Article |
Address correspondence to Carol C. Gregorio, Dept. of Cell Biology and Anatomy, University of Arizona, 1501 N. Campbell Ave., Tucson, AZ 85724. Tel.: (520) 626-8113. Fax.: (520) 626-2097. email: gregorio{at}email.arizona.edu
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Abstract |
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Key Words: sarcomere; myofibrillogenesis; cardiac muscle; actin; thin filament
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Introduction |
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At the Z-lines, the borders of individual sarcomeres, the barbed (plus or fast-growing) ends of the thin filaments are capped by CapZ; thereby, the thin filaments are prevented from elongating or shortening from their barbed ends. Along the length of the thin filaments, tropomyosin, an -helical rodlike molecule, forms hetero- and homodimers in a head-to-tail fashion. One well-established role of tropomyosin is to cooperate with the troponin complex in regulating the Ca2+-dependent actomyosin interaction (Huxley, 1969; for review see Cooke, 1997). However, mounting evidence from a number of in vitro studies indicate that tropomyosin also functions to stabilize the thin filaments. Tropomyosin increases filament stiffness, prevents fragmentation and bending, and prevents depolymerization of actin monomers from their pointed (minus or slow-growing) ends (Wegner, 1982; Broschat et al., 1989; Weigt et al., 1990; Adami et al., 2002). Tropomyosin also enhances actin filament assembly and physically protects thin filaments from the depolymerizing effects of ADF/cofilin or gelsolin (Ono and Ono, 2002; Nyakern-Meazza et al., 2002; for review see Cooper, 2002). In vivo studies have revealed a critical role for tropomyosin in proper muscle function. For example, homozygous
-tropomyosin null mice are embryonic lethal, whereas heterozygous knockout mice show no obvious phenotype (Blanchard et al., 1997; Rethinasamy et al., 1998). In Drosophila, mutations of one muscle tropomyosin isoform (Tm1) result in disruption of peripheral, but not central, myofibrillar organization, as well as alterations in force-generating properties (Kreuz et al., 1996). Studies in Caenorhabditis elegans demonstrate that suppression of tropomyosin expression leads to disorganized sarcomeric actin filaments and muscle paralysis (Ono and Ono, 2002). However, the precise mechanisms by which tropomyosin contributes to thin filament stability remain unclear, particularly in vertebrate muscle.
Another sarcomeric protein known to be critical for thin filament length regulation is tropomodulin 1 (Tmod1/E-Tmod), which caps the pointed ends of the thin filaments in cardiac muscle cells as well as in other cell types (for review see Weber, 1999). Unlike other actin capping proteins, Tmod1 also binds tropomyosin. Tmod1 completely blocks elongation and depolymerization from the pointed ends of actin filaments in the presence of tropomyosin in vitro (Kd < 50 pM). However, in the absence of tropomyosin, Tmod1 has a lower affinity for the pointed ends (Kd = 0.10.2 µM) and its capping activity is down-regulated >1,000-fold (Weber et al., 1994, 1999). Based on additional biochemical analyses, it was proposed that Tmod1 may also contribute to thin filament length regulation by preventing additional tropomyosin molecules from binding to the pointed ends of the actin filaments, thus acting as a tropomyosin capping molecule (Wegner, 1979; Fowler, 1990; Fowler et al., 1993).
Tmod1's dual interactions with tropomyosin and actin filaments are reflected by its distinct structure. Its COOH-terminal half is compact and tightly folded (Kostyukova et al., 2000). It is this half of the molecule that possesses the primary actin filament capping activity (Fowler et al., 2003). In contrast, Tmod1's NH2-terminal half is highly flexible and elongated (Kostyukova et al., 2000), and contains overlapping binding sites for both muscle and nonmuscle tropomyosins (Babcock and Fowler, 1994; Vera et al., 2000). Tmod1 also interacts with the extreme NH2-terminal modules of the giant striated muscle protein, nebulin. Although the significance of this interaction is unknown, it is intriguing to speculate that its role is consistent with nebulin's proposed function as a thin filament ruler that defines their uniform lengths (McElhinny et al., 2001). The existence of several Tmod1-interacting molecules in striated muscle suggests that Tmod1 may play multiple roles in thin filament length regulation.
The physiological roles of Tmod1 in cardiac muscle cells have been investigated in several studies. For example, decreasing levels of endogenous Tmod1 in rat cardiac myocytes resulted in abnormally long thin filaments (Sussman et al., 1998a). In contrast, overexpression of Tmod1 in rat and chick cardiac myocytes, or Sanpodo (a Tmod homologue) in Drosophila, resulted in shorter thin filaments (Sussman et al., 1998a; Littlefield et al., 2001; Mardahl-Dumesnil and Fowler, 2001). Transgenic mice overexpressing Tmod1 (TOT) in their myocardium exhibited myofibril disarray and dilated cardiomyopathy (Sussman et al., 1998b), and Tmod1-/- mouse embryos die at approximately day E10, suggesting an essential role for Tmod1 in myofibril assembly (Chu et al., 2003; unpublished data). Therefore, the levels of Tmod1 expression are important in maintaining the lengths of thin filaments and myofibril architecture in vivo.
The functional role of Tmod1's actin filament pointed end capping activity has been investigated directly in studies using primary cultures of chick cardiac myocytes. Microinjection of a monoclonal antibody that specifically blocked Tmod1's actin capping activity (but not its interaction with tropomyosin), resulted in an abnormal elongation of the actin filaments from their pointed ends and abolished contractile activity (Gregorio et al., 1995). Thus, Tmod1's actin filament capping activity is required to maintain the lengths of the mature thin filaments in cardiac muscle. Interestingly, in these studies, Tmod1 and tropomyosin remained associated with the thin filaments. Therefore, it has remained unclear what exactly is the function of Tmod1tropomyosin interactions in thin filament length regulation.
Here, we sought to investigate the functional properties of the interaction of Tmod1 with tropomyosin on thin filament length and stability in cardiac muscle. We determined that blocking the interaction of Tmod1 with tropomyosin in live cardiac myocytes resulted in a dramatic loss of thin filaments and subsequent contractile activity. The disappearance of thin filaments was visualized in real time and occurred from their pointed ends toward the Z-line. Our data indicate that the actin- and tropomyosin-binding activities of Tmod1 have unique and complementary functional roles. The actin capping activity of Tmod1 inhibits actin elongation and maintains the lengths of the thin filaments (Gregorio et al., 1995), whereas the tropomyosin-binding domain of Tmod1 appears to stabilize the thin filaments by preventing depolymerization from their pointed ends. These studies indicate that the regulated activity of Tmod1 is essential for proper muscle function.
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Results |
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Disrupting the interaction of Tmod1 with tropomyosin in live cardiac myocytes results in a reversible disassembly of thin filaments and cessation of beating
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 35 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 anticardiac 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 (7075%) 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).
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Next, to determine whether mAb17-induced Tmod1 and actin disassembly was accompanied by disassembly of tropomyosin, cells microinjected with mAb17 Fabs were stained for tropomyosin. A loss of tropomyosin staining was clearly observed, which is consistent with the loss of actin filaments (see Fig. 4 b), indicating that the entire thin filament had disassembled. This result suggested that the interaction of Tmod1 with tropomyosin is necessary to stabilize the association of tropomyosin with the actin filaments in cardiac myocyte sarcomeres.
As a complementary approach to using anti-Tmod1 antibodies to disrupt Tmod1's interaction with tropomyosin, we microinjected a recombinant NH2-terminal fragment of Tmod1 (1130) into cardiac myocytes, containing the tropomyosin binding site (Babcock and Fowler, 1994; Greenfield and Fowler, 2002). Again, a loss of actin filaments was observed (Fig. 2 o), likely due to a dominant-negative mechanism; that is, the injected Tmod1 fragment containing amino acids 1130, likely prevented endogenous Tmod1 from binding to endogenous tropomyosin. These experiments support the hypothesis that the interaction of Tmod1 with tropomyosin is critical for actin filament stability.
The dramatic disappearance of thin filaments in microinjected cells suggested that contractile activity of the cells would be inhibited. Indeed, beating activity was dramatically diminished in cells microinjected with mAb17. Only 5% of those myocytes injected with mAb17 were observed to beat 1 h after injection, compared with
88% of those cells injected with MOPC-21 (Table I). 100% of the uninjected cells that were beating at the onset of the experiment remained beating after 1 h (unpublished data). In summary, these data suggest that the Tmod1tropomyosin interaction is critical for thin filament stability and for contractile activity in cardiac myocytes.
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Discussion |
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Although this mechanism is an attractive model, it does not fully account for a surprising result from our cardiac myocyte thin filament reconstitution assays. Actin filaments appear to be relatively stable in extracted cells (myocyte ghosts) in the absence (or presence) of tropomyosin and Tmod1; however, the addition of the antiNH2-terminal Tmod1 antibodies mAb8 or mAb17 appear to transform Tmod1 (Fig. 7 III) such that it catalytically imparts instability on what would otherwise be a relatively stable structure. In other words, in these experiments, Tmod1 plus mAb8 or mAb17 appears to behave like a "depolymerase" for tropomyosinactin filaments. Perhaps the binding of these antibodies leads to a structural alteration in Tmod1 so that it now actively disrupts actinactin or actintropomyosin interactions. Alternatively, such an altered Tmod1 may interact differently with the giant protein nebulin (see Discussion), causing a propagated destabilization down the length of the filament, resulting in its disassembly. It is also plausible that a structural alteration occurs in the thin filament once Tmod1 has associated with the pointed ends, causing the filaments to become unstable if Tmod1tropomyosin interactions are perturbed. Our current experiments are addressing these intriguing issues.
An interesting, yet complex, question that arises is, how does Tmod1 function with tropomyosin in stabilizing the thin filaments given the fact that the pointed end components are dynamic? GFP-Tmod1 and fluorescently labeled actin monomers rapidly associate and dissociate from the pointed ends in chick embryonic cardiac myocytes (Littlefield et al., 2001). Additionally, epitope-tagged tropomyosin molecules, but not epitope-tagged troponin I molecules, preferentially assemble onto the pointed ends of the thin filaments in adult rat cardiac myocytes (Michele et al., 1999). These studies and our results indicate that the slow-growing, pointed ends of the thin filaments are not only dynamic with respect to the rapid exchange of actin, tropomyosin, and Tmod1 but are the critical site for both length regulation and overall stability of the thin filaments. Exactly how this occurs remains to be determined.
Although Tmod1 is a critical component for maintaining and stabilizing thin filament lengths, there is no evidence that Tmod1 is involved in specifying their lengths. However, several groups have identified nebulin, the Tmod1 and tropomyosin-binding protein, as the prime candidate molecule for functioning as a "ruler" to specify the precise lengths of the thin filaments in skeletal muscle and, more recently, in cardiac muscle (Fock and Hinssen, 2002; Kazmierski et al., 2003; for review see McElhinny et al., 2003). In support of this hypothesis, nebulin participates in the initial stages of assembly of I-Z-I bodies, precursor structures that mature into definitive Z-lines and I-bands (Ojima et al., 1999). Additionally, in many studies nebulin is observed in a striated pattern before the thin filaments attain their mature lengths, which is consistent with the idea that nebulin dictates thin filament architecture and restricts filament lengths (Shimada et al., 1996; Moncman and Wang, 1996). Nebulin may also act with tropomyosin as thin filament stabilizers during myofibril assembly. Importantly, recent reports have suggested a critical role for tropomyosin in myofibril assembly. Specifically, genetic analysis of the Unc-60B (homologue of cofilin/ADF) mutant in C. elegans (Ono and Ono, 2002) indicated that actin filament assembly depends on a balance between actin stabilization by tropomyosin and actin disassembly mediated by cofilin/ADF. Tropomyosin has also been implicated in playing an important role in zebrafish myofibrillogenesis based on analyses of cardiac troponin T (TNNT2) mutants; the loss of Tnnt2 expression in silent heart (sih) mutants resulted in a significant reduction in tropomyosin levels, causing sarcomere loss and myocyte disarray (Sehnert et al., 2002). Furthermore, tropomyosin is important for myofibrillogenesis in the amphibian, Mexican axolotl; that is, when mouse -tropomyosin was introduced into mutant hearts (that showed a reduction of overall tropomyosin expression), myofibrillogenesis and contractile activity were restored (Zajdel et al., 1998). Given these data, it is tempting to speculate the following model of thin filament assembly: when the actintropomyosin filaments attain their mature lengths, nebulin's NH2-terminal modules target Tmod1 to the pointed ends of the thin filaments. Once Tmod1 assembles, it functions to cap the actin and tropomyosin polymers, thus stabilizing and maintaining the lengths of the thin filaments at their pointed ends. We speculate that nebulin, tropomyosin, and Tmod1 play complementary, critical roles in controlling thin filament lengths and stability. Furthermore, the regulated interactions among these three thin filament components appear to be essential for proper myofibril assembly, structure, and function.
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Materials and methods |
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Western blot analysis
0.5 µg of full-length or fragments of Tmod1, and 20 ng of full-length Tmod1 and equivalent molar amounts of fragments, were loaded for Coomassie blue staining and Western blot analysis, respectively. Blots were probed as described previously (Gregorio et al., 1995).
The specificity of the anti-Tmod1 antibodies was determined from day 6 embryonic chick hearts. In brief, hearts were dissected, snap-frozen, and ground in liquid nitrogen. The powder was solubilized in 2x SDS sample buffer, run on a 10% gel, and transferred to nitrocellulose. Nitrocellulose strips were incubated with either 0.5 µg/ml of MOPC-21, mAb8, mAb17 or 1.0 µg/ml mAb17 Fabs followed by HRP-conjugated antimouse IgG (1:20,000). Blots were incubated in Super Signal chemiluminescent substrate (Pierce Chemical Co.) and exposed to film (Biomax MR; Kodak).
Antibody competition and dissociation assays
5 pmol of full-length recombinant chicken Tmod1 were absorbed onto nitrocellulose as spots using a dot blot apparatus and preincubated with 0700 nM of mAb8, 9, or 17 for 3 h at RT in a final volume of 200 µl (25 nM Tmod1). The molar ratio of mAb to Tmod1 varied from 1.3:1 at the lowest antibody concentration used (33 nM) to 28:1 at the highest concentration used (700 nM). After several washes, dots were incubated overnight at 4°C with 33 nM 125I-Bolton Hunterlabeled rabbit skeletal muscle tropomyosin in binding buffer (20 mM Hepes, 80 mM KCl, 2 mM MgCl2,1 mM DTT, 0.2% Triton X-100, and 20 mg/ml BSA), and washed to remove unbound tropomyosin. For the dissociation assay, 5 pmol Tmod1 were adsorbed onto nitrocellulose dots and incubated with 33 nM 125I-tropomyosin overnight at 4°C. After several washes, the Tmod1 dots were incubated with 330 nM mAb8 or MOPC-21 (molar ratio of mAb/Tmod = 13:1). The amount of bound 125I-tropomyosin was quantified in a counter.
Cell culture and microinjection procedures
Cardiac myocytes were isolated from day 6 embryonic chick hearts (Gregorio and Fowler, 1995). Isolated cells were plated at 106 cells/dish in 35-mm culture dishes containing CELLocate gridded coverslips (Eppendorf). Cells cultured for 35 d were injected with a 0.31.0-mg/ml solution of mAbs, Fabs, or purified Tmod1 fragments in injection buffer using a Transjector (model 5246; Eppendorf) and micromanipulator (model 5171; Eppendorf). Injected cells were incubated for 148 h before fixation.
Beating assays were performed by injecting beating cardiac myocytes cultured for 4 d with mAb17 or MOPC-21. Cells were incubated for 1 h at 37°C and the percentage of injected cells that were beating was determined. Greater than 60 cells/coverslip were analyzed and the experiment was performed in triplicate. Numbered grids on coverslips allowed for the identification of microinjected cells.
Indirect immunofluorescence and deconvolution microscopy
Cardiac myocytes were fixed 1 h after injection in 5% formaldehyde/PBS for 10 min, washed in PBS, and permeabilized in 0.2% Triton X-100/PBS for 15 min. Coverslips were blocked in 2% BSA/1% donkey serum/PBS for 30 min. Microinjected cells were stained with an AlexaFluor 594conjugated goat antimouse IgG (1:1,000) or a donkey Texas redconjugated antimouse IgG (Fab specific; 1:100) to detect the injected antibody. Actin was visualized using an AlexaFluor 488, 594 or 647 phalloidin, or a monoclonal anticardiac actin antibody (Ac1-20.4.2; 1:10; American Research Products) followed by a donkey Texas redconjugated antimouse IgG (Fc specific) antibody (1:100). For triple labeling, cells within numbered grids on the coverslip were injected with mAb17 Fabs or MOPC-21 Fabs and incubated for 1 h before fixation. Cells were incubated with monoclonal sarcomeric anti-actinin antibodies (1:1,500; EA-53; Sigma-Aldrich), antimyomesin B4 antibodies (1:50; provided by J-.C. Perriard and E. Ehler, Institute for Cell Biology, Zurich, Switzerland; Grove et al., 1984), or antimyosin F59 antibodies (1:10; provided by F. Stockdale, Stanford University, Stanford, CA) followed by FITC-conjugated antimouse Fc-specific IgG (1:100). Antititin A168-A170 (1:100) or titin N2A antibodies (10 µg/ml) (Centner et al., 2000) were added, followed by Cy5-conjugated donkey antirabbit IgG antibodies (1:600). Cells were incubated in AlexaFluor 594 phalloidin. AlexaFluor-conjugated antibodies and phalloidin were purchased from Molecular Probes. All other fluorescent antibodies were purchased from Jackson ImmunoResearch Laboratories. Cells were analyzed on a microscope (model IX70; Olympus). Micrographs were recorded as digital images (with Z-series containing 0.15-µm sections) using a CCD camera (model Series 300; Photometrics) and deconvolved using DeltaVision software (Applied Precision).
Transfection and live cell imaging
For live cell imaging, transfection was performed by incubating 1 µg pEGFPrat-tropomyosin (gift from J.-C. Perriard; Helfman et al., 1999) with 7 µl Cytofectene (Bio-Rad Laboratories) and 100 µl Opti-MEM (Life Technologies) for 15 min at RT and adding the mixture to cardiac myocytes cultured for 24 h 34 d after transfection, cells were injected with mAb17 or MOPC-21. Coverslips were placed into a Focht Live-cell Chamber apparatus (Bioptechs) and Z-series of injected cells were imaged every 15 min. Staining with anti
-actinin antibodies allowed us to determine the direction of depolymerization.
Cell permeabilization assay
The assay was performed as described previously (Gregorio and Fowler, 1995). Day 6 cardiac myocytes were permeabilized in relaxing buffer (0.12 M KCl, 4 mM MgCl2, 20 mM Tris-HCl, pH 6.8, 4 mM EGTA, 4 mM ATP, and 0.2 mg/ml saponin) for 10 min at 0°C, and then were extracted in a high salt buffer (0.5 M KCl, 10 mM sodium pyrophosphate, 5 mM MgCl2, 10 mM Tris-HCl, pH 6.5, 1 mM EGTA, and 0.2 mg/ml saponin) for 10 min at RT. Extracted cells were washed in incubation buffer (20 mM KCl, 5 mM Tris-HCl, pH 6.8, 0.1 mM CaCl2, 0.1 mM ATP, and 0.2 mg/ml saponin) for 15 s. To confirm the extraction, cells were fixed and stained with antimyosin F59, antitropomyosin CH1 (Lin et al., 1985), or anti-Tmod1 monoclonal 95 antibodies (1:50; Almenar-Queralt et al., 1999) followed by AlexaFluor 594conjugated goat antimouse IgG (1:800) and AlexaFluor 488 phalloidin. To reconstitute Tmod1 onto the myofibrils, cells were first incubated with 50 µg/ml biotinylated tropomyosin in 0.1 mM KCl, and 0.1 M Hepes, pH 7.5 for 10 min, rinsed, and then were incubated with recombinant Tmod1 at 70 µg/ml in 80 mM KCl, 2 mM MgCl2, 0.1 mM DTT, and 20 mM Hepes, pH 7.3, in the presence of either 0.2 mg/ml of mAb17, mAb8, MOPC-21 or 0.05 mg/ml mAb17 Fabs for 25 min. After rinsing the cells in rigor buffer (60 mM KCl, 5 mM MgCl2, 1 mM EGTA, 10 mM Tris-HCl, pH 6.8, and 0.2 mg/ml saponin) the cells were fixed and stained for biotinylated tropomyosin using FITC-conjugated avidin (1:200; Zymed Laboratories) and Tmod1 using rabbit anti-Tmod1 1844 antibodies (1:100) followed by Cy5-conjugated donkey antirabbit IgG antibodies (1:500). Actin was visualized using AlexaFluor 594 phalloidin. Stabilization of actin filaments was performed by adding either AlexaFluor 594 phalloidin for 20 min or 1 µM jasplakinolide (Molecular Probes) in DMSO for 7 min after the cells were extracted. Myocytes treated with actin-stabilizing agents were reconstituted with tropomyosin and Tmod1, fixed, and stained as described in the section Indirect immunofluorescence and deconvolution microscopy.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL57461, HL03985 (to C.C. Gregorio), and GM34125 (to V.M. Fowler), National Science Foundation (NSF) predoctoral fellowship (to R.E. Mudry), and NSF DBI 9912036 (to C.N. Perry).
Submitted: 7 May 2003
Accepted: 29 July 2003
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References |
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---|
Adami, R., O. Cintio, G. Trombetta, D. Choquet, and E. Grazi. 2002. Effects of chemical modification, tropomyosin, and myosin subfragment 1 on the yield strength and critical concentration of F-actin. Biochemistry. 41:59075912.[CrossRef][Medline]
Almenar-Queralt, A., A. Lee, C.A. Conley, L. Ribas de Pouplana, and V.M. Fowler. 1999. Identification of a novel tropomodulin isoform, skeletal tropomodulin, that caps actin filament pointed ends in fast skeletal muscle. J. Biol. Chem. 274:2846628475.
Babcock, G., and V.M. Fowler. 1994. Isoform specific interaction of tropomodulin with skeletal muscle and erythrocyte tropomyosins. J. Biol. Chem. 269:2751027518.
Blanchard, E.M., K. Iizuka, M. Christe, D.A. Conner, A. Geisterfer-Lowrance, F.J. Schoen, D.W. Maughan, C.E. Seidman, and J.G. Seidman. 1997. Targeted ablation of the murine -tropomyosin gene. Circ. Res. 81:10051010.
Broschat, K.O. 1990. Tropomyosin prevents depolymerization of actin filaments from the pointed end. J. Biol. Chem. 265:2132321329.
Broschat, K.O., A. Weber, and D.R. Burgess. 1989. Tropomyosin stabilizes the pointed end of actin filaments by slowing depolymerization. Biochemistry. 28:85018506.[Medline]
Centner, T., F. Fougerousse, A. Freiburg, C. Witt, J.S. Beckmann, H. Granzier, K. Trombitas, C.C. Gregorio, and S. Labeit. 2000. Molecular tools for the study of titin's differential expression. Adv. Exp. Med. Biol. 481:3549.[Medline]
Chu, X., J. Chen, M.C. Reedy, C. Vera, K.L. Sung, and L.A. Sung. 2003. E-Tmod capping of actin filaments at the slow-growing end is required to establish mouse embryonic circulation. Am. J. Physiol. Heart Circ. Physiol. 284:H1827H1838.
Cooke, R. 1997. Actomyosin interaction in striated muscle. Physiol. Rev. 77:671697.
Cooper, J.A. 2002. Actin dynamics: tropomyosin provides stability. Curr. Biol. 12:R523R525.[CrossRef][Medline]
Fock, U., and H. Hinssen. 2002. Nebulin is a thin filament protein of the cardiac muscle of the agnathans. J. Muscle Res. Cell Motil. 23:205213.[CrossRef][Medline]
Fowler, V.M. 1990. Tropomodulin: a cytoskeletal protein that binds to the end of the erythrocyte tropomyosin and inhibits tropomyosin binding to actin. J. Cell Biol. 111:471482.[Abstract]
Fowler, V.M., M.A. Sussmann, P.G. Miller, B.E. Flucher, and M.P. Daniels. 1993. Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle. J. Cell Biol. 120:411420.[Abstract]
Fowler, V.M., N.J. Greenfield, and J. Moyer. 2003. Tropomodulin contains two actin filament pointed end-capping domains. J. Biol. Chem. 10.1074/jbc.M306895200.
Greenfield, N.J., and V.M. Fowler. 2002. Tropomyosin requires an intact N-terminal coiled coil to interact with tropomodulin. Biophys. J. 82:25802591.
Gregorio, C.C., and V.M. Fowler. 1995. Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly. J. Cell Biol. 129:683695.[Abstract]
Gregorio, C.C., A. Weber, M. Bondad, C.R. Pennise, and V.M. Fowler. 1995. Requirement of pointed-end capping by tropomodulin to maintain actin filament length in embryonic chick cardiac myocytes. Nature. 377:8386.[Medline]
Grove, B.K., V. Kurer, C. Lehner, T.C. Doetschman, J.C. Perriard, and H.M. Eppenberger. 1984. A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies. J. Cell Biol. 98:518524.[Abstract]
Helfman, D.M., C. Berthier, J. Grossman, M. Leu, E. Ehler, E. Perriard, and J.C. Perriard. 1999. Nonmuscle tropomyosin-4 requires coexpression with other low molecular weight isoforms for binding to thin filaments in cardiomyocytes. J. Cell Sci. 112:371380.
Huxley, H.E. 1969. The mechanism of muscular contraction. Science. 164:13561365.[Medline]
Kazmierski, S.T., P.B. Antin, C.C. Witt, N. Huebner, A.S. McElhinny, S. Labeit, and C.C. Gregorio. 2003. The complete mouse nebulin gene sequence and the identification of cardiac nebulin. J. Mol. Biol. 328:835846.[CrossRef][Medline]
Kostyukova, A., K. Maeda, E. Yamauchi, I. Krieger, and Y. Maeda. 2000. Domain structure of tropomodulin: distinct properties of the N-terminal and C-terminal halves. Eur. J. Biochem. 267:64706475.
Kreuz, A.J., A. Simcox, and D. Maughan. 1996. Alterations in flight muscle ultrastructure and function in Drosophila tropomyosin mutants. J. Cell Biol. 135:673687.[Abstract]
Lin, J.J.-C., C.S. Chou, and J.L.C. Lin. 1985. Monoclonal antibodies against chicken tropomyosin isoforms: production, characterization, and application. Hybridoma. 4:223242.[Medline]
Linke, W.A., D.E. Rudy, T. Centner, M. Gautel, C. Witt, S. Labeit, and C.C. Gregorio. 1999. I-Band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure. J. Cell Biol. 146:631644.
Littlefield, R., A. Almenar-Queralt, and V.M. Fowler. 2001. Actin dynamics at pointed ends regulates thin filament length in striated muscle. Nat. Cell Biol. 3:544551.[CrossRef][Medline]
Mardahl-Dumesnil, M., and V.M. Fowler. 2001. Thin filaments elongate from their pointed ends during myofibril assembly in Drosophila indirect flight muscle. J. Cell Biol. 155:10431053.
McElhinny, A.S., B. Kolmerer, V.M. Fowler, S. Labeit, and C.C. Gregorio. 2001. The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments. J. Biol. Chem. 276:583592.
McElhinny, A.S., S.T. Kazmierski, S. Labeit, and C.C. Gregorio. 2003. Nebulin: the nebulous, multifunctional giant of striated muscle. Trends Cardiovasc. Med. 13:195201.[CrossRef][Medline]
Michele, D.E., F.P. Albayya, and J.M. Metzger. 1999. Thin filament protein dynamics in fully differentiated adult cardiac myocytes: toward a model of sarcomere maintenance. J. Cell Biol. 145:14831495.
Moncman, C.L., and K. Wang. 1996. Assembly of nebulin into the sarcomeres of avian skeletal muscle. Cell Motil. Cytoskeleton. 34:167184.[CrossRef][Medline]
Nyakern-Meazza, M., K. Narayan, C.E. Schutt, and U. Lindberg. 2002. Tropomyosin and gelsolin cooperate in controlling the microfilament system. J. Biol. Chem. 277:2877428779.
Ojima, K., Z.X. Lin, Z.Q. Zhang, T. Hijikata, S. Holtzer, S. Labeit, H.L. Sweeney, and H. Holtzer. 1999. Initiation and maturation of I-Z-I bodies in the growth tips of transfected. J. Cell Sci. 112:41014112.
Ono, S., and K. Ono. 2002. Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics. J. Cell Biol. 156:10651076.
Rethinasamy, P., M. Muthuchamy, T. Hewett, G. Boivin, B.M. Wolska, C. Evans, R.J. Solaro, and D.F. Wieczorek. 1998. Molecular and physiological effects of -tropomyosin ablation in the mouse. Circ. Res. 82:116123.
Sehnert, A.J., A. Huq, B.M. Weinstein, C. Walker, M. Fishman, and D.Y. Stainier. 2002. Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nat. Genet. 31:106110.[CrossRef][Medline]
Shimada, Y., M. Komiyama, S. Begum, and K. Maruyama. 1996. Development of connectin/titin and nebulin in striated muscles of chicken. Adv. Biophys. 33:223233.[CrossRef][Medline]
Sussman, M.A., S. Baque, C.S. Uhm, M.P. Daniels, R.L. Price, D. Simpson, L. Terracio, and L. Kedes. 1998a. Altered expression of tropomodulin in cardiomyocytes disrupts the sarcomeric structure of myofibrils. Circ. Res. 82:94105.
Sussman, M.A., S. Welch, N. Cambon, R. Klevitsky, T.E. Hewett, R. Price, S.A. Witt, and T.R. Kimball. 1998b. Myofibril degeneration caused by tropomodulin overexpression leads to dilated cardiomyopathy in juvenile mice. J. Clin. Invest. 101:5161.
Vera, C., A. Sood, K.M. Gao, L.J. Yee, J.J. Lin, and L.A. Sung. 2000. Tropomodulin-binding site mapped to residues 7-14 at the N-terminal heptad repeats of tropomyosin isoform 5. Arch. Biochem. Biophys. 378:1624.[CrossRef][Medline]
von Arx, P., S. Bantle, T. Soldati, and J.C. Perriard. 1995. Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function. J. Cell Biol. 131:17591773.[Abstract]
Weber, A. 1999. Actin binding proteins that change extent and rate of actin monomer-polymer distribution by different mechanisms. Mol. Cell. Biochem. 190:6774.[CrossRef][Medline]
Weber, A., C.R. Pennise, G.G. Babcock, and V.M. Fowler. 1994. Tropomodulin caps the pointed ends of actin filaments. J. Cell Biol. 127:16271635.[Abstract]
Weber, A., C.R. Pennise, and V.M. Fowler. 1999. Tropomodulin increases the critical concentration of barbed end-capped actin filaments by converting ADP.P(i)-actin to ADP-actin at all pointed filament ends. J. Biol. Chem. 274:3463734645.
Wegner, A. 1979. Equilibrium of the actin-tropomyosin interaction. J. Mol. Biol. 131:839853.[Medline]
Wegner, A. 1982. Kinetic analysis of actin assembly suggests that tropomyosin inhibits spontaneous fragmentation of actin filaments. J. Mol. Biol. 161:217227.[Medline]
Weigt, C., B. Schoepper, and A. Wegner. 1990. Tropomyosin-troponin complex stabilizes the pointed ends of actin filaments against polymerization and depolymerization. FEBS Lett. 260:266268.[CrossRef][Medline]
Zajdel, R.W., M.D. McLean, S.L. Lemanski, M. Muthuchamy, D.F. Wieczorek, L.F. Lemanski, and D.K. Dube. 1998. Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts. Dev. Dyn. 213:412420.[CrossRef][Medline]