Nebulin interacts with CapZ and regulates thin filament architecture within the Z-disc

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant "molecular ruler" nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160-164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160-164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.     

Nebulin regulates the assembly and lengths of the thin filaments in striated muscle

In many tissues, actin monomers polymerize into actin (thin) filaments of precise lengths. Although the exact mechanisms involved remain unresolved, it is proposed that "molecular rulers" dictate the lengths of the actin filaments. The giant nebulin molecule is a prime candidate for specifying thin filament lengths in striated muscle, but this idea has never been proven. To test this hypothesis, we used RNA interference technology in rat cardiac myocytes. Live cell imaging and triple staining revealed a dramatic elongation of the preexisting thin filaments from their pointed ends upon nebulin knockdown, demonstrating its role in length maintenance; the barbed ends were unaffected. When the thin filaments were depolymerized with latrunculin B, myocytes with decreased nebulin levels reassembled them to unrestricted lengths, demonstrating its importance in length specification. Finally, knockdown of nebulin in skeletal myotubes revealed its involvement in myofibrillogenesis. These data are consistent with nebulin functioning as a thin filament ruler and provide insight into mechanisms dictating macromolecular assembly.     

The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments

Strict regulation of actin thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with actin filament capping proteins to regulate thin filament lengths. Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity. These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle.     

Thin filament length regulation in striated muscle sarcomeres: pointed-end dynamics go beyond a nebulin ruler

The actin (thin) filaments in striated muscle are highly regulated and precisely specified in length to optimally overlap with the myosin (thick) filaments for efficient myofibril contraction. Here, we review and critically discuss recent evidence for how thin filament lengths are controlled in vertebrate skeletal, vertebrate cardiac, and invertebrate (arthropod) sarcomeres. Regulation of actin polymerization dynamics at the slow-growing (pointed) ends by the capping protein tropomodulin provides a unified explanation for how thin filament lengths are physiologically optimized in all three muscle types. Nebulin, a large protein thought to specify thin filament lengths in vertebrate skeletal muscle through a ruler mechanism, may not control pointed-end actin dynamics directly, but instead may stabilize a large core region of the thin filament. We suggest that this stabilizing function for nebulin modifies the lengths primarily specified by pointed-end actin dynamics to generate uniform filament lengths in vertebrate skeletal muscle. We suggest that nebulette, a small homolog of nebulin, may stabilize a correspondingly shorter core region and allow individual thin filament lengths to vary according to working sarcomere lengths in vertebrate cardiac muscle. We present a unified model for thin filament length regulation where these two mechanisms cooperate to tailor thin filament lengths for specific contractile environments in diverse muscles.     

A Nebulin Ruler Does Not Dictate Thin Filament Lengths

To generate force, striated muscle requires overlap between uniform-length actin and myosin filaments. The hypothesis that a nebulin ruler mechanism specifies thin filament lengths by targeting where tropomodulin (Tmod) caps the slow-growing, pointed end has not been rigorously tested. Using fluorescent microscopy and quantitative image analysis, we found that nebulin extended 1.01-1.03 μm from the Z-line, but Tmod localized 1.13-1.31 μm from the Z-line, in seven different rabbit skeletal muscles. Because nebulin does not extend to the thin filament pointed ends, it can neither target Tmod capping nor specify thin filament lengths. We found instead a strong correspondence between thin filament lengths and titin isoform sizes for each muscle. Our results suggest the existence of a mechanism whereby nebulin specifies the minimum thin filament length and sarcomere length regulates and coordinates pointed-end dynamics to maintain the relative overlap of the thin and thick filaments during myofibril assembly.     

Nebulin regulation of actin filament lengths: new angles

The highly organized arrays of thick and thin filaments found in striated muscles continue to be the subject of studies that yield groundbreaking concepts regarding cell motility. One example is the idea that massive, linearly extended polypeptides function as molecular rulers that set the length of polymeric filaments. Actin filaments that are polymerized in vitro exhibit wide variations in length, but many cells can assemble structures that contain actin filaments that are remarkably uniform. In striated muscles, the giant nebulin polypeptide extends the length of the actin filaments, and nebulin size has been correlated with actin filament lengths in muscles from different species. Here, I discuss a recent study by Gregorio and colleagues that demonstrates that nebulin knockdown leads to loss of actin filament-length regulation in cardiomyocytes, providing functional evidence that is consistent with the molecular ruler concept.     

Measurement of thin filament lengths by distributed deconvolution analysis of fluorescence images

The lengths of the actin (thin) filaments in sarcomeres directly influence the physiological properties of striated muscle. Although electron microscopy techniques provide the highest precision and accuracy for measuring thin filament lengths, significant obstacles limit their widespread use. Here, we describe distributed deconvolution, a fluorescence-based method that determines the location of specific thin filament components such as tropomodulin (Tmod) or probes such as phallacidin (a phalloidin derivative). Using Tmod and phallacidin fluorescence, we were able to determine the thin filament lengths of isolated chicken pectoralis major myofibrils with an accuracy and precision comparable to electron microscopy. Additionally, phallacidin fluorescence intensity at the Z line provided information about the width of Z lines. Furthermore, we detected significant variations in thin filaments lengths among individual myofibrils from chicken posterior latissimus dorsai and embryonic chick cardiac myocytes, suggesting that a ruler molecule (e.g., nebulin) does not strictly determine thin filament lengths in these muscles. This versatile method is applicable to myofibrils in living cells that exhibit significant variation in sarcomere lengths, and only requires a fluorescence microscope and a CCD camera.     

A Myopathy-linked Desmin Mutation Perturbs Striated Muscle Actin Filament Architecture

Desmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the desmin-E245D mutant in myocytes displaces endogenous desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated desmin mutations.     

Nebulin regulates thin filament length, contractility, and Z-disk structure in vivo

The precise assembly of the highly organized filament systems found in muscle is critically important for its function. It has been hypothesized that nebulin, a giant filamentous protein extending along the entire length of the thin filament, provides a blueprint for muscle thin filament assembly. To test this hypothesis, we generated a KO mouse model to investigate nebulin functions in vivo. Nebulin KO mice assemble thin filaments of reduced lengths and approximately 15% of their Z-disks are abnormally wide. Our data demonstrate that nebulin functions in vivo as a molecular ruler by specifying pointed- and barbed-end thin filament capping. Consistent with the shorter thin filament length of nebulin deficient mice, maximal active tension was significantly reduced in KO animals. Phenotypically, the murine model recapitulates human nemaline myopathy (NM), that is, the formation of nemaline rods combined with severe skeletal muscle weakness. The myopathic changes in the nebulin KO model include depressed contractility, loss of myopalladin from the Z-disk, and dysregulation of genes involved in calcium homeostasis and glycogen metabolism; features potentially relevant for understanding human NM.     

Microscopic analysis of the elastic properties of nebulin in skeletal myofibrils.

The elastic properties of nebulin were studied by measuring the elasticity of single skeletal myofibrils, from which the portion of the thin filament located at the I band had been selectively removed by treatment with plasma gelsolin under rigor conditions. In this myofibril model, a portion of each nebulin molecule at the I band was expected to be free of actin filaments and exposed. The length of the exposed portion of the nebulin molecule was controlled by performing the gelsolin treatment at various sarcomere lengths. The relation between the passive tension and extension of the exposed portion of the nebulin showed a convex curve starting from a slack length, apparently in a fashion similar to that of wool. The slack sarcomere length shifted depending on the length of the exposed portion of the nebulin, however, the relation being represented by a single master curve. The elastic modulus of nebulin was estimated to be two to three orders of magnitude smaller than that of an actin filament. Based on these results, we conclude that nebulin attaches to an actin filament in a side-by-side fashion and that it does not significantly contribute to the elastic modulus of thin filaments. The relation between the passive tension and extension of connectin (titin) was obtained for a myofibril from which thin filaments had been completely removed with gelsolin under contracting conditions; this showed a concave curve, consistent with the previous results obtained in single fibers.     

Nebulin: Does It Measure up as a Ruler?

A recent study has shown that the giant protein nebulin maintains the lengths of actin filaments in striated muscle cells. Although on the surface, nebulin looks like a molecular ruler, it may be playing a more complex role in regulating dynamics at the pointed end of actin filaments in striated muscle.     

Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size, and epitope profile

Nebulin, a family of giant proteins with size-variants from 600 to 900 kD in various skeletal muscles, have been proposed to constitute a set of inextensible filaments anchored at the Z line (Wang, K., and J. Wright. 1988. J. Cell Biol. 107:2199-2212). This newly discovered filament of the skeletal muscle sarcomere is an attractive candidate for a length-regulating template of thin filaments. To evaluate this hypothesis, we address the question of coextensiveness of nebulin and the thin filament by searching for a correlation between the size of nebulin variants and the length distribution of the thin filaments in several skeletal muscles. A positive linear correlation indeed exists for a group of six skeletal muscles that display narrow thin filament length distributions. To examine the molecular and architectural differences of nebulin size-variants, we carried out immunoelectron microscopic studies to map out epitope profiles of nebulin variants in these muscles. For this purpose, a panel of mAbs to distinct nebulin epitopes was produced against rabbit nebulin purified by an improved protocol. Epitope profiles of nebulin variants in three skeletal muscles revealed that (a) nebulin is inextensible since nebulin epitopes maintain a fixed distance to the Z line irrespective of the degree of sarcomere stretch; (b) a single nebulin polypeptide spans a minimal distance of 0.9 microns from the Z line; (c) nebulin contains repeating epitopes that are spaced at 40 nm or its multiples; (d) nebulin repeats coincide with thin filament periodicity; (e) nebulin variants differ mainly at either or both ends; and (f) nebulin remains in the sarcomere in actin-free sarcomeres produced by gelsolin treatment. Together, these data suggest that nebulin is an inextensible full-length molecular filament that is coextensive with thin filaments in skeletal muscles. We propose that nebulin acts as a length-regulating template that determines thin filament length by matching its large number of 40-nm repeating domains with an equal number of helical repeats of the actin filaments.     

The Nebulin family: an actin support group

Nebulin, a giant, actin-binding protein, is the largest member of a family of proteins (including N-RAP, nebulette, lasp-1 and lasp-2) that are assembled in a variety of cytoskeletal structures, and expressed in different tissues. For decades, nebulin has been thought to act as a molecular ruler, specifying the precise length of actin filaments in skeletal muscle. However, emerging evidence suggests that nebulin should not be viewed as a ruler but as an actin filament stabilizer required for length maintenance. Nebulin has also been implicated recently in an array of regulatory functions independent of its role in actin filament length regulation. In this review, we discuss the current evolutionary, biochemical, and functional data for the nebulin family of proteins - a family whose members, both large and small, function as cytoskeletal scaffolds and stabilizers.     

Electron microscopic study of actin polymerization in airway smooth muscle

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.     

Each actin subunit has three nebulin binding sites: implications for steric blocking

Nebulin is a giant protein that spans most of the muscle thin filament. Mutations in nebulin result in myopathies and dystrophies. Nebulin contains approximately 200 copies of approximately 35 residue modules, each believed to contain an actin binding site, organized into seven-module superrepeats. The strong correlation between the number of nebulin modules and the length of skeletal muscle thin filaments in different species suggests that nebulin determines thin filament length. Little information exists about the interactions between intact nebulin and F-actin. More insight has come from working with fragments of nebulin, containing from one to hundreds of actin binding modules. However, the observed stoichiometry of binding between these fragments and actin has ranged from 0.4 to 13 modules per actin subunit. We have used electron microscopy and a novel method of helical image analysis to characterize complexes of F-actin with a nebulin fragment. The fragment binds as an extended structure spanning three actin subunits and binding to different sites on each actin. Muscle regulation involves tropomyosin movement on the surface of actin, with binding in three states. Our results suggest the intriguing possibility that intact nebulin may also be able to occupy three different sites on F-actin.     

Thin filaments elongate from their pointed ends during myofibril assembly in Drosophila indirect flight muscle.

Tropomodulin (Tmod) is an actin pointed-end capping protein that regulates actin dynamics at thin filament pointed ends in striated muscle. Although pointed-end capping by Tmod controls thin filament lengths in assembled myofibrils, its role in length specification during de novo myofibril assembly is not established. We used the Drosophila Tmod homologue, sanpodo (spdo), to investigate Tmod's function during muscle development in the indirect flight muscle. SPDO was associated with the pointed ends of elongating thin filaments throughout myofibril assembly. Transient overexpression of SPDO during myofibril assembly irreversibly arrested elongation of preexisting thin filaments. However, the lengths of thin filaments assembled after SPDO levels had declined were normal. Flies with a preponderance of abnormally short thin filaments were unable to fly. We conclude that: (a) thin filaments elongate from their pointed ends during myofibril assembly; (b) pointed ends are dynamically capped at endogenous levels of SPDO so as to allow elongation; (c) a transient increase in SPDO levels during myofibril assembly converts SPDO from a dynamic to a permanent cap; and (d) developmental regulation of pointed-end capping during myofibril assembly is crucial for specification of final thin filament lengths, myofibril structure, and muscle function.     

Tropomodulin caps the pointed ends of actin filaments

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.     

A 7.5-kb 3'-Terminal cDNA Sequence of Chicken Skeletal Muscle Nebulin Reveals Its Actin Binding Regions

Nebulin is an approximately 700 kDa filamentous protein in vertebrate skeletal muscle. It binds to the Z line and also binds side-by-side to the entire thin (actin) filament in a sarcomere. Nebulin is currently thought to be a molecular ruler regulating the length of the thin filament to 1 mum. The complete sequence of human skeletal muscle nebulin was determined by . Because of its large size, only fragmental sequence information has been available for nebulins other than human skeletal muscle. This paper describes for the first time the sequence of about one third (C terminal region) of chicken skeletal muscle nebulin. It was found that the fundamental structure of human nebulin, consisting of 35 amino acid repeats (modules) plus C terminal serine-rich and SH3 domains linked to the Z line are well conserved with chicken nebulin. Sequence identity ranged from 74 to 91%. There were super-repeats (seven modules), a first linker repeat, simple repeat and a second linker repeat in addition to the Z line binding region as in human nebulin. However, there were 2 fewer modules in the first linker repeat and 6 fewer in the simple repeat in chicken nebulin as compared to human nebulin. Two isoforms of chicken nebulin were sequenced indicating insertion of approximately 6 or 11 modules to a structure similar to that of human nebulin. Recombinant first linker repeats M51 approximately 56 were shown to bind to actin using the ELISA technique as well as human nebulin recombinants.     

Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle.

Ca2(+)-regulated native thin filaments were extracted from sheep aorta smooth muscle. The caldesmon content determined by quantitative gel electrophoresis was 0.06 caldesmon molecule/actin monomer (1 caldesmon molecule per 16.3 actin monomers). Dissociation of caldesmon and tropomyosin from the thin filament and the depolymerization of actin was measured by sedimenting diluted thin filaments. Actin critical concentration was 0.05 microM at 10.1 and 0.13 at 10.05 compared with 0.5 microM for pure F-actin. Tropomyosin was tightly bound, with half-maximal dissociation at less than 0.3 microM thin filaments (actin monomer) under all conditions. Caldesmon dissociation was independent of tropomyosin and not co-operative. The concentration of thin filaments where 50% of the caldesmon was dissociated (CD50) ranged from 0.2 microM (actin monomer) at 10.03 to 8 microM at 10.16 in a 5 mM-MgCl2, pH 7.1, buffer. Mg2+, 25 mM at constant I, increased CD50 4-fold. CD50 was 4-fold greater at 10(-4) M-Ca2+ than at 10(-9) M-Ca2+. Aorta heavy meromyosin (HMM).ADP.Pi complex (2.5 microM excess over thin filaments) strongly antagonized caldesmon dissociation, but skeletal-muscle HMM.ADP.Pi did not. The behaviour of caldesmon in native thin filaments was indistinguishable from caldesmon in reconstituted synthetic thin filaments. The variability of Ca2(+)-sensitivity with conditions observed in thin filament preparations was shown to be related to dissociation of regulatory caldesmon from the thin filament.