STRUCTURE OF LIMULUS STRIATED MUSCLE : The Contractile Apparatus at Various Sarcomere Lengths

The musculature of the telson of Limulus polyphemus L. consists of three dorsal muscles: the medial and lateral telson levators and the telson abductor, and one large ventral muscle; the telson depressor, which has three major divisions: the dorsal, medioventral, and lateroventral heads. The telson muscles are composed of one type of striated muscle fiber, which has irregularly shaped myofibrils. The sarcomeres are long, with discrete A and I and discontinuous Z bands. M lines are not present. H zones can be identified easily, only in thick (1.0 µm) longitudinal sections or thin cross sections. In lengthened fibers, the Z bands are irregular and the A bands appear very long due to misalignment of constituent thick filaments. As the sarcomeres shorten, the Z lines straighten somewhat and the thick filaments become more aligned within the A band, leading to apparent decrease in A band length. Further A band shortening, seen at sarcomere lengths below 7.4 µm may be a function of conformational changes of the thick filaments, possibly brought about by alterations in the ordering of their paramyosin cores.     

The Z disc of skeletal muscle fibrils

Summary The structure of the Z disc has been studied in thin sections of striated muscle fibers from a wide variety of vertebrates. A common organization is found in all muscles examined. The disc shows a regular pattern made up of dense lines which seem to connect the actin filaments from adjacent sarcomeres. The lines are sometimes disposed to form a regular zigzag configuration; in other orientations with respect to the plane of the section the morphology is confused and, in still other images, the dense lines continuous with the actin filaments seem to go straight through the Z disc. In cross section this structure corresponds to a square pattern of considerable regularity. The intersections in the square pattern mark the location in the plane of the section of the actin filaments from adjacent sarcomeres. Dense lines form the edges of the squares and appear to represent condensations of Z-disc material, i.e., the lines in the zigzag. The possible origin of the structure as a product of the stretching of a membrane is discussed, together with functional interpretations of the Z disc.Postdoctoral fellow under USPHS Training Grant 2 G-707 to K. R. Porter.     

Sarcomere reorganization in mite muscle.

The number of sarcomeres in a given muscle of the mite Tarsonemus randsi was constant in both larval and adult stages, with the exception of the two medial dorsal metapodosomal muscles in males. These muscles have three sarcomeres in larvae and one sarcomere in adults. This change in sarcomere number within a muscle was observed in the living animal by polarized light microscopy using parthenogenetically derived male larvae. Initially the transforming muscles shortened slowly (hours) and the appearance of the sarcomeres was comparable to that seen during normal contraction. With continued shortening there was apposition of adjacent A bands and disappearance of clearly visible Z lines, but no loss of birefringence. Over the next 12 hr there was further shortening of the muscle and loss of birefringence. This was apparent as shortening of the three apposed A regions to the length of a single A band with a small increase in muscle width and no increase in the peak retardation of the birefringent region. The observations are discussed in terms of differential loss of the A filaments of the two terminal sarcomeres.     

An electron microscope cytochemical study of calcium ion localization in the honey-bee flight muscle.

The authors studied the sarcomere ultrastructure and distribution of calcium ions in the cross striated flight muscles (longitudinal-dorsal and tergosternal) of the honey bee after being fixed in osmium tetraoxide and buffered to 7.4 pH and also according to the method of Carasso and Favard (1966). they ascertained an intensive and constant reaction to the presence of calcium on the level of myofilaments within the periphery of the sarcomeres and light and dark mitochondria. Calcium concentrations are not detectable in the Z line and M line. The authors carried out a control of the results using the method of Carasso and Favard (1966).     

Fine structure in near-field and far-field laser diffraction patterns from skeletal muscle fibers.

Regions of muscle fibers that are many sarcomeres in length and uniform with regard to striation spacing, curvature, and tilt have been observed by light microscopy. We have investigated the possibility that these sarcomere domains can explain the fine structure in optical diffraction patterns of skeletal muscle fibers. We studied near-field and far-field diffraction patterns with respect to fiber translation and to masking of the laser beam. The position of diffracted light in the near-field pattern depends on sarcomere length and position of the diffracting regions within the laser beam. When a muscle fiber was translated longitudinally through a fixed laser beam, the fine structural lines in the near-field diffraction pattern moved in the same direction and by the same amount as the fiber movement. Translation of the muscle fiber did not result in fine structure movement in the far-field pattern. As the laser beam was incrementally masked from one side, some fine structural lines in both the near-field and far-field diffraction patterns changed in intensity while others remained the same. Eventually, all the fine structural lines broadened and decreased in intensity. Often a fine structural line increased in intensity or a dark area in the diffraction pattern became brighter as the laser beam was restricted. From these results we conclude that the fine structure in the laser diffraction pattern is due to localized and relatively uniform regions of sarcomeres (domains) and to cross interference among light rays scattered by different domains.     

Ultrastructural investigations on the muscular systems in the barnacle, Tetraclita squamosa japonica

Four muscular systems of the Tetraclita squamosa barnacle were observed by means of an electron microscope and it was revealed that these systems each bore different types of muscle cells. The four systems were the adductor (A), the lateral scutal depressor (LSD), the ventral scutal depressor (VSD), and the tergal depressor (TD). The A-system included cross stiated muscle cells which showed long sarcomeres (about 10 mum) and rather disordered arrays of myofilaments. The LSD-system included cross striated muscles which had medium length sarcomeres (about 6.7 mum) and rather ordered myofilamental arrays. The VSD-system was constructed of cross striated muscle cells which bore shorter sarcomeres (4.6 mum) than the previous three systems and ordered myofilamental arrays. This last type of cell also bore well-developed sarcoplasmic reticular systems. The TD-system included smooth muscle cells which showed rather ordered arrays of myofilaments and dense-bodies. Each muscular system, as described above, included to its advantage one type of cross striated or smooth muscle cell for its characteristic contraction. The relations between ultrastructures and functions of each muscular system will now be discussed.     

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.     

Formation and alignment of Z lines in living chick myotubes microinjected with rhodamine-labeled alpha-actinin

We have used fluorescence analogue cytochemistry in conjunction with time lapse recording to study the dynamics of alpha-actinin, a major component of the Z line, during myofibrillogenesis. Rhodamine-labeled alpha-actinin microinjected into living cultured chick skeletal myotubes became localized in discrete cellular structures within 1 h and remained specifically associated with structures for up to 4 d, allowing individual identified structures to be followed during development. In the most immature cells used, alpha-actinin was found in diffuse aggregates, some of which displayed sarcomeric periodicity. Aggregates were observed to coalesce into better defined structures (Z bands) that were approximately 1.0-micron wide. Z bands condensed into narrow, more intensely fluorescent Z lines in 4-48 h. During this period, Z lines grew laterally, primarily by the addition of small beads of alpha-actinin to existing Z lines or by the merging of small Z lines. In more mature cells, alpha-actinin added to Z lines without going through a visible intermediary structure. Mean sarcomere length did not change significantly during the stages examined, although the variability of sarcomere length did decrease markedly over time for identified sets of sarcomeres. At early stages, myofibrils frequently shifted position in both the longitudinal and lateral directions. Neighboring myofibrils were frequently associated for one or more sarcomeres sporadically along their length, such that the intervening sarcomeres were often misaligned. Associations between myofibrils were often transitory. Shifts in myofibril location in conjunction with the formation, breaking, and reformation of lateral associations between myofibrils facilitated the alignment of Z lines through a trial and error process.     

Ultrastructural investigations on the muscular systems in the barnacle, Tetraclita squamosa japonica

Four muscular systems of the Tetraclita squamosa barnacle were observed by means of an electron microscope and it was revealed that these systems each bore different types of muscle cells. The four systems were the adductor (A), the lateral scutal depressor (LSD), the ventral scutal depressor (VSD), and the tergal depressor (TD). The A-system included cross stiated muscle cells which showed long sarcomeres (about 10 μm) and rather disordered arrays of myofilaments. The LSD-system included cross striated muscles which had medium length sarcomeres (about 6.7 μm) and rather ordered myofilamental arrays. The VSD-system was constructed of cross striated muscle cells which bore shorter sarcomeres (4.6 μm) than the previous three systems and ordered myofilamental arrays. This last type of cell also bore well-developed sarcoplasmic reticular systems. The TD-system included smooth muscle cells which showed rather ordered arrays of myofilaments and dense-bodies. Each muscular system, as described above, included to its advantage one type of cross striated or smooth muscle cell for its characteristic contraction. The relations between ultrastructures and functions of each muscular system will now be discussed.     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Chromosomal deletion in isolates of Phytophthora infestans correlates with virulence on R3, R10, and R11 potato lines.

In Phytophthora infestans, a cluster of three dominant avirulence genes is located on the distal part of linkage group VIII. In a mapping population from a cross between two Dutch field isolates, probe M5.1, derived from an amplified fragment length polymorphism (AFLP) marker linked to the Avr3-Avr10-Avr11 cluster, hybridized only to DNA from the parent and F1 progeny that is avirulent on potato lines carrying the R3, R10, and R11 resistance gene. In the virulent parent and the virulent progeny, no M5.1 homologue was detected, demonstrating a deletion on that part of linkage group VIII. P. infestans is diploid, so the avirulent strains must be hemizygous for the region concerned. A similar situation was found in another mapping population from two Mexican strains. The deletion was also found to occur in many field isolates. In a large set of unique isolates collected in The Netherlands from 1980 to 1991, 37% had no M5.1 homologue and the deletion correlated strongly with gain of virulence on potato lines carrying R3, R10, and R11. Also, in some old isolates that belong to a single clonal lineage (US-1) and are thus highly homogenous, deletions at the M5.1 locus were detected, indicating that this region is unstable.     

粘虫蛾飞行肌超微结构的研究

应用电子显微镜对粘虫雌蛾Mythimna separata(Walker)飞行(背纵)肌的研究结果表明,其肌原纤维由500-700根肌球蛋白丝(粗丝)组成,每根粗丝由6根肌动蛋白丝(细丝)环绕排列成六角形,每根细丝精确地位于两根粗丝间1/2处,从而使粗丝和细丝的比为1：3。肌节较短,长度约2.2-2.6μm。肌原纤维之间充满着线粒体和横管。每个肌节约有线粒体三个,横管二根。线粒体约占肌纤维体积的40%,而横管为7%。每根横管准确地位于肌节的1/4、3/4处,或Z线和中膈的中央,并与肌质网交接形成二位体(dyads)或三位体(triads)。肌质网相当不发达,约占肌纤维体积的2.5%。但其分布很有特色,即除了紧贴于肌原纤维周围的由单层液泡组成的肌质网以外,在中膈处还有一层横穿于肌原纤维的肌质网。和其它同步飞行肌的结构和功能分析比较的结果还表明,粘虫蛾飞行肌具有较善于飞行的结构。     

Interaction of force transmission and sarcomere assembly at the muscle-tendon junctions of carp (Cyprinus carpio): ultrastructure and distribution of titin (connectin) and α-actinin

At muscle-tendon junctions of red and of white axial muscle fibres of carp, new sarcomeres are found adjacent to existing sarcomeres along the bundles of actin filaments that connect the myofibrils with the junctional sarcolemma. As the filament bundles that transmit force to the junction originate proximal to new sarcomeres, they probably relieve these new sarcomeres from premature loading. In red fibres, these filament bundles are long (up to 20 m) and dense, permitting light-microscopical immunohistochemistry (double reactions: anti-titin or anti--actinin and phalloidin). New sarcomeres have clear I bands; their A band lengths are similar to those of older sarcomeres and the thick filaments lie in register. T tubules are found at the distal side of new sarcomeres but terminal Z lines are absent. The late addition of -actinin suggests that -actinin mainly has a stabilizing role in sarcomere formation. The presence of titin in the terminal fibre protrusions is in agreement with its supposed role in sarcomere formation, viz. the integration of thin and thick filaments. The absence of a terminal Z line from sarcomeres with well-registered A bands suggests that this structure is not essential for the anchorage of connective (titin) filaments.     

Differences in I band structure,sarcomere extensibility,and electrophoresis of titin between two muscle fiber types of the perch (Perca fluviatilis L.)

I bands of perch muscle contain crosslinks that interconnect the thin filaments as well as the connecting filaments and link them to the membrane systems and to other intermyofibrillar material. In shortened sarcomeres cross connections seem to pile up in the I band and at the A/I band junction. Sarcomeres of red fibers, which in another study are reported to have steep passive stress-strain curves, were pulled apart when they were stretched by about 40% of their slack length. Sarcomeres of white fibers could be stretched by almost 100% and still display filament alignment. Sarcomeres of the red fibers also differed from those of white fibers in the distance over which connecting filaments could be discerned, the presence of an extra transverse filament in their NI lines, the electrophoretic mobility of titin, and the titin/actin ratio.     

Optical Diffraction Studies of Muscle Fibers

A new technique to monitor light diffraction patterns electrically is applied to frog semitendinosus muscle fibers at various levels of stretch. The intensity of the diffraction lines, sarcomere length change, and the length-dispersion (line width) were calculated by fast analogue circuits and displayed in real time. A heliumneon laser (wavelength 6328 Å) was used as a light source. It was found that the intensity of the first-order diffraction line drops significantly (30-50%) at an optimal sarcomere length of 2.8 μm on isometric tetanic stimulation. Such stimulation produced contraction of half-sarcomeres by about 22 nm presumably by stretching inactive elements such as tendons. The dispersion of the sarcomere lengths is extremely small, and it is proportional to the sarcomere length (less than 4%). The dispersion increases on stimulation. These changes on isometric tetanic stimulation were dependent on sarcomere length. No vibration or oscillation in the averaged length of the sarcomeres was found during isometric tetanus within a resolution of 3 nm; however, our observation of increased length dispersion of the sarcomeres together with detection of the averaged shortening of the sarcomere lengths suggests the presence of asynchronous cyclic motions between thick and thin filaments. An alternative explanation is simply an increase of the length dispersion of sarcomeres without cyclic motions.     

Structural fluctuations in the steady state of muscular contraction.

Recent studies of the intensity fluctuation spectra of coherent light scattered from striated muscle have demonstrated the existence of large scale fluctuations in position and polarizability at the level of the myofibrillar sarcomere and its major structural subunits during the steady state of contraction. The existence of these fluctuations implies a fluctuating driving force. Various possible fluctuating motions of the thick and thin filaments, A and I bands, and entire sarcomeres are described. The magnitude of the fluctuating forces associated with the making and breaking of cross bridges is estimated. A mechanical model is proposed for coupling structural elements of a single sarcomere to one another and for coupling myofibrillar sarcomeres to one another. It is shown that the fluctuating force generated by the spontaneous making and breaking of cross bridges in conjunction with the model accounts for some of the features of the observed intensity fluctuation spectra.     

Muscle diffraction theory. Relationship between diffraction subpeaks and discrete sarcomere length distributions.

A theoretical discussion is presented that describes the diffraction on monochromatic light by a three-dimensional sarcomere array having the following properties. The basic repetitive diffracting unit is the sarcomere. The contiguous arrangement of physically attached serial sarcomeres in the myofibril is contained within the model so that relative position of sarcomeres depend upon the lengths of intervening ones. Sarcomere length is described by a distribution function. This function may be discrete or continuous and contain one or more subpopulations. Two arrangements of sarcomeres are considered: (a) when sarcomeres of different lengths are arranged randomly in myofibrils the amplitude and width of mth order (m greater than or equal to 1) peaks and associated secondary diffraction maxima decrease and increase monotonically, respectively, as the standard deviation of the length distribution increases. No subpeaks are present regardless of the number of subpopulations within the distribution function. This behavior is shown to follow from the dependence of sarcomere position on the length of intervening sarcomeres. (b) When sarcomeres belonging to the same length subpopulation are arranged in serial contiguous fashion to form domains and more than one length subpopulation is present, then mth order diffraction peaks split to form subpeaks. The theoretical basis for this behavior is developed for the first time and may explain the subpeaks evident in diffraction patterns from cardiac and skeletal muscle.     

Localization of beta-actinin in the Z lines of chicken breast muscle, as detected with a monoclonal antibody specific to the beta I subunit (Cap Z alpha).

Immunofluorescence microscopy showed that a monoclonal antibody, 2F3, specific to the beta I subunit (Cap Z alpha) of beta-actinin (Cap Z) bound to the Z lines of chicken breast muscle. When myofibrils were briefly extracted with 0.6 M KI, the reactivity of the Z lines with 2F3 was very weak, but on subsequent treatment with purified beta-actinin, the antibody binding recovered. beta-Actinin inhibited elongation of the actin filaments of isolated I-Z-I brushes, myosin-extracted sarcomeres, on the addition of G-actin. However, when an increased concentration of G-actin was added, the inhibitory action of beta-actinin became negligible, suggesting that beta-actinin did not cap the pointed end of an actin filament in a myofibril.     

Fine Structure of Cardiac Sarcomeres in the Black Widow Spider Latrodectus mactans

Fine structural characteristics of the cardiac muscle and its sarcomere organization in the black widow spider, Latrodectus mactans were examined using transmission electron microscopy. The arrangement of cardiac muscle fibers was quite similar to that of skeletal muscle fibers, but they branched off at the ends and formed multiple connections with adjacent cells. Each cell contained multiple myofibrils and an extensive dyadic sarcotubular system consisting of sarcoplasmic reticulum and T‐tubules. Thin and thick myofilaments were highly organized in regular repetitive arrays and formed contractile sarcomeres. Each repeating band unit of the sarcomere had three apparent striations, but the H‐zone and M‐lines were not prominent. Myofilaments were arranged into distinct sarcomeres defined by adjacent Z‐lines with relatively short lengths of 2.0 μm to 3.3 μm. Cross sections of the A‐band showed hexagon‐like arrangement of thick filaments, but the orbit of thin filaments around each thick filament was different from that seen in other vertebrates. Although each thick filament was surrounded by 12 thin filaments, the filament ratio of thin and thick myofilaments varied from 3:1 to 5:1 because thin filaments were shared by adjacent thick filaments.     

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.