Ultrastructure of the aortic diverticula of the adult dragonfly Sympetrum danae (Odonata: anisoptera)

Summary The aorta of Sympetrum danae possesses two dorsal diverticula: one in the mesothorax and one in the metathorax. They are very similar in form and position. Each diverticulum has a dorsal valve through which blood is pumped from the wings down into the aorta. The wall of the aortic diverticula consists of two simple cell layers: an outer epidermis-like layer and an inner muscle layer. The nuclei of the muscle cells are situated close to the lumen of the diverticula. The mitochondria are evenly dispersed between the myofibrils and are often paired up on either side of the Z-band. The Z-bands are thick and fragmented. The length of the sarcomeres varies from 3.3 to 6.1 . The A-band length is about 3 . The myofibrils consist of thick (250 Å) and thin (85 Å) filaments. Each thick filament is surrounded by 9–12 thin filaments. The sarcoplasmic reticulum is well developed and separates the myofibrils with one or two layers. The T-tubules are flattened and branch irregularly like a two-dimensional tree between the lamellar myofibrils. Intercalated discs are observed.The peculiarities of the muscle of aortic diverticula in S. danae are discussed in relation to various muscles of other insects and arthropods.     

Ultrastruktur glycerinextrahierter Dünndarmmuskelzellen der Ratte vor und nach Kontraktion

Zusammenfassung Die Tunica muscularis des Dünndarms der Ratte wurde elektronenmikroskopisch vor und nach Glycerinextraktion und nach verschieden lang andauernder ATP-Behandlung untersucht. Vor und nach der Extraktion sind nur 50–80 Å breite F-Actin-Filamente in den glatten Muskelzellen nachzuweisen. Die extrahierten glatten Muskelzellen kontrahieren sich nach Zugabe von ATP. Gleichzeitig treten in der Längsrichtung der Zelle verlaufende 150–200 Å dicke Myosinfilamente auf. Während langanhaltender Inkubation mit ATP trennen sich Actin- und Myosinfilamente zunächst voneinander durch eine Art Gleitmechanismus, da die Actinfilamente noch an der Zellmembran verhaftet bleiben, die Myosinfilamente sich aber verschieben. Dann lösen sich die Actinfilamente von der Zellmembran und Actin- und Myosinfilamente bilden ein dichtes Netzwerk im Zentrum der Zelle. In der Umgebung dieses Netzwerkes verbleiben feine Filamente mit einem Durchmesser von 20–30 Å.

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Ultrastructure of glycerinated small intestine muscle cells of the rat before and after contraction

Summary Tunica muscularis of the rat's small intestine was studied electron microscopically before and after glycerol-extraction and at various times after ATP treatment. Before and after extraction only F-actin-filaments with a diameter of 50–80 Å could be found in smooth muscle cells. Dense bodies disappear during extraction. Glycerinated smooth muscle cells contract when ATP is added. At the same time thick filaments with a diameter of 150–200 Å appear, which probably represent myosin filaments, running longitudinally within the cells. During prolonged ATP treatment actin and myosin filaments first separate from each other by a sort of sliding mechanism because actin filaments are still bound to the cell membrane while myosin filaments move. Then actin filaments are drawn off from the cell membrane and actin and myosin filaments assemble in an intricate network of filaments in the central part of the cell. Around this network fine filaments with a diameter of 20–30 Å remain.

Für die technische Mithilfe danke ich Frau Karla Struwe.     

Ultrastructure of the heart muscle cells of the cuttlefish Rossia macrosoma (Delle Chiaje) (Mollusca: Cephalopoda)

Summary The muscle cells of the ventricle, the branchial heart and the branchial heart appendages of Rossia macrosoma (Delle Chiaje) are studied. The ventricle myocardium has three muscle layers, while the other two organs exhibit a loose arrangement of muscle cells. The muscle cells of the ventricle, the branchial heart and the branchial heart appendages are similar in structure. The nuclei are surrounded by myofibrils. In the myofibrils A-, I- and discontinuous Z-bands are seen. The diameters of the thick filaments are 300–400Å, their length varies from 1.7 to 3.9 . Thin filaments have a diameter of approximately 85Å. The ratio between thick and thin filaments is roughly 1 to 11.The SR runs mostly as a longitudinal network within the myofibrils. A few short T-tubules are observed in the Z-regions. Peripheral and internal couplings exist. The latter are few in number.Intercalated discs are small and rarely observed. They have been found in all three organs. A difference in the function of these organs is not reflected in the ultrastructure of the intercalated discs. These discs are often of the interdigitating type with interfibrillar junctions and unspecialized regions. Peripheral couplings are seen at the unspecialized regions. The intercalar surfaces of the muscle cells shoulder off into the lateral surface, and the transition between the two surfaces is not a sharp one. Attachment plaques are found scattered over the whole sarcolemma.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.     

Elektronenmikroskopische Untersuchungen an Spinnenmuskeln

Zusammenfassung Die Pasern aus den Beinmuskeln der Vogelspinne Dugesiella hentzi sind zwischen 100 und 250 m dick und durch tiefe Einfaltungen des Sarcolemms in Untereinheiten gegliedert. Die meist bandförmigen Myofibrillen liegen darin in radiärer Anordnung. A-Bandbreite und Sarcomerenlänge variieren sehr stark (Extremwerte 2,8 und 5,6 bzw. 3,0 und 7,3 m). Ausrichtung und Anordnung der Myofilamente sind wenig exakt. Auf ein Primärfilament (Durchmesser 230–235 Å) kommen durchschnittlich 4–4,5 Sekundärfilamente (70–80 Å).Das sarcoplasmatische Reticulum (SR) ist extensiv und in Form eines unregelmäßigen Netzes aus schlauchartigen Elementen ausgebildet. Im Bereich des A-Bandes erweitern sich einzelne Schläuche zu Cisternen, die mit den Tubuli des Transversalsystems Dyaden bilden. Die SR-Membran zeigt dabei im Dyadenbereich charakteristische Strukturen: punktförmige Membranverdickungen, die ein Muster von großer Regelmäßigkeit bilden. Lage und Zahl der Dyaden sind sehr variabel (Durchschnitt 3–4 pro Sarcomer).

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An electron microscopical study of spider muscles

Summary Four different leg muscles of the tarantula Dugesiella hentzi were investigated electron microscopically. The fibers measure 100 to 250 m in diameter. They are divided into subunits by deep invaginations of the sarcolemma. The myofibrils have the shape of irregular ribbons which are arranged radially within the fiber subunits. The length of the A band as well as the sarcomer length varies from 2.8 to 5.6 and 3.0 to 7.3 n respectively. The myofilaments do not form very regular patterns. The ratio thick filaments (diameter 230 to 235 Å) to thin filaments (70 to 80 Å) is approximately 1 to 4 or 4.5. The sarcoplasmic reticulum (SE) is extensively developed. It consists of an irregular network of tubular elements surrounding the myofibrils and frequently crossing the Z discs. In the A band region some of the SR tubules widen. These cisternae form dyads with the tubules of the transversal system. In the dyads the membrane of the cisternae shows a characteristic structure: i.e. an exact pattern of small, point-like membrane thickenings. The position and the number of the dyads vary widely. Usually there are 3 to 4 in each sarcomer.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft. Fräulein Jördis Behrsing danken wir für ausgezeichnete technische Mithilfe.     

COORDINATED DEVELOPMENT OF THE SARCOPLASMIC RETICULUM AND T SYSTEM DURING POSTNATAL DIFFERENTIATION OF RAT SKELETAL MUSCLE

An electron microscope study has been carried out on rat psoas muscle, during the early postnatal stages of development. Among the several subcellular components, the sarcotubular system undergoes the most striking modifications during this period. In muscle fibers of the newborn rat, junctional contacts between the T system and the SR are sparse and are, mostly, longitudinally or obliquely oriented. The T tubules do not penetrate deeply into the muscle cell, as indicated by the predominantly peripheral location of the triads and the persistence, at these stages of development, of a highly branched subsarcolemmal system of tubules. Diadic associations of junctional SR elements with the plasma membrane are also occasionally observed. The early SR elaborations incompletely delineate the myofibrils, at both the A- and I-band level. Longitudinal sections show irregularly oriented SR tubules, running continuously over successive sarcomeres. Flattened junctional cisterns filled with granular material are sparse and laterally interconnected, at circumscribed sites, with the SR tubules. Between 1 and 2 wk postpartum, transversal triadic contacts are extensively established, at the A-I band level, and the SR network differentiates into two portions in register with the A and I band, respectively. At 10–15 days after birth, the SR provides a transversely continuous double sheet around the myofibrils at the I-band level, whereas it forms a single discontinuous layer at the A-band level. The relationship that these morphological modifications of the sarcotubular system may bear to previously described biochemical and physiological changes of rat muscle fibers after birth is discussed.     

The cardiac ultrastructure of Chimaera monstrosa L. (Elasmobranchii: Holocephali)

Summary The ultrastructure of the heart in Chimaera monstrosa L. is described. The endocardial and the epicardial cells are similar in the three cardiac regions. Myocardial cells show small variations.The myofibre, 4–6 m thick, contains one or a few myofibrils. Each myosin filament is surrounded by six actin filaments. The sarcomere banding pattern includes the Z-, A-, I-, M-, N-, and H-band. End-to-end attachments between myofibres are composed of alternating desmosomes and fasciae adhaerentes. Desmosomes and nexuses occur between longitudinally oriented cell surfaces. The sarcoplasmic reticulum is poorly developed but well defined. Peripheral coupling-like structures are common, T-tubules are absent. Membrane bound dense bodies occur in all regions. Areas with ribosomes and single myosin filaments are often seen.The epicardial cells have a regular hexagonal surface and are much thicker than the endocardial cells. Numerous short and a few longer cytoplasmic extensions face the pericardial cavity.The fiat endocardial cells contain a large nucleus and small amounts of cytoplasm.     

Ultrastructure of the neurogenic heart of Limulus polyphemus

Summary The ultrastructure of Limulus cardiac muscle was examined. The hearts were fixed in situ by perfusion with isotonic glutaraldehyde solution while in relaxed, contracted, or stretched states. The sarcomeres are relatively long, varying in length from about 2.5 to 6.6 . The average A-band length is 2.46 . M lines are absent, and H zones are poorly distinguished. Thick and thin filament diameters average about 200 Å and 50 Å, respectively; each thick filament is surrounded by 8–12 thin ones. Superficial invaginations of the sarcolemma occur, making contact with the Z lines of the outermost myofibrils. There is an extensive sarcoplasmic reticulum and transverse (T) tubules. Some T tubules run longitudinally and some open into deep sarcolemmal invaginations which extend into the fiber interior. The T tubules swell markedly in hypertonic solution. Single neurons and small bundles of neurons are observed in close apposition with myocardial cells. Intercalated disks are found in Limulus heart at regions of contact between contiguous myocardial cells lying end to end; semitight or gap junctions are essentially absent. Prominent differences in sarcomere lengths sometimes occur across the disk, thus indicating that the disks demarcate cells functionally. Hence, in addition to direct motoneuron activation, there may be some transfer of excitation across the intercalated disks in accord with our previous finding that propagating, overshooting action potentials can be induced in this heart.Supported by grants from the American Heart Association and from the Public Health Service (HE-11155 and HE-05815). I thank Mrs. Jan Redick for expert technical assistance.     

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。     

Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor

Tryptic digestion of rabbit skeletal myofibrils at physiological ionic strength and pH results in cleavage of the myosin heavy chain at one site giving two bands (M_r = 200,000 and 26,000) on sodium dodecyl sulfate/polyacrylamide gels. Following addition of sodium pyrophosphate (to 1 mm) to dissociate the myosin heads from actin, tryptic proteolysis results in production of three bands, 160K², 51K and 26K, with a 74K band appearing as a precursor of the 51K and 26K species. Under these conditions, there is insignificant cleavage of heavy chain to the heavy and light meromyosins. Trypsin-digested myofibrils yield the same amount of rod as native myofibrils when digested with papain. These results indicate that actin blocks tryptic cleavage of the myosin heavy chain at a site 74K from the N terminus. From measurements of the amount of 51K species formed by digestion of rigor fibers at various sarcomere lengths, we estimate that at least 95% of the myosin heads are bound to actin at 100% overlap of thick and thin filaments. Hence all myosin molecules can bind to actin, and consequently both heads of a myosin molecule can interact simultaneously with actin filaments under rigor conditions.     

Cytoskeletal filaments in embryonic chick myocardial cells as revealed by the quick-freeze deep-etch method combined with immunocytochemistry

Summary The three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12–14 nm filaments formed a loose network surrounding nascent myofibrils. These 12–14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8–11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3–5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)-treated samples, subsarcolemmal 8–11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.     

Localization of myosin and actin in ocular nonmuscle cells

Summary Myosin and actin were localized by indirect immunofluorescence microscopy using specific antibodies prepared in rabbits against highly purified gizzard myosin and actin. A strong fluorescence staining with both antibodies was observed in rat corneal epithelial cells, anterior lens epithelial cells, rod inner segments, and in rat and frog pigment epithelial cells. The immunohistochemical localization of myosin in corneal epithelial cells was further supported by the electrophoretic and immunological identification of smooth muscle type myosin heavy chain in pure corneal epithelial abrasions. Electron-microscopic observations revealed a clear correlation between staining with actin antibodies and the presence of numerous thin cytoplasmic filaments (50–80 Å in diameter). The functional and biochemical nature of 90–110 Å filaments occurring in corneal and lens epithelial cells, as well as the ultrastructural localization of myosin in ocular nonmuscle cells under study remains obscure.     

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions of connectin and was compared to the incorporation of -actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with -actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.     

Localization of paratropomyosin in skeletal muscle myofibrils and its translocation during postmortem storage of muscles

Using polyclonal antibodies against paratropomyosin, which is believed to modify the actin-myosin interaction in postrigor skeletal muscles, we studied the localization of paratropomyosin in chicken breast muscle myofibrils. Intact myofibrils stained with fluorescent antibodies showed that paratropomyosin was exclusively located at the A-I junction region of sarcomeres. In stretched myofibrils (3.7 micron in sarcomere length), the approximate width of the fluorescent stripes and their relation to the A band remained constant. Removal of the A band from myofibrils led to loss of stainability. During postmortem storage of muscles, on the other hand, paratropomyosin was translocated from its original position at the A-I junction region onto thin filaments. The translocation of paratropomyosin was successfully induced with a calcium ion concentration of 10(-4) M in the presence of protease inhibitors. We therefore conclude that in postrigor muscles, paratropomyosin is released from the A-I junction region following the increase in the sarcoplasmic calcium ion concentration to 10(-4) M, and then binds to thin filaments, which results in weakening of rigor linkages formed between actin and myosin.     

Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A- band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha- actinin and, if actin is added subsequently, the exogenous alpha- actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.     

Fine structure and differentiation of Ascidian muscle. I. Differentiated caudal musculature of Distaplia occidentalis tadpoles

The structure of the caudal muscle in the tadpole larva of the compound ascidian Distaplia occidentalis has been investigated with light and electron microscopy. The two muscle bands are composed of about 1500 flattened cells arranged in longitudinal rows between the epidermis and the notochord. The muscle cells are mononucleate and contain numerous mitochondria, a small Golgi apparatus, lysosomes, proteid-yolk inclusions, and large amounts of glycogen. The myofibrils and sarcoplasmic reticulum are confined to the peripheral sarcoplasm. Myofibrils are discrete along most of their length but branch near the tapered ends of the muscle cell, producing a Felderstruktur. The myofibrils originate and terminate at specialized intercellular junctional complexes. These myomuscular junctions are normal to the primary axes of the myofibrils and resemble the intercalated disks of vertebrate cardiac muscle. The myofibrils insert at the myomuscular junction near the level of a Z-line. Thin filaments (presumably actin) extend from the terminal Z-line and make contact with the sarcolemma. These thin filaments frequently appear to be continuous with filaments in the extracellular junctional space, but other evidence suggests that the extracellular filaments are not myofilaments. A T-system is absent, but numerous peripheral couplings between the sarcolemma and cisternae of the sarcoplasmic reticulum (SR) are present on all cell surfaces. Cisternae coupled to the sarcolemma are continuous with transverse components of SR which encircle the myofibrils at each I-band and H-band. The transverse component over the I-band consists of anastomosing tubules applied as a single layer to the surface of the myofibril. The transverse component over the H-band is also composed of anastomosing tubules, but the myofibrils are invested by a double or triple layer. Two or three tubules of sarcoplasmic reticulum interconnect consecutive transverse components. Each muscle band is surrounded by a thin external lamina. The external lamina does not parallel the irregular cell contours nor does it penetrate the extracellular space between cells. In contracted muscle, the sarcolemmata at the epidermal and notochordal boundaries indent to the level of each Z-line, and peripheral couplings are located at the base of the indentations. The external lamina and basal lamina of the epidermis are displaced toward the indentations. The location, function, and neuromuscular junctions of larval ascidian caudal muscle are similar to vertebrate somatic striated muscle. Other attributes, including the mononucleate condition, transverse myomuscular junctions, prolific gap junctions, active Golgi apparatus, and incomplete nervous innervation are characteristic of vertebrate cardiac muscle cells.     

The cytoskeleton of Cobaea seed hairs:

The cell wall of Cobaea scandens seed hairs developed in a characteristic sequence, with the deposition of a cellulose thread onto a pectic swelling layer was the final event. The cellulose thread was intracellularly accompanied by a band of 10–18 microtubules. During the formation of the swelling layer the microtubules were homogeneously distributed; they ran circumferentially normal to the cell axis. When cellulose-thread formation started, the microtubules became arranged in a helical band. The density of the microtubules varied during the different phases of development. The highest density was observed before cellulosethread formation and ranged from 6–15 m·m^-2. The length of the microtubules, 20–30 m, was determined by direct measurements, as well as estimated from the total microtubular length in a given area and the counted free ends. With the indirect immunofluorescence technique the microtubules of the band stained inhomogeneously. Those which were located at the edges of the band fluoresced more intensely than those of the central part. Attempts to visualize actin filaments in the hair cells with rhodaminyl-conjugated phalloidin resulted in a homogeneous staining of the area of the microtubular band, indicating that actin filaments may be present in this region. Though, in thin sections and dry-cleaved cells, filamentous structures were observed between the microtubules, caution is expressed that the observed fluorescence was, indeed, due to actin filaments. The role of the filamentous structures is discussed with respect to formation and maintenance of the microtubular band. Microtubules apparently did not cross coated pits which were visualized in the plasma membrane through the dry-cleaving technique.Abbreviations IFT indirect immunofluorescence technique - RP rhodaminyl-conjugated phalloidin - SEM scanning electron microscopy     

AN ELECTRON MICROSCOPE STUDY OF MYOFIBRIL FORMATION IN EMBRYONIC CHICK SKELETAL MUSCLE

The formation of myofibrils in the developing leg muscle of the 12-day chick embryo was studied by electron microscopy. Myofilaments of two varieties, thick (160–170 A in diameter) and thin (60–70 A in diameter), which have been designated myosin and actin filaments, respectively, on the basis of their similarity to natural and synthetic myosin and actin filaments, appear in the cytoplasm of developing muscle cells. There is a greater than 7:1 ratio of thin to thick filaments in these young myofibers. The free myofilaments become aligned in the long axis of the cells, predominantly in subsarcolemmal locations, and aggregate into hexagonally packed arrays of filaments. The presence of Z band material or M band cross-bridges do not appear to be essential for the formation or spacing of these aggregates of filaments. Formation of the Z band lattices occurs coincidentally with the back-to-back apposition of thin filaments. An hypothesis concerning myofibril growth, based on the self-assembly characteristics of the filaments, is presented.     

Immunofluorescence staining of thin-filament sections not participating in actomyosin crossbridges: Studies by use of a monoclonal antibody specific to actin

Summary Monoclonal antibodies (mcab) were produced in vitro by fusing mouse X63-Ag8.653 plasmacytoma cells with spleen cells from a Balb/c mouse immunized with primary cultures of chick skeletal muscle (pmcc). After cloning on agar, stable clones were obtained, the antibodies of which stain specifically the I-band of myofibrils in the immunofluorescence (IF) procedure. For further characterization of these mcab their affinities to muscle proteins were tested by immunoblotting and by enzyme-linked immunosorbent assay (ELISA). Mcab specific for actin were revealed by these criteria. One of the anti-actin antibodies, mcab 647, reveals a variety of IF-staining patterns on myofibrils. On rest-length myofibrils the I-band is labeled only. However, at sarcomere lengths below 2 m, where the thin filaments meet in the middle of the A-band and form a region of double overlap, an additional fluorescent band appears in this position. The fluorescence intensity of this band is increased significantly in shorter sarcomeres. Finally, when the I-band has disappeared at a sarcomere length of 1.5 m, fluorescence is located exclusively in the middle of the A-band. These IF-staining patterns suggest that only those sections of the thin filament are stained that do not participate in actomyosin crossbridges.     

Ca2+-induced fragmentation of actin filaments in pollen tubes

Summary To control the intracellular free Ca²⁺ concentration from the cell exterior, pollen tubes ofLilium longiflorum were treated with a Ca²⁺ ionophore, A23187. Cytoplasmic streaming was inhibited when the free Ca²⁺ concentration of the external medium ([Ca²⁺]) was raised to 5×10^–6 M or higher. At [Ca²⁺] below 1×10^–6 M, the rhodamine-phalloidin stained actin filaments appeared straight and thin. However, at [Ca²⁺] which inhibited cytoplasmic streaming, the actin filaments appeared fragmented. In pollen tubes, Ca²⁺ regulation of cytoplasmic streaming may be linked not only to myosin (Shimmen 1987) but also to actin.Abbreviations ATP adenosine-5-triphosphoric acid - [Ca²⁺] concentration of free Ca²⁺ - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - Rh-ph rhodamine-conjugated phalloidin