod1 and tropomyosin in live cardiac myocytes results in a loss of actin (thin) filaments. Cardiac myocytes were microinjected with MOPC-21 (a and b), mAb17 (c and d), mAb17 Fab fragments (i and j), mAb8 (e and f), or mAb95 (g and h). Injected cells were observed using an AlexaFluor 594–conjugated anti–mouse IgG to detect the injected antibody (a, c, and e). Microinjection of mAb17 or mAb8 resulted in a loss of actin filaments as detected by AlexaFluor 488 phalloidin (d and f, arrowheads) or using anti–α-actin antibodies (j, arrowheads). Normal actin filaments were seen in myocytes injected with MOPC-21 (b, arrows) or with mAb95 (h). Fibroblasts were also microinjected with mAb17 (k and l); no perturbation of actin filaments was seen (l). To test for recovery, injected cells were incubated for 48 h before staining. Actin filaments were easily visualized at this time point (m, arrows). A recombinant Tmod1 fragment containing residues 1–130, mixed with MOPC-21 (n, to identify injected cells) was microinjected into cardiac myocytes and incubated for 1 h. Microinjection of the Tmod1 fragment containing the tropomyosin binding site resulted in a loss of actin filaments (o). Bars, 10 μm.

Mentions: We next used mAb17 and mAb8 in microinjection studies designed to investigate the interaction of Tmod1 with tropomyosin in the context of living cardiac myocytes. Day 3–5 cardiac myocytes were microinjected with mAb17 or mAb8 and incubated for 1 h after injection. The myocytes were fixed and stained for thin filament components. A few cells microinjected with mAb17 or mAb8 demonstrated a thin filament pointed end striated staining pattern for Tmod1 and unperturbed actin filaments (unpublished data). However, the majority (>80%) of myocytes injected with mAb17 (Fig. 2 c) or mAb8 (Fig. 2 e) exhibited a dramatic loss of actin filaments, as determined by staining with fluorescently conjugated phalloidin (Fig. 2, d and f), compared with the normal striated appearance of sarcomeric actin staining in cells injected with the control antibody MOPC-21 (Fig. 2 b) or in surrounding uninjected cells (Fig. 2 d, bottom). Additionally, staining with anti–cardiac actin antibodies demonstrated that the absence of actin staining was not due to an artifact from inhibition of phalloidin staining (Fig. 2 j). Notably, no Tmod1 striations were detected (i.e., Tmod appeared diffused in the cytoplasm) in the cells microinjected with mAb17 or mAb8, suggesting that disrupting the interaction of Tmod1 with tropomyosin promoted Tmod1's dissociation from thin filament pointed ends (Fig. 2, c and e). In the majority of cells (70–75%) perturbed by the microinjection of mAb17 or mAb8, no actin filaments were detected (not depicted), whereas in other cells remnants remained (Fig. 2, d, f, and j). Cells injected with Fab fragments of mAb17 exhibited an identical phenotype, indicating that the loss of sarcomeric actin filament staining was not due to Tmod1 cross-linking or sequestration in the cells (Fig. 2, i and j). The effect of mAb8 and mAb17 was also not due to nonspecific effects of introducing any anti-Tmod1 monoclonal antibody into cardiac myocytes. Microinjection of mAb95, another anti-Tmod1 antibody that recognizes an epitope close to the middle of the molecule (unpublished data), or microinjection of mAb9, that recognizes the COOH-terminal region of Tmod1 and disrupts its capping activity, did not result in the loss of actin filament striations (Fig. 2, g and h; Gregorio et al., 1995). In additional control experiments to examine for any nonspecific effects on actin filaments, we found that microinjection of mAb17 into fibroblasts that contain actin stress fibers but no detectable Tmod1 (Gregorio and Fowler, 1995) resulted in no observable effects on actin staining (Fig. 2, k and l).


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)

Disruption of the interaction between Tmod1 and tropomyosin in live cardiac myocytes results in a loss of actin (thin) filaments. Cardiac myocytes were microinjected with MOPC-21 (a and b), mAb17 (c and d), mAb17 Fab fragments (i and j), mAb8 (e and f), or mAb95 (g and h). Injected cells were observed using an AlexaFluor 594–conjugated anti–mouse IgG to detect the injected antibody (a, c, and e). Microinjection of mAb17 or mAb8 resulted in a loss of actin filaments as detected by AlexaFluor 488 phalloidin (d and f, arrowheads) or using anti–α-actin antibodies (j, arrowheads). Normal actin filaments were seen in myocytes injected with MOPC-21 (b, arrows) or with mAb95 (h). Fibroblasts were also microinjected with mAb17 (k and l); no perturbation of actin filaments was seen (l). To test for recovery, injected cells were incubated for 48 h before staining. Actin filaments were easily visualized at this time point (m, arrows). A recombinant Tmod1 fragment containing residues 1–130, mixed with MOPC-21 (n, to identify injected cells) was microinjected into cardiac myocytes and incubated for 1 h. Microinjection of the Tmod1 fragment containing the tropomyosin binding site resulted in a loss of actin filaments (o). Bars, 10 μm.
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Related In: Results  -  Collection

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fig2: Disruption of the interaction between Tmod1 and tropomyosin in live cardiac myocytes results in a loss of actin (thin) filaments. Cardiac myocytes were microinjected with MOPC-21 (a and b), mAb17 (c and d), mAb17 Fab fragments (i and j), mAb8 (e and f), or mAb95 (g and h). Injected cells were observed using an AlexaFluor 594–conjugated anti–mouse IgG to detect the injected antibody (a, c, and e). Microinjection of mAb17 or mAb8 resulted in a loss of actin filaments as detected by AlexaFluor 488 phalloidin (d and f, arrowheads) or using anti–α-actin antibodies (j, arrowheads). Normal actin filaments were seen in myocytes injected with MOPC-21 (b, arrows) or with mAb95 (h). Fibroblasts were also microinjected with mAb17 (k and l); no perturbation of actin filaments was seen (l). To test for recovery, injected cells were incubated for 48 h before staining. Actin filaments were easily visualized at this time point (m, arrows). A recombinant Tmod1 fragment containing residues 1–130, mixed with MOPC-21 (n, to identify injected cells) was microinjected into cardiac myocytes and incubated for 1 h. Microinjection of the Tmod1 fragment containing the tropomyosin binding site resulted in a loss of actin filaments (o). Bars, 10 μm.
Mentions: We next used mAb17 and mAb8 in microinjection studies designed to investigate the interaction of Tmod1 with tropomyosin in the context of living cardiac myocytes. Day 3–5 cardiac myocytes were microinjected with mAb17 or mAb8 and incubated for 1 h after injection. The myocytes were fixed and stained for thin filament components. A few cells microinjected with mAb17 or mAb8 demonstrated a thin filament pointed end striated staining pattern for Tmod1 and unperturbed actin filaments (unpublished data). However, the majority (>80%) of myocytes injected with mAb17 (Fig. 2 c) or mAb8 (Fig. 2 e) exhibited a dramatic loss of actin filaments, as determined by staining with fluorescently conjugated phalloidin (Fig. 2, d and f), compared with the normal striated appearance of sarcomeric actin staining in cells injected with the control antibody MOPC-21 (Fig. 2 b) or in surrounding uninjected cells (Fig. 2 d, bottom). Additionally, staining with anti–cardiac actin antibodies demonstrated that the absence of actin staining was not due to an artifact from inhibition of phalloidin staining (Fig. 2 j). Notably, no Tmod1 striations were detected (i.e., Tmod appeared diffused in the cytoplasm) in the cells microinjected with mAb17 or mAb8, suggesting that disrupting the interaction of Tmod1 with tropomyosin promoted Tmod1's dissociation from thin filament pointed ends (Fig. 2, c and e). In the majority of cells (70–75%) perturbed by the microinjection of mAb17 or mAb8, no actin filaments were detected (not depicted), whereas in other cells remnants remained (Fig. 2, d, f, and j). Cells injected with Fab fragments of mAb17 exhibited an identical phenotype, indicating that the loss of sarcomeric actin filament staining was not due to Tmod1 cross-linking or sequestration in the cells (Fig. 2, i and j). The effect of mAb8 and mAb17 was also not due to nonspecific effects of introducing any anti-Tmod1 monoclonal antibody into cardiac myocytes. Microinjection of mAb95, another anti-Tmod1 antibody that recognizes an epitope close to the middle of the molecule (unpublished data), or microinjection of mAb9, that recognizes the COOH-terminal region of Tmod1 and disrupts its capping activity, did not result in the loss of actin filament striations (Fig. 2, g and h; Gregorio et al., 1995). In additional control experiments to examine for any nonspecific effects on actin filaments, we found that microinjection of mAb17 into fibroblasts that contain actin stress fibers but no detectable Tmod1 (Gregorio and Fowler, 1995) resulted in no observable effects on actin staining (Fig. 2, k and l).

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