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.     

A Highlights from MBoC Selection: Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle

Duchenne muscular dystrophy (DMD) induces sarcolemmal mechanical instability and rupture, hyperactivity of intracellular calpains, and proteolytic breakdown of muscle structural proteins. Here we identify the two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, as novel proteolytic targets of m-calpain, with Tmod1 exhibiting ∼10-fold greater sensitivity to calpain-mediated cleavage than Tmod4 in situ. In mdx mice, increased m-calpain levels in dystrophic soleus muscle are associated with loss of Tmod1 from the thin filament pointed ends, resulting in ∼11% increase in thin filament lengths. In mdx/mTR mice, a more severe model of DMD, Tmod1 disappears from the thin filament pointed ends in both tibialis anterior (TA) and soleus muscles, whereas Tmod4 additionally disappears from soleus muscle, resulting in thin filament length increases of ∼10 and ∼12% in TA and soleus muscles, respectively. In both mdx and mdx/mTR mice, both TA and soleus muscles exhibit normal localization of α-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic γ-actin, indicating that m-calpain does not cause wholesale proteolysis of other sarcomeric and actin cytoskeletal proteins in dystrophic skeletal muscle. These results implicate Tmod proteolysis and resultant thin filament length misspecification as novel mechanisms that may contribute to DMD pathology, affecting muscles in a use- and disease severity–dependent manner.     

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.     

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.     

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.     

鸣鸣蝉发声肌的结构观察

鸣鸣蝉Onvotympana maculaticollit Motsch的发声肌平均含193个初级肌束,多数初级肌束含9-10条肌纤维,其顶、底瑞的附着结构仅由柱状粘和细胞层组成。每条肌纤维约含1 900根肌原纤维,多数肌原纤维的长,宽和截面分别约0.77μm、0.68μm和0.53μm².井约含200根粗肌丝,其粗细肌丝的比值一般为3∶1。肌小节的长度和z线的宽度分别约3μm 和0.2μm.三联管分别位于距两端z线约0.75μm处。肌原纤维、线粒体和微气管-肌质网的面积系数分别约31.3%、46.O%和11.9%。肌小节中粗肌丝纵贯两端z线,中间无1带;细肌丝由z线相向延伸到肌小节中央,其空区约0.15-0.25μm,并无M线。这些结构特征不仅使发声肌能够利用有限的几何空间产生最大的张力,并可适应高速串的收缩运动。     

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.     

Actin dynamics at pointed ends regulates thin filament length in striated muscle

Regulation of actin dynamics at filament ends determines the organization and turnover of actin cytoskeletal structures. In striated muscle, it is believed that tight capping of the fast-growing (barbed) ends by CapZ and of the slow-growing (pointed) ends by tropomodulin (Tmod) stabilizes the uniform lengths of actin (thin) filaments in myofibrils. Here we demonstrate for the first time that both CapZ and Tmod are dynamic on the basis of the rapid incorporation of microinjected rhodamine-labelled actin (rho-actin) at both barbed and pointed ends and from the photobleaching of green fluorescent protein (GFP)-labelled Tmod. Unexpectedly, the inhibition of actin dynamics at pointed ends by GFP-Tmod overexpression results in shorter thin filaments, whereas the inhibition of actin dynamics at barbed ends by cytochalasin D has no effect on length. These data demonstrate that the actin filaments in myofibrils are relatively dynamic despite the presence of capping proteins, and that regulated actin assembly at pointed ends determines the length of thin filaments.     

The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes

Actin (thin) filament length regulation and stability are essential for striated muscle function. To determine the role of the actin filament pointed end capping protein, tropomodulin1 (Tmod1), with tropomyosin, we generated monoclonal antibodies (mAb17 and mAb8) against Tmod1 that specifically disrupted its interaction with tropomyosin in vitro. Microinjection of mAb17 or mAb8 into chick cardiac myocytes caused a dramatic loss of the thin filaments, as revealed by immunofluorescence deconvolution microscopy. Real-time imaging of live myocytes expressing green fluorescent protein-alpha-tropomyosin and microinjected with mAb17 revealed that the thin filaments depolymerized from their pointed ends. In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments. These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability. These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.     

Thin-filament length correlates with fiber type in human skeletal muscle

Force production in skeletal muscle is proportional to the amount of overlap between the thin and thick filaments, which, in turn, depends on their lengths. Both thin- and thick-filament lengths are precisely regulated and uniform within a myofibril. While thick-filament lengths are essentially constant across muscles and species (～1.65 μm), thin-filament lengths are highly variable both across species and across muscles of a single species. Here, we used a high-resolution immunofluorescence and image analysis technique (distributed deconvolution) to directly test the hypothesis that thin-filament lengths vary across human muscles. Using deltoid and pectoralis major muscle biopsies, we identified thin-filament lengths that ranged from 1.19 ± 0.08 to 1.37 ± 0.04 μm, based on tropomodulin localization with respect to the Z-line. Tropomodulin localized from 0.28 to 0.47 μm further from the Z-line than the NH(2)-terminus of nebulin in the various biopsies, indicating that human thin filaments have nebulin-free, pointed-end extensions that comprise up to 34% of total thin-filament length. Furthermore, thin-filament length was negatively correlated with the percentage of type 2X myosin heavy chain within the biopsy and shorter in type 2X myosin heavy chain-positive fibers, establishing the existence of a relationship between thin-filament lengths and fiber types in human muscle. Together, these data challenge the widely held assumption that human thin-filament lengths are constant. Our results also have broad relevance to musculoskeletal modeling, surgical reattachment of muscles, and orthopedic rehabilitation.     

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.     

Nebulin regulates actin filament lengths by a stabilization mechanism

Efficient muscle contraction requires regulation of actin filament lengths. In one highly cited model, the giant protein nebulin has been proposed to function as a molecular ruler specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of nebulin (mini-nebulin). When endogenous nebulin was replaced with mini-nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-nebulin were remarkably maintained at lengths either matching or longer than mini-nebulin. This indicates that mini-nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of nebulin also resulted in more dynamic populations of thin filament components, whereas expression of mini-nebulin decreased the dynamics at both filament ends (i.e., recovered loss of endogenous nebulin). Thus, nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.     

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-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle

Nebulin is a giant modular sarcomeric protein that has been proposed to play critical roles in myofibrillogenesis, thin filament length regulation, and muscle contraction. To investigate the functional role of nebulin in vivo, we generated nebulin-deficient mice by using a Cre knock-in strategy. Lineage studies utilizing this mouse model demonstrated that nebulin is expressed uniformly in all skeletal muscles. Nebulin-deficient mice die within 8-11 d after birth, with symptoms including decreased milk intake and muscle weakness. Although myofibrillogenesis had occurred, skeletal muscle thin filament lengths were up to 25% shorter compared with wild type, and thin filaments were uniform in length both within and between muscle types. Ultrastructural studies also demonstrated a critical role for nebulin in the maintenance of sarcomeric structure in skeletal muscle. The functional importance of nebulin in skeletal muscle function was revealed by isometric contractility assays, which demonstrated a dramatic reduction in force production in nebulin-deficient skeletal muscle.     

Leiomodin and tropomodulin in smooth muscle

Evidence isaccumulating to suggest that actin filament remodeling is critical forsmooth muscle contraction, which implicates actin filament ends asimportant sites for regulation of contraction. Tropomodulin (Tmod) andsmooth muscle leiomodin (SM-Lmod) have been found in many tissuescontaining smooth muscle by protein immunoblot and immunofluorescencemicroscopy. Both proteins cofractionate with tropomyosin in theTriton-insoluble cytoskeleton of rabbit stomach smooth muscle and aresolubilized by high salt. SM-Lmod binds muscle tropomyosin, abiochemical activity characteristic of Tmod proteins. SM-Lmod stainingis present along the length of actin filaments in rat intestinal smoothmuscle, while Tmod stains in a punctate pattern distinct from that ofactin filaments or the dense body marker -actinin. After smoothmuscle is hypercontracted by treatment with 10 mM Ca²⁺,both SM-Lmod and Tmod are found near -actinin at the periphery ofactin-rich contraction bands. These data suggest that SM-Lmod is anovel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth musclemay be capped by Tmod in localized clusters.

     

Tropomodulin contains two actin filament pointed end-capping domains

Tropomodulin 1 (Tmod1) is a approximately 40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd approximately 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd approximately 0.1 microM). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160-359) contains the principal actin-capping activity (Kd approximately 0.4 microM), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1-130) contains a second, weaker actin-capping activity (Kd approximately 1.8 microM). Interestingly, 160-359 but not 1-130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity.     

Genetic dissection of novel myopathy models reveals a role of CapZα and Leiomodin 3 during myofibril elongation

Myofibrils within skeletal muscle are composed of sarcomeres that generate force by contraction when their myosin-rich thick filaments slide past actin-based thin filaments. Although mutations in components of the sarcomere are a major cause of human disease, the highly complex process of sarcomere assembly is not fully understood. Current models of thin filament assembly highlight a central role for filament capping proteins, which can be divided into three protein families, each ascribed with separate roles in thin filament assembly. CapZ proteins have been shown to bind the Z-disc protein α-actinin to form an anchoring complex for thin filaments and actin polymerisation. Subsequent thin filaments extension dynamics are thought to be facilitated by Leiomodins (Lmods) and thin filament assembly is concluded by Tropomodulins (Tmods) that specifically cap the pointed end of thin filaments. To study thin filament assembly in vivo, single and compound loss-of-function zebrafish mutants within distinct classes of capping proteins were analysed. The generated lmod3- and capza1b-deficient zebrafish exhibited aspects of the pathology caused by variations in their human orthologs. Although loss of the analysed main capping proteins of the skeletal muscle, capza1b, capza1a, lmod3 and tmod4, resulted in sarcomere defects, residual organised sarcomeres were formed within the assessed mutants, indicating that these proteins are not essential for the initial myofibril assembly. Furthermore, detected similarity and location of myofibril defects, apparent at the peripheral ends of myofibres of both Lmod3- and CapZα-deficient mutants, suggest a function in longitudinal myofibril growth for both proteins, which is molecularly distinct to the function of Tmod4.     

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.     

Flightin Is Necessary for Length Determination, Structural Integrity, and Large Bending Stiffness of Insect Flight Muscle Thick Filaments

Despite the fundamental role of thick filaments in muscle contraction, little is known about the mechanical behavior of these filaments and how myosin-associated proteins dictate differences between muscle types. In this study, we used atomic force microscopy to study the morphological and mechanical properties of fully hydrated native thick filaments isolated from indirect flight muscle (IFM) of normal and mutant Drosophila lacking flightin (fln⁰). IFM thick filaments from newly eclosed (0-1 h old) wild-type flies have a mean length of 3.04 ± 0.05 μm. In contrast, IFM thick filaments from newly eclosed fln⁰ flies are more variable in length and, on average, are significantly longer (3.90 ± 1.33 μm) than wild-type filaments from flies of the same age. In the absence of flightin, thick filaments can attain lengths > 300% of wild-type filaments, indicating that flightin is required for setting the proper filament length in vivo. Filaments lacking flightin are structurally compromised, and filament preparations from fully matured 3- to 5-day-old adult fln⁰ IFM yielded fragments of variable length much shorter than 3.20 ± 0.04 μm, the length obtained from wild-type flies of similar age. The persistence length, an index of bending stiffness, was calculated from measurements of filament end-to-end length and contour length. We show that the presence of flightin increases persistence length by more than 40% and that wild-type filaments increase in stiffness with age. These results indicate that flightin fulfills an essential role in defining the structural and mechanical properties of IFM thick filaments.