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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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Depolymerization of thin filaments from their pointed ends was visualized in live cells expressing GFP–α-tropomyosin and microinjected with mAb17. Cardiac myocytes expressing GFP–α-tropomyosin were microinjected with MOPC-21 (a–c) or mAb17 (d–f). Images were recorded every 15 min for 1 h (data shown for preinjection, 30 and 60 min). After 30 and 60 min, cells injected with mAb17 displayed thin filament shortening from their pointed ends (a–f, arrows). No depolymerization was observed in cells microinjected with MOPC-21. Bar, 5 μm.
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fig3: Depolymerization of thin filaments from their pointed ends was visualized in live cells expressing GFP–α-tropomyosin and microinjected with mAb17. Cardiac myocytes expressing GFP–α-tropomyosin were microinjected with MOPC-21 (a–c) or mAb17 (d–f). Images were recorded every 15 min for 1 h (data shown for preinjection, 30 and 60 min). After 30 and 60 min, cells injected with mAb17 displayed thin filament shortening from their pointed ends (a–f, arrows). No depolymerization was observed in cells microinjected with MOPC-21. Bar, 5 μm.

Mentions: The data presented thus far indicated that thin filament stability was compromised in cells in which the interaction of Tmod1 with tropomyosin was disrupted. To address the mechanism of how this disassembly occurred, we collected time-lapse images of cells expressing GFP–α-tropomyosin that were microinjected with mAb17. GFP–α-tropomyosin assembled along the entire length of the thin filaments in all myocytes where it was expressed (Helfman et al., 1999). After injection with mAb17, striated GFP–α-tropomyosin was observed to dissociate directly from the pointed ends of the thin filaments in live cells (i.e., the GFP–α-tropomyosin detectable at the Z-lines remained at similar intensities, whereas the intensity from the pointed ends greatly diminished). This dissociation from the pointed ends was first noticeable at 30 min, with progressive dissociation only from the pointed ends up to 60 min (Fig. 3); i.e., no detectable GFP–α-tropomyosin intensity was lost from the sides of the filaments or from the Z-lines. In contrast, injection of MOPC-21 had no effect on the fluorescence intensity of GFP–α-tropomyosin. Interestingly, the depolymerization effect on the thin filaments appeared to occur with slower kinetics than that observed in myocytes injected with mAb17, fixed, and then stained. We speculate that this slower depolymerization effect was due to overexpression of α-tropomyosin in myocytes that typically do not have a considerable pool of soluble tropomyosin (Gregorio and Fowler, 1995) and/or alterations in the stoichiometry of tropomyosin isoform expression that may have further stabilized the thin filaments. The results from this experiment directly show that the loss of thin filaments is due to the depolymerization of the filaments from their pointed ends in cells. Furthermore, this observation also suggests that Tmod1 acts as a “cap” to stabilize tropomyosin's association with the actin filament pointed ends (Fowler et al., 1993), functioning with tropomyosin to prevent actin filament depolymerization.


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)

Depolymerization of thin filaments from their pointed ends was visualized in live cells expressing GFP–α-tropomyosin and microinjected with mAb17. Cardiac myocytes expressing GFP–α-tropomyosin were microinjected with MOPC-21 (a–c) or mAb17 (d–f). Images were recorded every 15 min for 1 h (data shown for preinjection, 30 and 60 min). After 30 and 60 min, cells injected with mAb17 displayed thin filament shortening from their pointed ends (a–f, arrows). No depolymerization was observed in cells microinjected with MOPC-21. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172850&req=5

fig3: Depolymerization of thin filaments from their pointed ends was visualized in live cells expressing GFP–α-tropomyosin and microinjected with mAb17. Cardiac myocytes expressing GFP–α-tropomyosin were microinjected with MOPC-21 (a–c) or mAb17 (d–f). Images were recorded every 15 min for 1 h (data shown for preinjection, 30 and 60 min). After 30 and 60 min, cells injected with mAb17 displayed thin filament shortening from their pointed ends (a–f, arrows). No depolymerization was observed in cells microinjected with MOPC-21. Bar, 5 μm.
Mentions: The data presented thus far indicated that thin filament stability was compromised in cells in which the interaction of Tmod1 with tropomyosin was disrupted. To address the mechanism of how this disassembly occurred, we collected time-lapse images of cells expressing GFP–α-tropomyosin that were microinjected with mAb17. GFP–α-tropomyosin assembled along the entire length of the thin filaments in all myocytes where it was expressed (Helfman et al., 1999). After injection with mAb17, striated GFP–α-tropomyosin was observed to dissociate directly from the pointed ends of the thin filaments in live cells (i.e., the GFP–α-tropomyosin detectable at the Z-lines remained at similar intensities, whereas the intensity from the pointed ends greatly diminished). This dissociation from the pointed ends was first noticeable at 30 min, with progressive dissociation only from the pointed ends up to 60 min (Fig. 3); i.e., no detectable GFP–α-tropomyosin intensity was lost from the sides of the filaments or from the Z-lines. In contrast, injection of MOPC-21 had no effect on the fluorescence intensity of GFP–α-tropomyosin. Interestingly, the depolymerization effect on the thin filaments appeared to occur with slower kinetics than that observed in myocytes injected with mAb17, fixed, and then stained. We speculate that this slower depolymerization effect was due to overexpression of α-tropomyosin in myocytes that typically do not have a considerable pool of soluble tropomyosin (Gregorio and Fowler, 1995) and/or alterations in the stoichiometry of tropomyosin isoform expression that may have further stabilized the thin filaments. The results from this experiment directly show that the loss of thin filaments is due to the depolymerization of the filaments from their pointed ends in cells. Furthermore, this observation also suggests that Tmod1 acts as a “cap” to stabilize tropomyosin's association with the actin filament pointed ends (Fowler et al., 1993), functioning with tropomyosin to prevent actin filament depolymerization.

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