Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study

The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments.     

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Electrophoretic and autoradiographic analyses of the incorporation of ³⁵S-methionine into newly synthesized proteins during myogenesis reveal that presumptive chicken myoblasts synthesize primarily one intermediate filament protein: vimentin. Desmin synthesis is initiated at the onset of fusion. Synthesis rates of both filament subunits increase during the first three days in culture, relative to the total protein synthesis rate. The observed increase in the rate of desmin synthesis (at least 10 fold) is significantly greater than that observed for vimentin, and is responsible for a net increase in the cellular desmin content relative to vimentin. Both filament subunits continue to be synthesized through at least 20 days in culture. Immunofluorescent staining using desmin- and vimentin-specific antisera supports the conclusion that desmin is synthesized only in fusing or multinucleate cells. These results indicate that the synthesis of the two filament subunits is not coordinately regulated during myogenesis. The distributions of desmin and vimentin in multinucleate chicken myotubes are indistinguishable, as determined by double immunofluorescence techniques. In early myotubes, both proteins are found in an intricate network of free cytoplasmic filaments. Later in myogenesis, several days after the appearance of α-actinin-containing Z line striations, both filament proteins become associated with the Z lines of newly assembled myofibrils, with a corresponding decrease in the number of cytoplasmic filaments. This transition corresponds to the time when the a-actinin-containing Z lines become aligned laterally. These data suggest that the two intermediate filament systems, desmin and vimentin, have an important role in the lateral organization and registration of myofibrils and that the synthesis of desmin and assembly of desmin-containing intermediate filaments during myogenesis is directly related to these functions. These results also indicate that the Z disc is assembled in at least two distinct steps during myogenesis.     

Myoseverin disrupts sarcomeric organization in myocytes: an effect independent of microtubule assembly inhibition

Although disruption of the microtubule (MT) array inhibits myogenesis in myocytes, the relationship between the assembly of microtubules (MT) and the organization of the contractile filaments is not clearly defined. We now report that the assembly of mature myofibrils in hypertrophic cardiac myocytes is disrupted by myoseverin, a compound previously shown to perturb the MT array in skeletal muscle cells. Myoseverin treated cardiac myocytes showed disruptions of the striated Z-bands containing alpha-actinin and desmin and the localization of tropomyosin, titin and myosin on mature sarcomeric filaments. In contrast, MT depolymerization by nocodazole did not perturb sarcomeric filaments. Similarly, expression of constitutively active stathmin as a non-chemical molecular method of MT depolymerization did not prevent sarcomere assembly. The extent of MT destabilization by myoseverin and nocodazole were comparable. Thus, the effect of myoseverin on sarcomere assembly was independent of its capacity for MT inhibition. Furthermore, we found that upon removal of myoseverin, sarcomeres reformed in the absence of an intact MT network. Sarcomere formation in cardiac myocytes therefore, does not appear to require an intact MT network and thus we conclude that a functional MT array appears to be dispensable for myofibrillogenesis.     

Dystonin-Deficient Mice Exhibit an Intrinsic Muscle Weakness and an Instability of Skeletal Muscle Cytoarchitecture

Dystonia musculorum (dt) was originally described as a hereditary sensory neurodegeneration syndrome of the mouse. The gene defective in dt encodes a cytoskeletal linker protein, dystonin, that is essential for maintaining neuronal cytoskeletal integrity. In addition to the nervous system, dystonin is expressed in a variety of other tissues, including muscle. We now show that dystonin cross-links actin and desmin filaments and that its levels are increased during myogenesis, coinciding with the progressive reorganization of the intermediate filament network. A disorganization of cytoarchitecture in skeletal muscle from dt/dt mice was observed in ultrastructural studies. Myoblasts from dt/dt mice fused to form myotubes in culture; however, terminally differentiated myotubes contained incompletely assembled myofibrils. Another feature observed in dt/dt myotubes in culture and in skeletal muscle in situ was an accumulation and abnormal distribution of mitochondria. The diaphragm muscle from dt/dt mice was weak in isometric contractility measurements in vitro and was susceptible to contraction-induced sarcolemmal damage. Altogether, our data indicate that dystonin is a cross-linker of actin and desmin filaments in muscle and that it is essential for establishing and maintaining proper cytoarchitecture in mature muscle.     

Excitation-contraction uncoupling in the developing skeletal muscle of the muscular dysgenesis mouse embryo

The muscular dysgenesis recessive autosomal mutation is characterized by a total lack of muscular contraction and a myofibrillar non-organization. Many abnormalities involved in the excitation-contraction coupling are found in mdg/mdg myotubes: 1) the internal structural organization of the membrane coupling between the sarcoplasmic reticulum (SR) and the transverse (T)-tubule forming the triadic association is defective: the triad number is decreased in the muscle and there are a lack of periodic densities between the SR and T-tubule apposed membranes. 2) the voltage-dependent Ca2+ channel contents, identified by binding with the specific blocker PN 200-110, are decreased. The two fast (30 ms) and slow (100 ms) Ca2+ currents present in normal myotubes are absent in mdg/mdg myotubes in vitro. 3) the Ca2+-dependent K+ conductance triggering an action potential followed by a long lasting after hyperpolarization (ahp) is absent in mdg/mdg myotubes. This indicates a lack of the free intracellular Ca2+ increased by the action potential. These results suggest that: 1) the lack of differentiated triadic junctions is directly correlated with very low amounts of voltage-dependent Ca2+ channels; 2) the low amount of Ca2+ channels results directly in decreased Ca2+ currents; 3) the decreased Ca2+ currents are the consequence of the low intracellular Ca2+ concentration which is not sufficient to trigger a contraction. However, the addition of normal motoneurones to mdg/mdg myotubes in culture induces, few days later, an increase in Ca2+ currents.     

Effects of 12-O-tetradecanoyl-phorbol- 13-acetate on myofibril integrity and Ca2+ content in developing myotubes

The cocarcinogen 12-O-tetradecanoyl-phorbol-13-acetate (TPA) has a prompt and selective effect on striated myofibrils in well-formed, multinucleated myotubes. The myofibrillar apparatus is totally disrupted and largely degraded after 3 days in TPA. Fluorescent-tagged antibodies to muscle-specific light meromyosin and electron microscopy document this catabolic effect of TPA. This selective degradation of fully assembled striated myofibrils is readily reversible. This reassembly of myofibrils requires de novo protein synthesis. The TPA-treated myotubes are unusually rich in autophagosomes, organelles rarely observed in normal myotubes. TPA has no discernable effect on the morphology of the subsarcolemmal microfilaments, mitochondria, microtubules, or sarcoplasmic reticulum (SR). The programmed disappearance of the fibroblast-type, intermediate 10-nm filaments (FIF) that occurs as normal myotubes mature is not altered by TPA. However, the TPA-treated myotubes depleted of myofibrils are filled with an extensive meshwork of the muscle-type intermediate 10-nm filaments (MIF). The drug has no obvious effect on the constitutive contractile proteins comprising the submembranous microfilaments, or the FIF in presumptive myoblasts or fibroblasts, nor does it affect the motility associated with these replicating cells. The striking increase in total calcium content which occurs as normal myotubes mature is absent in myotubes treated with TPA.     

The electrophysiological expression of Ca2+ channels and of apamin sensitive Ca2+ activated K+ channels is abolished in skeletal muscle cells from mice with muscular dysgenesis

Action potentials of myotubes in culture prepared from 18-19 day -old mouse embryos have a contractile activity and action potentials that are followed by a long lasting after hyperpolarization (ahp) which is blocked by apamin. Myotubes prepared from embryos of mice with muscular dysgenesis (mdg/mdg) did not contract and had action potentials which were never followed by a.h.p.'s. Voltage-clamp experiments have shown that Na+ channel activity was identical in mutant and control muscles and that the activity of fast and slow Ca2+ channels was nearly absent in the mutant.     

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro.

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.     

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.     

Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis

An expression vector was prepared containing a cDNA coding for a truncated version of the intermediate filament (IF) protein desmin. The encoded truncated desmin protein lacks a portion of the highly conserved alpha-helical rod region as well as the entire nonhelical carboxy-terminal domain. When transiently expressed in primary fibroblasts, or in differentiating postmitotic myoblasts and multinucleated myotubes, the truncated protein induces the complete dismantling of the preexisting vimentin or desmin/vimentin IF networks, respectively. Instead, in both cell types vimentin and desmin are packaged into hybrid spheroid bodies scattered throughout the cytoplasm. Despite the complete lack of intact IFs, myoblasts and myotubes expressing truncated desmin assemble and laterally align normal striated myofibrils and contract spontaneously in a manner indistinguishable from that of control myogenic cells. In older cultures the spheroid bodies shift from a longitudinal to a predominantly transverse orientation and loosely align along the I-Z-I-regions of striated myofibrils (Bennett, G.S., S. Fellini, Y. Toyama, and H. Holtzer. 1979. J. Cell Biol. 82:577-584), analogous to the translocation of intact desmin/vimentin IFs in control muscle. These results suggest the need for a critical reexamination of currently held concepts regarding the functions of desmin IFs during myogenesis.     

Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle

Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.     

The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.     

The association of desmin with the developing myofibrils of cultured embryonic rat heart myocytes

The association of desmin, a 55,000-dalton intermediate-filament protein, with the developing cardiac myofibril was studied by immunocytochemical methods in primary cultured myocytes isolated from embyronic rat hearts at different ages. In the earliest contractile myocytes obtained from 10-day-old embryonic hearts, desmin exists as an extensive cytoskeletal network with little or no association with the myofibrils. As the heart develops the cytoskeletal desmin undergoes the myofibrils. Initially, the cytoskeletal desmin appears to outline the developing myofibril as short, discontinuous filaments. At intermediate stages of heart development, desmin filaments in 12- to 16-day-old embryonic myocytes continue to outline the forming myofibrils. Associated with these filaments are crossbridges and foci of desmin spaced at a frequency equal to that of the Z-line spacing. Desmin becomes progressively associated with the myofibril from the central region of the cell toward the cell margin. Desmin filaments at this stage begin to coalesce in the region of the intercalated disk. In the early neonatal heart, desmin of the Z lines becomes continuous across the sarcomere and appears to integrate the myofibrils into a unit. These observations suggest that desmin is not required in the early stages of mammalian heart development for the initial assembly of cardiac sarcomeres or the initiation of cardiac myofibrillar contractions. In later stages of mammalian heart development, desmin is found associated with the cardiac myofibrils in such a manner as to stably integrate these elements into the cytoplasm. Additionally, desmin, in the Z lines of the more mature myocytes appears to maintain the myofibrils in close registry to each other and to the intercalated disk.     

Nestin is expressed during development and in myotendinous and neuromuscular junctions in wild type and desmin knock-out mice.

Desmin, the main component of intermediate filaments (IFs) in mature skeletal muscle, forms an interlinking scaffold around myofibrils with connections to the sarcolemma and the nuclear membrane. Desmin is enriched in neuromuscular and myotendinous junctions. Mice lacking the desmin gene develop normally and reproduce. However, postnatally they develop a cardiomyopathy and a dystrophy in highly used muscles. We have investigated whether and how neuromuscular and myotendinous junctions are affected and whether nestin compensates for the lack of desmin in the knock-out (K/O) mice. We show that neither neuromuscular nor myotendinous junctions were markedly affected in the desmin K/O mice. In neuromuscular junctions nestin was present between the postjunctional folds and the subneural nuclei and between the nucleus and the myofibrillar cytoskeleton. In myotendinous junctions nestin was present between myofibrils at the Z-disc level and in longitudinal strands close to and at the junction. Nestin expression at these specialized sites, as well as during myogenesis and myofibrillogenesis, is independent of the presence of desmin. In desmin K/O mice nestin was also found in regenerating myofibers. The presence of nestin at neuromuscular and myotendinous junctions might provide enough strength for preservation and organization of the junctional areas, although desmin is lacking.     

Visualization of intermediate filaments in living cells using fluorescently labeled desmin

Fluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10-nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells.     

Expression of desmin cDNA in PtK2 cells results in assembly of desmin filaments from multiple sites throughout the cytoplasm.

The assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co-localized with filaments of vimentin just as it does with fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co-localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, co-localizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells.     

Mitosis and intermediate-sized filaments in developing skeletal muscle

A new class of filaments intermediate in diameter between actin and myosin filaments has been demonstrated in skeletal muscle cells cultured from chick embryos. These filaments, which account for the majority of free filaments, average 100 A in diameter. They may run for more than 2 µ in a single section and can be distinguished in size and appearance from the thick and thin filaments assembled into myofibrils. The 100-A filaments are seen scattered throughout the sarcoplasm at all stages of development and show no obvious association with the myofibrils. The 100-A filaments are particularly conspicuous in myotubes fragmented by the mitotic inhibitors, colchicine and Colcemid. In addition, filaments similar in size and appearance to those found in myotubes are present in fibroblasts, chondrocytes, and proliferating mononucleated myoblasts. The 100-A filaments are present in cells arrested in metaphase by mitotic inhibitors. Definitive thick (about 150 A) or thin (about 60 A) myofilaments are not found in skeletal myogenic cells arrested in metaphase. Myogenic cells arrested in metaphase do not bind fluorescein-labeled antibody directed against myosin or actin. For these reasons, it is concluded that not all "thin" filaments in myogenic cells are uniquely associated with myogenesis.     

Gelsolin sensitivity of microfilaments as a marker for muscle differentiation

The ability of porcine smooth muscle gelsolin to sever actin filaments was used to study alterations in the organization of F-actin containing structures during skeletal myogenesis. In permeabilized fibroblasts and unfused myoblasts, gelsolin induced complete degradation of the actin cytoskeleton. After fusion of myoblasts to multinucleated myotubes, gelsolin removed a substantial amount of actin, revealing fibers with a sarcomere-like arrangement of gelsolin-insensitive actin. These fibrils were much thinner and had shorter sarcomeres than fully differentiated myofibrils. The proportion of gelsolin-resistant fibrils increased during differentiation, resulting in almost complete inertness of mature myofibrils. Fibrils isolated from adult muscle were also found nearly resistant to gelsolin. Extraction of tropomyosin and myosin in buffer of high ionic strength prior to gelsolin treatment reestablished the susceptibility to the severing protein, both in myotubes and isolated myofibrils. Only small remnants of phalloidin-stainable material were retained. We therefore conclude that during myotube differentiation either an increased interaction of actin with actin-binding proteins (e.g., myosin and tropomyosin), or the assembly of muscle-specific isoforms of these proteins protect the filaments against degradation by actin severing proteins.     

THE EFFECT OF COLCHICINE ON MYOGENESIS IN VIVO IN RANA PIPIENS AND RHODNIUS PROLIXUS (HEMIPTERA)

The effect of colchicine on myogenesis in vivo has been studied in the regenerating tadpole tail of the frog, Rana pipiens, and in the abdominal molting muscles of a blood-sucking bug, Rhodnius prolixus Stål. Colchicine is shown to disrupt microtubules in the differentiating muscle cells of both these organisms. The disruption of microtubules is correlated with a loss of longitudinal anisometry in the myoblasts and myotubes of the regeneration blastema in the tadpole tail. Before colchicine treatment, the myotubes contain longitudinally oriented myofibrils. After colchicine treatment, rounded, multinucleate myosacs containing randomly oriented myofibrils are present. It is suggested that the primary function of microtubules in myogenesis in the Rana pipiens tadpole is the maintenance of cell shape. The abdominal molting muscles of Rhodnius undergo repeated phases of differentiation and dedifferentiation of the sarcoplasm. However, the longitudinal anisometry of the muscle fibers is maintained in all phases by the attachments of the ends of the fibers to the exoskeleton, and microtubule disruption does not alter cell shape. The orientation of the developing myofibrils is also unaltered, indicating that the microtubules do not directly align or support the myofibrils in this system.     

Monoclonal antibodies to intermediate filaments in chick muscle cell cultures

Two monoclonal antibodies, FIFI and PHIL, have been prepared using detergent-washed myogenic cells as immunogen. On Western blots of total protein extracts of muscle cells, both antibodies bind to vimentin (52 kD) and its degradation products (major band at 42 kD), but do not bind to mouse proteins or to actin (42 kD). Specificity for a determinant common to vimentin and desmin was confirmed by 2-D gel electrophoresis of muscle cell extracts and purified desmin. Western blots with FIFI reveal particularly well the extreme sensitivity of intermediate filaments (IFs) to proteolysis, which was preventable in brain tissue only by boiling in 1% SDS, although it could be reduced in both brain and muscle by less extreme methods. Western blots suggest a large increase in IF content of differentiating myoblast cell cultures at the time of cell fusion and an increase of at least 4-fold is confirmed by a quantitative immunoassay using a direct ELISA method. Immunofluorescence microscopy shows that this increase is due to the appearance of high concentrations of the intermediate filament antigen at the ends of early myotubes, preceding the appearance of cross-striations in myofibrils. Furthermore, whereas the polar filaments detected by FIFI run right to the ends of the early myotubes and only sparingly penetrate the central area, cross-striated myofibrils (as detected by the monoclonal antibody, SAM) run the length of the myotube but do not reach the ends. Colcemid and colchicine cause the vimentin filaments in fibroblasts to collapse into perinuclear rings or caps, but do not have this effect on the polar fluorescence in early myotubes. Heat shock (2 h at 45 degrees C) has a similar differential effect. The results suggest that early in muscle differentiation intermediate filament proteins accumulate rapidly at myotube ends, where they are organized differently from those in fibroblasts.