Correspondence to Carol C. Gregorio: gregorio{at}u.arizona.edu
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several studies investigating thin filament length regulation have focused on capping proteins, which prevent depolymerization and elongation of actin filaments in vitro (for review see Littlefield and Fowler, 1998). Capping protein (CapZ) caps the barbed ends of the filaments and likely contributes to their alignment within the Z-discs (Schafer et al., 1995). Tropomodulin (Tmod) is critical for maintaining the lengths of the thin filaments at their pointed ends (Weber et al., 1994; Gregorio et al., 1995), the region of the filament where highly regulated actin dynamics influence their mature lengths (Littlefield et al., 2001). In vitro and cell culture studies indicate that Tmod's interaction with actin prevents the filaments from elongating, whereas its interaction with tropomyosin prevents depolymerization (Gregorio et al., 1995; Mudry et al., 2003). The levels of Tmod present in the cell are critical for thin filament length regulation because its overexpression results in filament shortening, whereas its reduction causes filament elongation across the H zone, in the center of the sarcomere (Sussman et al., 1998a; Littlefield et al., 2001; Mardahl-Dumesnil and Fowler, 2001). Interestingly, although it is known that a large portion (40%) of Tmod is present in a soluble pool (Gregorio and Fowler, 1995), exactly how this molecule's dynamic properties and targeting to the pointed ends are regulated remains unclear.
Although pivotal for understanding thin filament length maintenance and dynamics, investigations into the capping proteins have not revealed how filament lengths are specified. It has long been proposed that molecular templates dictate thin filament lengths, but their identities have remained elusive. Since its discovery over two decades ago (Wang and Williamson, 1980), the molecular properties of the giant protein nebulin (mol wt = 500900 kD) have implicated it as a prime candidate to act as a ruler in specifying thin filament lengths in striated muscle. First, single molecules of nebulin span the entire length of the thin filaments: its COOH termini localize to the Z-lines, and its NH2 termini extend to the thin filament pointed ends (Wang and Wright, 1988; Millevoi et al., 1998; McElhinny et al., 2001). Second, nebulin interacts with thin filament components, including tropomyosin, actin, troponins, and Tmod (Jin and Wang, 1991; Pfuhl et al., 1994; McElhinny et al., 2001). Third, nebulin's modular structure, composed of repeating motifs that are susceptible to alternative splicing events, suggests that its isoforms dictate thin filament architecture (Labeit and Kolmerer, 1995; Wang et al., 1996). Consistent with this idea, the molecular masses of nebulin isoforms correlate with thin filament lengths that differ among muscle fibers, during development, and with disease (for reviews see Trinick, 1994; McElhinny et al., 2003). Finally, many studies have reported that nebulin assembles in a striated pattern before the thin filaments attain their mature, defined lengths during myofibrillogenesis, which is also consistent with the idea that it restricts their lengths (Moncman and Wang, 1996; Shimada et al., 1996; Nwe et al., 1999). Although intriguing, these studies are based on correlative evidence because nebulin's massive size, coupled with the challenges of isolating it in its native form, has precluded definitive testing of its function.
In addition to its proposed role as a thin filament ruler, several investigations using recombinant nebulin fragments suggest the intriguing possibility that it may have other functions (for review see McElhinny et al., 2003). Nebulin may contribute to actin nucleation and enhance filament stability (Chen et al., 1993; Gonsior et al., 1998). Biochemical evidence also suggests that it regulates the interaction of actin with myosin in response to calcium levels, perhaps in cooperation with the tropomyosintroponin complex (Root and Wang, 1994, 2001). Finally, although speculative, nebulin may function in myofibril-based signaling events because it contains an SH3 domain and Ser-rich domain in its COOH-terminal, Z-line region (Labeit and Kolmerer, 1995; Wang et al., 1996).
Given the fact that nebulin may have these critical functions, a conundrum that existed in the field for years was that nebulin was reportedly expressed in skeletal muscle but not in heart or other tissues (Stedman et al., 1988; Labeit and Kolmerer, 1995; Zhang et al., 1996). Cardiac muscle contains nebulette, a smaller (107 kD) protein that has nebulin-like motifs but is encoded by a separate gene (Moncman and Wang, 1995; Millevoi et al., 1998). If extended, nebulette would span
25% of the lengths of the mature thin filaments and would not be expected to function in length specification. The apparent absence of nebulin in cardiac muscle was puzzling because although the thin filament lengths are more variable than those in skeletal muscle (Robinson and Winegrad, 1979), the length distributions in both tissues are Gaussian, not exponential, as actin filaments in vitro have been observed to be (for review see Littlefield and Fowler, 1998). In resolution to this conundrum, nebulin recently was detected in cardiac muscle in a molecular layout identical to skeletal muscle nebulin, although at lower levels (Fock and Hinssen, 2002; Kazmierski et al., 2003; Donner et al., 2004).
We investigated the functional properties of nebulin in living myocytes. Using RNA interference technology, we found that the preexisting thin filaments in rat cardiomyocytes dramatically elongated from their pointed ends immediately upon nebulin knockdown. Other myofibril components, including the barbed end marker -actinin, were unaffected. When the thin filaments were depolymerized by latrunculin B (Lat B), they reassembled to unrestricted lengths in the absence of nebulin. Finally, knockdown of nebulin in skeletal myocytes delayed myofibril assembly. Thus, a primary function for nebulin is in thin filament length regulation and assembly, and our data support the hypothesis that this giant molecule acts as a ruler to specify thin filament lengths.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Nebulin is required for thin filament reassembly after depolymerization
So far, the results have indicated that nebulin is required for thin filament length maintenance because the filaments were already in their normal, striated pattern before nebulin was knocked down (unpublished data). Therefore, we aimed to determine whether nebulin is also involved in thin filament assembly. We first treated myocytes with nebulin siRNA for 18 h and then added Lat B for 4 h to specifically depolymerize the thin filaments. At this time, only remnants of total cellular actin filaments were visible by phalloidin staining, whereas cardiac actin, nebulin, and Tmod1 staining were diffuse throughout the cytoplasm in both control and nebulin siRNAtreated myocytes (Fig. 5, ah). Not surprisingly, in >70% of both populations of myocytes, -actinin was severely perturbed (Fig. 5, i and j), indicating that the thin filaments had completely depolymerized (i.e., both barbed and pointed ends were affected). In contrast, no discernible effects on titin or thick filament integrity were detected (Fig. 5, kn), indicating that the thin filaments specifically were depolymerized by Lat B treatment. It also should be noted that no difference in the rates of depolymerization were observed between the nebulin and control siRNAtreated myocytes, suggesting that nebulin expression is not immediately essential for thin filament stability.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data reveal that although thin filament components polymerize in the absence of nebulin, the giant molecule is required for restricting the thin filaments to their proper lengths at the pointed ends. Importantly, it is the pointed ends of the filament that undergo dynamic exchange of the actin monomers Tmod1 and tropomyosin (Suzuki et al., 1998; Michele et al., 1999; Littlefield et al., 2001). Not surprisingly, then, the pointed ends are where the variability in mature thin filament lengths is apparent and where mechanisms for length determination and regulation have been indicated (Robinson and Winegrad, 1979; Littlefield et al., 2001; Luther et al., 2003). Based on data to date, we propose that one important mechanism by which nebulin participates in thin filament length regulation is that its NH2-terminal region, which contains a high affinity binding site for Tmod1 (McElhinny et al., 2001), targets Tmod1 to the pointed ends. Thus, immediately upon nebulin knockdown, the thin filaments became permanently "uncapped" at their pointed ends and elongate across the H zone. In fact, thin filament elongation across the H zone is known to occur upon inhibition of Tmod's capping activity (Gregorio et al., 1995) and upon reduction of Tmod levels (Sussman et al., 1998b). It is likely that nebulin specifies thin filament lengths and then regulates Tmod dynamics for maintaining and "fine-tuning" the lengths of the filaments. In this regard, mounting evidence has led to the proposal that thin filament length specification is not simply a one-time event (Littlefield et al., 2001). Instead, the ongoing, coordinated activity of caps (Tmod and CapZ), a ruler (nebulin), and potentially other proteins that regulate thin filament dynamics are likely involved in linking length specification with maintenance during development and throughout the lifetime of the cell.
Our studies using Lat B to depolymerize the preexisting thin filaments also implicate nebulin as an essential regulator of thin filament assembly. In the absence of proper nebulin levels, depolymerized thin filament components were unable to reassemble to their uniform lengths and lacked assembled Tmod1. These data are also consistent with the idea of nebulin's role in specification of their mature lengths and targeting Tmod1 to their pointed ends for maintenance. However, our studies also indicate that nebulin plays a critical role in the assembly and organization of thin filament barbed ends because nebulin siRNAtreated myocytes were unable to reorganize -actinin into its normal, striated pattern after Lat B treatment. This important finding raises many questions concerning nebulin's mechanism of action in the Z-lines.
Why is nebulin required for barbed end reassembly but appears to be nonessential for barbed end structural maintenance in the preexisting thin filaments? One idea is that nebulin is critical to the initial assembly of I-Z-I bodies (precursor Z-line and thin filament structures; Ojima et al., 2000). It has been hypothesized that these structures are more dynamic and compliant (i.e., contain unsaturated binding sites) than the tightly packed, mature Z-lines (Schroeter et al., 1996; Ojima et al., 2000). Thus, the thin filament components depolymerized by Lat B may have been rendered to a configuration similar to their organization in I-Z-I bodies (consistent with their staining patterns; Fig. 6 j and Fig. 7 d), and may require nebulin's binding sites and/or potential signaling capabilities in its COOH terminus to attain their mature structure. This is particularly interesting given the fact that proteins containing SH3 and Ser domains are implicated in the assembly of specialized cytoskeletal structures (for reviews see Mayer, 2001; Kioka et al., 2002) and these domains in nebulin's Z-line region have long been proposed to function in assembly (for review see McElhinny et al., 2003).
In contrast, in the preexisting thin filaments, the barbed ends may have been securely "bolted down" via interactions with other sarcomeric components in mature Z-lines. Thus, nebulin may not be immediately required for maintaining their stability. For example, nebulette, which is anchored in the Z-lines (Moncman and Wang, 1995), may have compensated for the loss of nebulin. Consistent with this idea, both nebulin and nebulette SH3 domains bind myopalladin, which also interacts with -actinin, perhaps forming a docking site for the barbed ends (Bang et al., 2001). In fact, SH3 domains are known to exhibit broad binding specificity for several target proteins (Buday, 1999; Mayer, 2001). It should be emphasized, however, that the barbed ends maintained their structure in the absence of nebulin for only a few hours after elongation from their pointed ends was first observed, eventually widening and then becoming less defined (unpublished data). Thus, we cannot rule out that a reduction of nebulin influenced barbed end dynamics or stability in the preexisting thin filaments, even though
-actinin organization was not immediately affected. Finally, it is important to note that nebulin knockdown perturbed thin filament reassembly at both barbed and pointed ends after Lat B treatment yet did not affect the integrity of the thick filaments or titin's Z-line region (Figs. 6 and 7). This is consistent with studies demonstrating that the integrity of titin and thick and thin filaments are not dependent on each other in cardiomyocytes (Linke et al., 1999; McElhinny et al., 2002; Moncman and Wang, 2002).
Is nebulin a ruler for thin filament length regulation?
Our studies support the long-proposed hypothesis that nebulin is a thin filament ruler to specify the uniform thin filament lengths in muscle. We favor the following model for nebulin's role in thin filament length regulation: (a) Nebulin assembles early in myofibrillogenesis (Moncman and Wang, 1996; Shimada et al., 1996) and aids in aligning and organizing thin filament components at their barbed ends. This may occur in cooperation with CapZ and -actinin. (b) Nebulin isoform sizes specify the precise lengths of the thin filaments. (c) Nebulin's NH2-terminal modules target Tmod to the pointed ends of mature thin filaments, where it functions as a dynamic cap to maintain their specified lengths. (d) Nebulin and Tmod1 work together to maintain (and perhaps fine-tune) the lengths at the pointed ends throughout the lifetime of the myocyte.
This model incorporates a myriad of biochemical and structural data suggesting that nebulin's modules dictate the number of actin monomers per thin filament. However, we cannot rule out the possibility that nebulin functions in thin filament assembly and length regulation via another mechanism. For example, nebulin may mediate the activities or expression of downstream molecules required for actin filament length regulation. Another possibility is that perturbation of nebulin and/or its associated proteins may alter the critical concentration for actin polymerization, which would change thin filament lengths via thermodynamic and kinetic considerations. Definitively proving a ruler function requires complex, future studies using model systems expressing mutant nebulins, both larger and smaller.
Important questions remain concerning our model and the results from our investigations. First, how are nebulin isoform sizes controlled during development and in different muscle tissues? Perhaps chronic demands placed on muscles modulate nebulin's splicing machinery via nebulin-interacting proteins proposed to affect gene expression. Additionally, what is the mechanism by which the contractile activity of cardiomyocytes was severely inhibited upon nebulin knockdown? We hypothesize that this resulted from the elongated thin filaments, which crossed the H zone and thus were of opposite polarity with respect to the thick filaments from the opposing half-sarcomere. This idea is consistent with studies suggesting that misregulated thin filament lengths affect contractile activity (Gregorio et al., 1995; Sussman et al., 1998a; Mudry et al., 2003). Another possibility is that absence of nebulin disrupted the positioning of the tropomyosintroponin complex along the thin filament, an idea based on the proposal that nebulin dictates the architecture of the regulatory complex (Labeit and Kolmerer, 1995; Wang et al., 1996). On the other hand, nebulin may participate in contractile regulation more directly. For example, fragments of nebulin affect actin sliding over myosin filaments in vitro (Root and Wang, 1994, 2001). Clearly, further investigations into nebulin's role in contractile regulation are warranted.
Nebulin is involved in myofibrillogenesis
Our final experiments revealed that, upon nebulin knockdown during myofibrillogenesis in skeletal myotubes, no components examined assembled into mature, striated patterns during the 5 d in culture. Therefore, nebulin is involved in the formation of myofibrils during differentiation. Given the reduction of the I-Z-I complexes in myocytes with reduced nebulin, as demonstrated by staining for -actinin and other thin filament components (Fig. 8), it is not surprising that myofibril assembly was abrogated (Ojima et al., 2000). Our data is consistent with studies revealing that perturbation of key sarcomeric components is deleterious to normal muscle differentiation. For example, the indirect flight muscles of flies defective in myosin lack thick filaments, and their myofibrils are perturbed (Mogami et al., 1986; O'Donnell and Bernstein, 1988). Furthermore, a functional knockout of titin impairs thick filament formation and blocks myofibrillogenesis (van der Ven et al., 2000).
However, other reports suggest that thick and thin filament assembly occur independently in skeletal myocytes. For example, perturbation of CapZ function in myotubes delays thin filament formation but does not affect thick filament assembly (Schafer et al., 1995). Additionally, Drosophila melanogaster actin mutants lack thin filaments and Z-lines but have normal thick filaments, and -actinin mutants have normal myofibrils (Beall et al., 1989; Roulier et al., 1992). Thus, it appears that during muscle development, perturbation of some sarcomeric components is deleterious to myofibril assembly, whereas disruption of others has more subtle consequences. In summary, our data indicate that nebulin is involved in myofibrillogenesis. However, it remains to be determined whether thin filament assembly and length regulation are required processes for differentiation and whether nebulin is a multifunctional giant, perhaps involved in gene expression and signaling.
In conclusion, our results demonstrate that more than two decades after its discovery, a primary function for nebulin is indeed thin filament length regulation and assembly. An exciting implication is that the precise lengths of actin filaments in numerous cellular structures are regulated by molecular templates. Thus, there may be an entire nebulin family in cells that contain actin filaments of defined lengths, an idea supported by an in situ hybridization study that detected nebulin in other tissues (Kazmierski et al., 2003), as well as a study identifying nebulin-like molecules in many species (for review see Clark et al., 2002). Future investigations into ruler proteins can serve as a precedent for investigating molecular length regulation and macromolecular assembly in many types of biological systems and will provide insight into actin dynamics in most, if not every, cell type.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For skeletal myotube cultures, myoblasts were isolated from rat hind limbs. The deboned tissue was suspended in 10 ml trypsin/EDTA (Life Technologies) and incubated for 10 min at 37°C. After centrifugation, the pellets were resuspended in MEM + 15% chick embryo extract + 10% horse serum, and cells were preplated at 37°C for 1 h on 100-mm dishes. Collected cells were plated at 4 x 105 cells/ml in 35-mm culture dishes containing 12-mm round coverslips coated with rat-tail collagen (Sigma-Aldrich). Cells were transfected with siRNA 24 h later, except this time using 10 nM siRNA. After 3 d, cells were treated with 1 µg/ml 1-[ß-D-arabinofuranosyl]-cytosine (Ara-; Sigma-Aldrich) in MEM with 2.5% chick embryo extract and 10% horse serum and fixed on days 5 and 6.
RT-PCR
Total RNA was harvested from cardiac myocytes within 24 h of siRNA treatment, or from skeletal myotubes within 48 h, using the RNeasy Mini kit (QIAGEN), or with Trizol (Sigma-Aldrich), and quantified using a Biophotometer (Eppendorf). cDNA was synthesized from 13 µg of total RNA using SuperScript II Reverse Transcriptase (Invitrogen). 0.53 µl of template were used per 25 µl PCR reaction. Reagents were from the TripleMaster Taq kit (Eppendorf). For nebulin amplification, at least 2830 cycles were performed (with the threshold of detection being 30 cycles in cDNA from cardiac myocytes and
27 from skeletal myotubes), and for other transcripts, 2630 cycles were used. Primers used included rat nebulin (forward, 5'-ACTGTCTTCCATCCCGTCAC-3', and reverse, 5'-GCCATACATCCAGCCTTCAT-3', to amplify a 202-bp product; rat nebulette (forward, 5'-ATTGGGAAGGGCTACAGCTT-3', and reverse, 5'-GAAGCCTCTTCCCTTCGTCT-3' to amplify a 196-bp product); rat GAPDH (forward, 5'-CCAGTATGATTCTACCCACGGC-3', and reverse, 5'-CGGAGATGATGACCCTTTTGGC-3', to amplify a 227-bp product); and Tmod1 (forward, 5'-ACTGTAAGGCCATGGACAGC-3', and reverse, 5'-GCTGCAGTTGTGTTTCAAGG-3', to amplify a 141-bp product). All PCR products were sequenced.
Western blotting
Myotube lysates were solubilized in SDS sample buffer, sonicated, and incubated at 70°C for 5 min before loading onto a 420% gradient SDS-PAGE gel. After transfer to nitrocellulose, strips were probed with antiCOOH- and antiNH2-terminal nebulin antibodies (1 µg/ml), followed by antirabbit IgG-conjugated HRP (1:25,000; Jackson ImmunoResearch Laboratories). After incubation in SuperSignal chemiluminescent substrate (Pierce Chemical Co.), the strips were exposed to BioMax MR film (Eastman Kodak Co.), and band intensity was quantified using National Institutes of Health image.
Immunofluorescence microscopy
Cells were stained as described previously (Gregorio and Fowler, 1995). All cultures were double or triple stained to distinguish myocytes from fibroblasts and/or to evaluate the intensity of nebulin staining. Myocytes were treated with relaxing buffer (150 mM KCl, 5 mM MgCl2, 10 mM MOPS, pH 7.4, 1 mM EGTA, and 4 mM ATP) and fixed in 3% PFA for 15 min. Cells were incubated with affinity purified rabbit antiNH2 and antiCOOH-terminal nebulin antibodies (5 µg/ml), monoclonal antimyosin F59 antibodies (1:10 of culture supernatant; provided by F. Stockdale, Stanford University, Stanford, CA), rabbit antititin antibodies (A168-170 at 1:500; Z1-Z1 at 1:100; Centner et al., 2000), monoclonal antisarcomeric
-actinin antibodies (1:1,500; EA-53; Sigma-Aldrich), affinity-purified rabbit antihuman Tmod1 antibodies (10 µg/ml), monoclonal anticardiac actin antibodies (1:10; Ac1-20.4.2; American Research Products, Inc.), and monoclonal antitropomyosin CH1 antibodies (1 µg/ml; Developmental Studies Hybridoma Bank). The following secondary antibodies were obtained from Jackson ImmunoResearch Laboratories and Invitrogen: goat antimouse AlexaFluor 488 (1:1,000), goat antimouse Texas red (1:600), donkey antirabbit Texas red (1:600), goat antirabbit AlexaFluor 350 (1:300), and goat antimouse AlexaFluor 350 (1:200) IgG. AlexaFluor phalloidin 488 (Invitrogen) or Texas red phalloidin (Sigma-Aldrich) labeled thin filaments, and DAPI (5 µg/ml; Sigma-Aldrich) labeled myotube nuclei. Coverslips were mounted using Aqua Poly/Mount (Polysciences, Inc.) and analyzed on a microscope (Axiovert; Carl Zeiss MicroImaging, Inc.) using a 63 (NA 1.4) or 100x (NA 1.25) objective, and micrographs were collected as digital images on a camera (Orca-ER; Hamamatsu) using OpenLab software (Improvision). Imaging was also performed using a microscope (DeltaVision Deconvolution model D-OL; Olympus) with a 100x objective (1.3 NA) using a charge-coupled device camera (series 300; Photometrics), and on a multiphoton microscope (model 510; Carl Zeiss MicroImaging, Inc.) using a 100x objective (1.4 NA). For pixel intensity plots, images of phalloidin-stained thin filaments were oriented parallel to the long axis in Adobe Photoshop, and intensity levels along the lengths of two adjacent sarcomeres were quantified using boxes drawn to encompass 69 x 6 total pixels (SoftWorx Data Inspector). Images were processed using Adobe Photoshop 7.0, and statistical analyses were performed using Microsoft Excel.
![]() |
Acknowledgments |
---|
This work was supported by an American Heart Association grant (0435316N) to A.S. McElhinny, a Howard Hughes Medical Institute grant to the University of Arizona for the Undergraduate Biology Research Program (71195-521304) to M. Valichnac, and National Institutes of Health grants (HL57461 and HL63926) to C.C. Gregorio.
Submitted: 28 February 2005
Accepted: 4 August 2005
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bang, M.L., R.E. Mudry, A.S. McElhinny, K. Trombitas, A.J. Geach, R. Yamasaki, H. Sorimachi, H. Granzier, C.C. Gregorio, and S. Labeit. 2001. Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies. J. Cell Biol. 153:413427.
Beall, C.J., M.A. Sepanski, and E.A. Fyrberg. 1989. Genetic dissection of Drosophila myofibril formation: effects of actin and myosin heavy chain null alleles. Genes Dev. 3:131140.[Abstract]
Buday, L. 1999. Membrane-targeting of signalling molecules by SH2/SH3 domain-containing adaptor proteins. Biochim. Biophys. Acta. 1422:187204.[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]
Chen, M.J., C.L. Shih, and K. Wang. 1993. Nebulin as an actin zipper. A two-module nebulin fragment promotes actin nucleation and stabilizes actin filaments. J. Biol. Chem. 268:2032720334.
Clark, K.A., A.S. McElhinny, M.C. Beckerle, and C.C. Gregorio. 2002. Striated muscle cytoarchitecture: an intricate web of form and function. Annu. Rev. Cell Dev. Biol. 18:637706.[CrossRef][Medline]
Donner, K., M. Sandbacka, V.L. Lehtokari, C. Wallgren-Pettersson, and K. Pelin. 2004. Complete genomic structure of the human nebulin gene and identification of alternatively spliced transcripts. Eur. J. Hum. Genet. 12:744751.[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]
Gonsior, S.M., M. Gautel, and H. Hinssen. 1998. A six-module human nebulin fragment bundles actin filaments and induces actin polymerization. J. Muscle Res. Cell Motil. 19:225235.[CrossRef][Medline]
Granzier, H.L.M., and K. Wang. 1993. Gel electrophoresis of giant proteins: solubilization and silver staining of titin and nebulin from single muscle fiber segments. Electrophoresis. 14:5664.[Medline]
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.[CrossRef][Medline]
Jin, J.P., and K. Wang. 1991. Nebulin as a giant actin-binding template protein in skeletal muscle sarcomere. Interaction of actin and cloned human nebulin fragments. FEBS Lett. 281:9396.[CrossRef][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]
Kioka, N., K. Ueda, and T. Amachi. 2002. Vinexin, CAP/ponsin, ArgBP2: a novel adaptor protein family regulating cytoskeletal organization and signal transduction. Cell Struct. Funct. 27:17.[CrossRef][Medline]
Labeit, S., and B. Kolmerer. 1995. The complete primary structure of human nebulin and its correlation to muscle structure. J. Mol. Biol. 248:308315.[CrossRef][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., and V.M. Fowler. 1998. Defining actin filament length in striated muscle: rulers and caps or dynamic stability? Annu. Rev. Cell Dev. Biol. 14:487525.[CrossRef][Medline]
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]
Luther, P.K., R. Padron, S. Ritter, R. Craig, and J.M. Squire. 2003. Heterogeneity of Z-band structure within a single muscle sarcomere: implications for sarcomere assembly. J. Mol. Biol. 332:161169.[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.
Mayer, B.J. 2001. SH3 domains: complexity in moderation. J. Cell Sci. 114:12531263.
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., K. Kakinuma, H. Sorimachi, S. Labeit, and C.C. Gregorio. 2002. Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1. J. Cell Biol. 157:125136.
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.
Millevoi, S., K. Trombitas, B. Kolmerer, S. Kostin, J. Schaper, K. Pelin, H. Granzier, and S. Labeit. 1998. Characterization of nebulette and nebulin and emerging concepts of their roles for vertebrate Z-discs. J. Mol. Biol. 282:111123.[CrossRef][Medline]
Mogami, K., P.T. O'Donnell, S.I. Bernstein, T.R. Wright, and C.P. Emerson Jr. 1986. Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function. Proc. Natl. Acad. Sci. USA. 83:13931397.
Moncman, C.L., and K. Wang. 1995. Nebulette: a 107 kD nebulin-like protein in cardiac muscle. Cell Motil. Cytoskeleton. 32:205225.[CrossRef][Medline]
Moncman, C.L., and K. Wang. 1996. Assembly of nebulin into the sarcomeres of avian skeletal muscle. Cell Motil. Cytoskeleton. 34:167184.[CrossRef][Medline]
Moncman, C.L., and K. Wang. 2002. Targeted disruption of nebulette protein expression alters cardiac myofibril assembly and function. Exp. Cell Res. 273:204218.[CrossRef][Medline]
Mudry, R.E., C.N. Perry, M. Richards, V.M. Fowler, and C.C. Gregorio. 2003. The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes. J. Cell Biol. 162:10571068.
Nwe, T.M., K. Maruyama, and Y. Shimada. 1999. Relation of nebulin and connectin (titin) to dynamics of actin in nascent myofibrils of cultured skeletal muscle cells. Exp. Cell Res. 252:3340.[CrossRef][Medline]
O'Donnell, P.T., and S.I. Bernstein. 1988. Molecular and ultrastructural defects in a Drosophila myosin heavy chain mutant: differential effects on muscle function produced by similar thick filament abnormalities. J. Cell Biol. 107:26012612.[Abstract]
Ojima, K., Z.X. Lin, M. Bang, S. Holtzer, R. Matsuda, S. Labeit, H.L. Sweeney, and H. Holtzer. 2000. Distinct families of Z-line targeting modules in the COOH-terminal region of nebulin. J. Cell Biol. 150:553566.
Pfuhl, M., S.J. Winder, and A. Pastore. 1994. Nebulin, a helical actin binding protein. EMBO J. 13:17821789.[Abstract]
Robinson, T.F., and S. Winegrad. 1979. The measurement and dynamic implications of thin filament lengths in heart muscle. J. Physiol. 286:607619.[Abstract]
Root, D.D., and K. Wang. 1994. Calmodulin-sensitive interaction of human nebulin fragments with actin and myosin. Biochemistry. 33:1258112591.[CrossRef][Medline]
Root, D.D., and K. Wang. 2001. High-affinity actin-binding nebulin fragments influence the actoS1 complex. Biochemistry. 40:11711186.[CrossRef][Medline]
Roulier, E.M., C. Fyrberg, and E. Fyrberg. 1992. Perturbations of Drosophila -actinin cause muscle paralysis, weakness, and atrophy but do not confer obvious nonmuscle phenotypes. J. Cell Biol. 116:911922.[Abstract]
Schafer, D.A., C. Hug, and J.A. Cooper. 1995. Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments. J. Cell Biol. 128:6170.[Abstract]
Schroeter, J.P., J.P. Bretaudiere, R.L. Sass, and M.A. Goldstein. 1996. Three-dimensional structure of the Z band in a normal mammalian skeletal muscle. J. Cell Biol. 133:571583.[Abstract]
Shimada, Y., M. Komiyama, S. Begum, and K. Maruyama. 1996. Development of connectin/titin and nebulin in striated muscles of chicken. Adv. Biophys. 33:223234.[CrossRef][Medline]
Shimada, Y., H. Suzuki, and A. Konno. 1997. Dynamics of actin in cardiac myofibrils and fibroblast stress fibers. Cell Struct. Funct. 22:5964.[Medline]
Stedman, H., K. Browning, N. Oliver, M. Oronzi-Scott, K. Fischbeck, S. Sarkar, J. Sylvester, R. Schmickel, and K. Wang. 1988. Nebulin cDNAs detect a 25-kilobase transcript in skeletal muscle and localize to human chromosome 2. Genomics. 2:17.[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.
Suzuki, H., M. Komiyama, A. Konno, and Y. Shimada. 1998. Exchangeability of actin in cardiac myocytes and fibroblasts as determined by fluorescence photobleaching recovery. Tissue Cell. 30:274280.[CrossRef][Medline]
Trinick, J. 1994. Titin and nebulin protein rulers in muscle? Trends Biochem. Sci. 19:405408.[CrossRef][Medline]
van der Ven, P.F., J.W. Bartsch, M. Gautel, H. Jockusch, and D.O. Furst. 2000. A functional knock-out of titin results in defective myofibril assembly. J. Cell Sci. 113:14051414.
Wang, K., and C.L. Williamson. 1980. Identification of an N2-line protein of striated muscle. Proc. Natl. Acad. Sci. USA. 77:32543258.
Wang, K., and J. Wright. 1988. Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line. J. Cell Biol. 107:21992212.[Abstract]
Wang, K., M. Knipfer, Q.Q. Huang, A. van Heerden, L.C. Hsu, G. Gutierrez, X.L. Quian, and H. Stedman. 1996. Human skeletal muscle nebulin sequence encodes a blueprint for thin filament architecture. Sequence motifs and affinity profiles of tandem repeats and terminal SH3. J. Biol. Chem. 271:43044314.
Weber, A., C.C. Pennise, G.G. Babcock, and V.M. Fowler. 1994. Tropomodulin caps the pointed ends of actin filaments. J. Cell Biol. 127:16271635.[Abstract]
Zhang, J.Q., G. Luo, A.H. Herrera, B. Paterson, and R. Horowits. 1996. cDNA cloning of mouse nebulin. Evidence that the nebulin-coding sequence is highly conserved among vertebrates. Eur. J. Biochem. 239:835841.[Abstract]
Related Article