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.     

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。     

Myosin mRNA accumulation and myofibrillogenesis at the myotendinous junction of stretched muscle fibers

Myofiber growth and myofibril assembly at the myotendinous junction (MTJ) of stretch-hypertrophied rabbit skeletal muscle was studied by in situ hybridization, immunofluorescence, and electron microscopy. In situ hybridization identified higher levels of myosin heavy chain (MHC) mRNA at the MTJ of fibers stretched for 4 d. Electron microscopy at the MTJ of these lengthening fibers revealed a large cytoplasmic space devoid of myofibrils, but containing polysomes, sarcoplasmic reticulum and T-membranes, mitochondria, Golgi complexes, and nascent filament assemblies. Tallies from electron micrographs indicate that myofibril assembly in stretched fibers followed a set sequence of events. (a) In stretched fiber ends almost the entire sarcolemmal membrane was electron dense but only a portion had attached myofibrils. Vinculin, detected by immunofluorescence, was greatly increased at the MTJ membrane of stretched muscles. (b) Thin filaments were anchored to the sarcolemma at the electron dense sites. (c) Thick filaments associated with these thin filaments in an unregistered manner. (d) Z-bodies splice into thin filaments and subsequently thin and thick filaments fall into sarcomeric register. Thus, the MTJ is a site of mRNA accumulation which sets up regional protein synthesis and myofibril assembly. Stretched muscles also lengthen by the addition of myotubes at their ends. After 6 d of stretch these myotubes make up the majority of fibers at the muscle ends. Essentially all these myotubes repeat the developmental program of primary myotubes and express slow MHC. MHC mRNA distribution in myotubes is disorganized as is the distribution of their myofibrils.     

Vertebrate isoforms of actin capping protein beta have distinct functions In vivo

Actin capping protein (CP) binds barbed ends of actin filaments to regulate actin assembly. CP is an alpha/beta heterodimer. Vertebrates have conserved isoforms of each subunit. Muscle cells contain two beta isoforms. beta1 is at the Z-line; beta2 is at the intercalated disc and cell periphery in general. To investigate the functions of the isoforms, we replaced one isoform with another using expression in hearts of transgenic mice.Mice expressing beta2 had a severe phenotype with juvenile lethality. Myofibril architecture was severely disrupted. The beta2 did not localize to the Z-line. Therefore, beta1 has a distinct function that includes interactions at the Z-line. Mice expressing beta1 showed altered morphology of the intercalated disc, without the lethality or myofibril disruption of the beta2-expressing mice.The in vivo function of CP is presumed to involve binding barbed ends of actin filaments. To test this hypothesis, we expressed a beta1 mutant that poorly binds actin. These mice showed both myofibril disruption and intercalated disc remodeling, as predicted.Therefore, CPbeta1 and CPbeta2 each have a distinct function that cannot be provided by the other isoform. CPbeta1 attaches actin filaments to the Z-line, and CPbeta2 organizes the actin at the intercalated discs.     

The site of paramyosin in insect flight muscle and the presence of an unidentified protein between myosin filaments and Z-line.

The position of paramyosin in insect flight muscle was determined by labelling myofibrils with antibody to paramyosin and examining them by fluorescent and electron microscopy.Antiserum to dung beetle paramyosin had antibodies to another protein as well as to paramyosin. Specific anti-paramyosin bound to the H-zone of Lethocerus myofibrils showing paramyosin was exposed only in that region. Antibodies to the other protein bound at the ends of the A-band.The exposure of antigenic sites in the two regions of the myofibril depended on the extent of contraction in the myofibril: the sites at the end of the A-band were most exposed in rest-length myofibrils and those at the H-zone in shortened ones.Antibody-labelling in stretched bee muscle showed that the protein at the ends of the sarcomere extended from myosin filaments to Z-line.The high resting elasticity of insect flight muscle and hence its capacity for oscillatory contraction may be due to the protein between myosin filaments and Z-line.     

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.     

Does actin bind to the ends of thin filaments in skeletal muscle?

We examined whether or not purified actin binds to the ends of thin filaments in rabbit skeletal myofibrils. Phase-contrast, fluorescence, and electron microscopic observations revealed that actin does not bind to the ends of thin filaments of intact myofibrils. However, in I-Z-I brushes prepared by dissolving thick filaments at high ionic strength, marked binding of actin to the free ends, i.e., the pointed ends, of thin filaments was observed when actin was added at an early phase of polymerization. As the polymerization of actin proceeded, the binding efficiency decreased. The critical actin concentration for this binding was higher than that for polymerization in solution. The binding of G-actin was not observed at low ionic strength. On the basis of these results, we suggest that a particular structure suppressing the binding of actin is present at the free ends of thin filaments in intact myofibrils and that a part of the end structure population is eliminated or modified at high ionic strength so that further binding of actin becomes possible. The myofibril and I-Z-I brush appear to be useful systems for studies aimed at elucidating the organizational mechanisms of actin filaments in vivo.     

Drosophila paramyosin is important for myoblast fusion and essential for myofibril formation

Paramyosin is a major structural protein of thick filaments in invertebrate muscles. Coiled-coil dimers of paramyosin form a paracrystalline core of these filaments, and the motor protein myosin is arranged on the core surface. To investigate the function of paramyosin in myofibril assembly and muscle contraction, we functionally disrupted the Drosophila melanogaster paramyosin gene by mobilizing a P element located in its promoter region. Homozygous paramyosin mutants die at the late embryo stage. Mutants display defects in both myoblast fusion and in myofibril assembly in embryonic body wall muscles. Mutant embryos have an abnormal body wall muscle fiber pattern arising from defects in myoblast fusion. In addition, sarcomeric units do not assemble properly and muscle contractility is impaired. We confirmed that these defects are paramyosin-specific by rescuing the homozygous paramyosin mutant to adulthood with a paramyosin transgene. Antibody analysis of normal embryos demonstrated that paramyosin accumulates as a cytoplasmic protein in early embryo development before assembling into thick filaments. We conclude that paramyosin plays an unexpected role in myoblast fusion and is important for myofibril assembly and muscle contraction.     

Extraction of Thick Filaments in Individual Sarcomeres Affects Force Production by Single Myofibrils

It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area but not on the number of active sarcomeres because they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study, we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated and relaxed using a perfusion system. An extra microperfusion needle filled with a high-ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by myofibrils with intact sarcomeres was significantly higher than the force produced by myofibrils with one or more sarcomeres lacking thick filaments (p < 0.0001) irrespective of the number of contractions imposed on the myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.     

Intermediate filaments connect z-discs in adult chicken muscle.

When adult chicken skeletal myofibrils are treated with a myosin-extracting solution, the Z-discs with attached actin filaments retain their linear connections with one another in the extracted myofibril. The sarcomere length increases in the extracted myofibrils from a control lenght of 2.5 micrometer up to 6 micrometer. In a sarcomere, eight to fifty 10 nm filaments can be seen in parallel array in the H-zone. The 10 nm-wide filaments do not bind heavy meromyosin and are two to four micrometers in length. These intermediate filaments are postulated to be an integral part of the sarcomere, connecting Z-bands along the length of the myofibril.     

Supercontraction in crayfish muscle: correlation with a peculiar actin localization.

Crayfish muscle, like muscles from some other invertebrates, can supercontract. This muscle shortening is characterized by an overlap of thin filaments with crossing of thick filaments through the Z discs. In intact muscle cells, supercontraction does not seem to induce irreversible structural modifications in the tissue. Isolated crayfish myofibrils in the relaxed state cannot be distinguished from vertebrate myofibrils under light microscope, either by phase contrast or by immunofluorescence, with antiactin antibodies, actin being localized in the I bands. However, when isolated crayfish myofibrils are supercontracted, irreversible dammage occurs, most thin filaments being lost. Actin becomes then hardly detectable, being visible, by immunofluorescence, either in the Z discs or evenly distributed in the whole myofibril. During myofibril supercontraction, high amounts of denatured actin, become soluble as shown by SDS-PAGE, by double immunodiffusion, and by DNAse inhibition.     

鸣鸣蝉（Oncotympana maculaticollis Motsch）发声肌中肌原纤维的双阵列结构及莫发声原初过程的分析

给出了鸣鸣蝉发声肌肌原纤维的双阵列结构,其肌纤维中并存两种不同阵列的“快”和“慢”动肌原纤维（ＦＳＭ和ＳＳＭ）．ＦＳＭ和ＳＳＭ虽然由粗肌丝构成相同的阵列骨架,但细肌丝对粗肌丝的比例（ＲＴＩＦ）不同,分别为３：１和５：１．明显区别于单音调鸣声的蝉类发声肌肌原纤维的ＲＴＩＦ为３：１的单阵列结构,即与鸣鸣蝉变音调声产生的原初机制相适应．     

鸣鸣蝉（Oncotympana maculaticollis Motsch）发声肌中肌原纤维的双阵列结构及莫发声原初过程的分析

给出了鸣鸣蝉发声肌肌原纤维的双阵结构,其肌纤维中并存两种不同阵列的“快”和“慢”劝肌原纤维不同,分别为３：１和５：１。明显区别于单音调鸣声的蝉类发声肌肌原纤维的ＲＴＴＦ为３：１的单阵列,即与鸣鸣蝉变音调声产生的原初机制相适应。     

FINE STRUCTURE AND SIZE DISTRIBUTION OF FREE THICK FILAMENTS IN EARLY FIBRILLOGENESIS OF ASCIDIAN TADPOLE

The development and the size distribution of free thick filaments which accumulate in the early stages of myofibril formation in somitic myoblasts of the ascidian tadpole were studied by electron microscopy. Such filaments appeared in the cell cortex but, rather dominantly, the aggregates of these thick filaments and filamentous structures were observed in the interior of the cell. The aggregate consisted of some of the following elements: filamentous structures (20–60 A in diameter); free thick filaments (60–220 A); dense Z-band precursor materials; bundles of thick (140–160 A) and thin (60–70 A) filaments; and ribosomal clusters. The free thick filaments were variable in diameter and showed long lateral projections (300–600 A) and tapered ends.
The variation curve in diameter of the free thick filaments indicates a continuous size distribution, suggesting the continuous growth of these filaments by polymerization of myosin molecules. Free thick filaments thicker than myosin filaments which were found within myofibrils were present; their significance is discussed in relation to myosin filament formation.     

Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart

The cardiomyopathic (CM) Syrian golden hamster (strain UM-X7.1) exhibits a hereditary cardiomyopathy, which causes premature death resulting from congestive heart failure. The CM animals show extensive cardiac myofibril disarray and myocardial calcium overload. The present study has been undertaken to examine the role of desmin phosphorylation in myofibril disarray observed in CM hearts. The data from skinned myofibril protein phosphorylation assays have shown that desmin can be phosphorylated by protein kinase C (PKC). There is no significant difference in the content of desmin between CM and control hamster hearts. However, the desmin from CM hearts has a higher phosphorylation level than that of the normal hearts. Furthermore, we have examined the distribution of desmin and myofibril organization with immunofluorescent microscopy and immunogold electron microscopy in cultured cardiac myocytes after treatment with the PKC-activating phorbol ester, 12-O-tetradecanylphorbol-13-acetate (TPA). When the cultured normal hamster cardiac cells are treated with TPA, desmin filaments are disassembled and the myofibrils become disarrayed. The myofibril disarray closely mimics that observed in untreated CM cultures. These results suggest that disassembly of desmin filaments, which could be caused by PKC-mediated phosphorylation, may be a factor in myofibril disarray in cardiomyopathic cells and that the intermediate filament protein, desmin, plays an important role in maintaining myofibril alignment in cardiac cells.     

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.     

Specificity of desmin to avian and mammalian muscle cells.

The extent of invariance and heterogeneity in desmin, the major component of the muscle form of 100 Å filaments, has been investigated in avian and mammalian muscle and nonmuscle cells with two-dimensional gel electrophoresis and indirect immunofluorescence. Desmin from chick, duck and quail, smooth, skeletal and cardiac muscle cells is resolved into two isoelectric variants, α and β, with each possessing the same charge and electrophoretic mobility in all three avian species irrespective of muscle type. Guinea pig and rat muscle desmin resolves into only one variant; it also possesses the same charge and electrophoretic mobility in the two mammalian species, but it is more acidic and slower in electrophoretic mobility than the two avian variants.In immunofluorescence, desmin is localized together with α-actinin along myofibril Z lines. Antibodies to chick smooth muscle desmin, prepared against the protein purified by preparative SDS gel electrophoresis prior to immunization, cross-react with myofibril Z lines in all three avian species. These antibodies do not cross-react with either rat or guinea pig myofibril Z lines. Similarly, they do not cross-react with avian or mammalian nonmuscle cells grown in tissue culture and known to contain cytoplasmic 100 Å filaments.These results demonstrate that desmin is highly conserved within avian muscle cells and within mammalian muscle cells. It is, however, both biochemically and immunologically distinguishable between avian and mammalian muscle cells, and between muscle and nonmuscle cells. We conclude that there are biochemically and immunologically specific forms of desmin for avian and mammalian muscle cells. Furthermore, within a particular vertebrate species, there are at least two separate classes of 100 Å filaments: the muscle class whose major component is desmin, and the nonmuscle class whose major component is distinct from desmin. Taking into consideration the immunological specificity reported by other laboratories for the 100 Å filaments in glial cells, for neurofilaments and for the epidermal 80 Å keratin filaments, we propose that a given vertebrate species contains at least four major distinguishable classes of 100 Å filaments: muscle 100 Å filaments (desmin filaments), glial filaments, neurofilaments and epidermal keratin filaments.     

Mechanism of myofibril growth and proliferation in fish muscle.

The mechanisms of myofibril growth proliferation were investigated in the red and white muscles of fish. In both types of muscle the ratio of lattice filament spacings between the Z disk and M line was found to be greater than that required for perfect transformation of a square into a hexagonal lattice. This mismatch was considered to result in the thin filaments being pulled obliquely instead of at right angles to the Z disk. The angle of pull of the thin filaments was measured in longitudinal sections. The splitting process was found to decrease the degree of pull. Splitting was also observed in transverse sections of the peripheral myofibrils. In both red and white fibres these myofibrils were found to commence splitting when they reached a size of approximately 1-2 mum diameter. Evidence from ultrastructural and autoradiographical studies suggested that growth of the myofibrils within the fibres is centrifugal. The outermost myofibrils appear to be the ones which are being built up and which split. The data indicated that in fish muscle a considerable number of filaments may be added to the daughter regions whilst splitting of the myofibril is still continuing.     

Extra actin filaments at the periphery of skeletal muscle myofibrils.

Myofibrils isolated from a variety of vertebrate muscle fibers have a set of peripheral filaments associated with the periphery of the Z line free to move away from the surface of the myofibril. Decoration with myosin subfragment 1 shows that these are actin filaments.     

Untersuchungen über das Längenwachstum der Skelettmuskulatur von Fischen

Zusammenfassung Ein Längenwachstum der Myofibrillen erscheint an terminalen, an das Interstitium grenzenden Myofibrillenabschnitten möglich. Diese Myofibrillenabschnitte weisen unterschiedliche Längen auf. Am kleinsten terminalen Myofibrillenabschnitt ist die Ribosomenkonzentration am höchsten. Mit zunehmender Verlängerung des Abschnittes erkennt man Aktinfilamente, die zwischen den dicht gelagerten Ribosomen liegen. Erreicht der terminale Myofibrillenabschnitt etwa die Länge einer Sarkomere, hat die Ribosomenkonzentration abgenommen, die Filamentanzahl zugenommen. In diesem Stadium treten zwischen den Aktinfilamenten erste Myosinfilamente auf. Eine neue Z-Scheibe entsteht in engem Kontakt mit dem Sarkolemm. Letztere löst sich nach und nach über ihre ganze Länge von der stets schräg ansetzenden Zellmembran. Die Frage nach der Spannungsübertragung an undifferenzierten terminalen Myofibrillenabschnitten wird diskutiert.

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Investigations on the longitudinal growth of fish skeletal muscle

Summary Longitudinal growth of myofibrils in the skeletal muscle of Macropodus opercularis appears to take place at their terminal parts. Since the Z-disks are arranged in lines and perpendicularly to the longitudinal axis, there are repeated terminal myofibril regions of different lengths following the last complete sarcomere. At the shortest terminal myofibril region, which is apparently the youngest one, ribosome concentration is very high. In the adjacent terminal regions of greater length, which probably represent older ones, actin filaments can be detected among the ribosomes. As soon as the terminal myofibril region approaches the full sarcomere length, the concentration of ribosomes is found to be reduced and the number of filaments increased. At this stage the first myosin filaments are clearly observed among actin filaments. Therefore, during longitudinal growth myosin filaments morphologically appear after the formation of actin filaments, whereas during myofibrillogenesis in the same muscle both types appear simultaneously in the differentiating sarcomere. After the arrangement of actin and myosin filaments into the A- and I-bands, a new Z-disk is formed in close contact with the sarcolemma and gradually detached over its entire length from the inclined cell membrane.The problem of tension transmission via the undifferentiated terminal myofibril regions is discussed in relation to these findings.