Cytoskeletal features in longitudinal and circular smooth muscles during development of the rat portal vein

Immunohistochemistry of -smooth muscle actin and desmin, two markers of smooth muscle cell differentiation, and electron-microscopic observation of thick filaments of myosin were performed on the media of the developing rat hepatic portal vein to gain insights into the chronology of differentiation of its longitudinal and circular smooth muscles. In accordance with the ultrastructural distribution of thin filaments, staining of -smooth muscle actin is lightly positive in the myoblasts at postnatal day 1 and then extends in probably all muscle cells of the developing vessel. Desmin, which appears later than -smooth muscle actin in the two muscles, is distributed throughout the longitudinal layer at day 8, whereas the first arrangements of thick filaments are detectable in most longitudinal muscle cells; at this stage, desmin and thick filaments are absent from the poorly differentiated circular muscle cells. The longitudinal muscle cells differentiate in a strikingly synchronized way from day 8 onwards, conferring a homogeneous structure to the developing and mature longitudinal layer. Several desmin-positive cells and a heterogeneous distribution of thick filaments occur in the circular muscle at day 14; the subsequent extension of these filaments in this layer results in a persisting heterogeneous distribution in the young 7-week-old adult. Many features of the mature smooth muscle cells are established within the third week in the longitudinal muscle, approximately one week before those of the circular layer. These results are consistent with the function of the longitudinal muscle as a spontaneously contractile smooth muscle unit, and emphasize the need for its fast maturation to fulfil its major role in the control of portal blood flow.     

Forces measured with micro-fabricated cantilevers during actomyosin interactions produced by filaments containing different myosin isoforms and loop 1 structures

Background

There is evidence that the actin-activated ATP kinetics and the mechanical work produced by muscle myosin molecules are regulated by two surface loops, located near the ATP binding pocket (loop 1), and in a region that interfaces with actin (loop 2). These loops regulate force and velocity of contraction, and have been investigated mostly in single molecules. There is a lack of information of the work produced by myosin molecules ordered in filaments and working cooperatively, which is the actual muscle environment.

Methods

We use micro-fabricated cantilevers to measure forces produced by myosin filaments isolated from mollusk muscles, skeletal muscles, and smooth muscles containing variations in the structure of loop 1 (tonic and phasic myosins). We complemented the experiments with in-vitro assays to measure the velocity of actin motility.

Results

Smooth muscle myosin filaments produced more force than skeletal and mollusk myosin filaments when normalized per filament overlap. Skeletal muscle myosin propelled actin filaments in a higher sliding velocity than smooth muscle myosin. The values for force and velocity were consistent with previous studies using myosin molecules, and suggest a close correlation with the myosin isoform and structure of surface loop 1.

General significance

The technique using micro-fabricated cantilevers to measure force of filaments allows for the investigation of the relation between myosin structure and contractility, allowing experiments to be conducted with an array of different myosin isoforms. Using the technique we observed that the work produced by myosin molecules is regulated by amino-acid sequences aligned in specific loops.     

Ultrastructure of the myocardial cell and its membrane systems in the adult fly Calliphora erythrocephala (Insecta: Diptera)

Summary Calliphora erythrocephala has cross-striated cardiac muscle cells with A, I and Z-bands. The diameters of the myosin and actin filaments are 200–250 Å and 85 Å respectively and the length of the myosin filaments (A-band) is approximately 1.5 . Usually 8–10 actin filaments surround each myosin filament.The myocardial cells show a well-developed membrane system and interior couplings. A perforated sheet of SR envelopes the myofibrils at the A-band, dilates into flattened cisternae at both A-I band levels before it merges into a three-dimensional net-work between the actin filaments of the I-bands and between the dense bodies of the discontinuous Z-discs. The T-system consists of broad flattened tubules running between the myofibrils at the A-I band levels forming dyads with the SR-cisternae. Longitudinal connections between the transverse (T-) tubules often occur.It is suggested that this well-developed SR may be an adaptation to facilitate a rapid contraction/relaxation frequency by an effective Ca²⁺ uptake.     

Demonstration of ‘cardiac-specific’ myosin heavy chain in masticatory muscles of human and rabbit

Summary Human and rabbit masticatory muscles were analyzed immuno-and enzyme-histochemically using antibodies specific to cardiac , slow and fast myosin heavy chain isoforms. In human masseter, temporalis, and lateral pterygoid muscle cardiac myosin heavy chain is found in fibres that contain either fast, or fast and slow myosin heavy chain. In rabbit masseter, temporalis and digastric muscles, fibres are present that express cardiac myosin heavy chain either exclusively, or concomitantly with slow myosin heavy chain or fast myosin heavy chain. Our results demonstrate a much broader distribution of cardiac myosin heavy chain than hitherto recognized and these might explain in part the specific characteristics of masticatory muscles. The cardiac myosin heavy chain is only found in skeletal muscles originating from the cranial part of the embryo (including the heart muscle) suggesting that its expression might be determined by the developmental history of these muscles.     

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.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Expression of an alpha cardiac-like myosin heavy chain in muscle spindle fibres

Summary In the present study we have investigated the reactivity of rat muscle to a specific monoclonal antibody directed against alpha cardiac myosin heavy chain. Serial cross sections of rat hindlimb muscles from the 17th day in utero to adulthood, and after neonatal denervation and de-efferentation, were studied by light microscope immunohistochemistry. Staining with anti- myosin heavy chain was restricted to intrafusal bag fibres in all specimens studied. Nuclear bag₂ fibres were moderately to strongly stained in the intracapsular portion and gradually lost their reactivity towards the ends, whereas nuclear bag₁ fibres were stained for a short distance in each pole. Nuclear bag₂ fibres displayed reactivity to anti- myosin heavy chain from the 21st day of gestation, whereas nuclear bag₁ fibres only acquired reactivity to anti- myosin heavy chain three days after birth. After neonatal de-efferentation, the reactivity of nuclear bag₂ fibres to anti- myosin heavy chain was decreased and limited to a shorter portion of the fibre, whereas nuclear bag₁ fibres were unreactive. We showed that a myosin heavy chain isoform hitherto unknown for skeletal muscle is specifically expressed in rat nuclear bag fibres. These findings add further complexity to the intricate pattern of isomyosin expression in intrafusal fibres. Furthermore, we show that motor innervation influences the expression of this isomyosin along the length of the fibres.     

Myosin Heavy Chain Profiles in Regenerated Fast and Slow Muscles Innervated by the Same Motor Nerve Become Nearly Identical

Plasticity of mature muscles exposed to different activation patterns is limited, probably due to restricted adaptive range of their muscle fibres. In this study, we tested whether satellite cells derived from slow muscles can give rise to a normal fast muscle, if transplanted to the fast muscle bed. Marcaine-treated rat soleus and extensor digitorum longus (EDL) muscles were transplanted to the EDL muscle bed and innervated by the EDL nerve. Six months later expression of myosin heavy chain isoforms was analysed by areal densities of fibres, binding specific monoclonal antibodies, and by SDS gel electrophoresis. Both regenerated muscles closely resembled each other. Their myosin heavy chain profiles were similar to those in fast muscles although they were not identical to that in the control EDL muscle. Since not even regenerated EDL was able to reach the myosin heavy chain isoform profile of mature EDL muscle, our experimental model did not permit studying the adaptive capacity of satellite cells in different muscles in its whole extent. However, the results favour the multipotential myoblast stem cell population in rat muscles and underline the importance of the extrinsic regulation of muscle phenotype.     

Objective characterization of cells in terms of microscopical parameters: An example from muscle histochemistry

Summary A quantitative histochemical analysis of almost 200 mouse triceps surae muscle fibres is presented, together with some data from similar surveys of rabbit skeletal muscles. In the main work, ten parameters are considered: mean diameter, and reaction intensities (estimated as apparent absorbances) of three markers for oxidative-lipolytic metabolism, three for metabolism associated with glycolysis and three for the type of myosin. Cytoarchitecture of one deposit (succinate dehydrogenase) is also noted.Frequency histograms for each parameter and correlation analyses for all possible pairings demonstrate that markers within the same metabolic system are not truly equivalent. Therefore, fibre typing in terms of just one marker from each system cannot be independent of the markers used. Selecting the most sharply discriminatory pair-myosin ATPase (following formalin and alkaline pretreatment) and glycogen phosphorylasea — one isolates four more-or-less discrete clusters of points. Taking succinate dehydrogenase also into account, to indicate characteristic oxidative levels within clusters, one can label the indicated fibre types (following a well-established muscle terminology) as fast, essentially glycolytic (FG), fast, oxidative and glycolytic (FOG), fast, essentially-oxidative (FO) and slow, essentially oxidative (SO). However, with either -glycerophosphate dehydrogenase or the periodic acid-Schiff reaction (PAS) as glycolytic marker, the FO-group is not separated from the FOG; and with oxidative level as a primary clustering criterion FG and FOG groups are continuous. An entirely different basis for classification-cytoarchitecture-also suggests four fibre types but the divisions most comparable to the FG/FOG and FOG/FO boundaries are best located somewhat differently again.Some of the techniques of formal cluster analysis are next introduced. These are ways of searching for similarities (defined in terms of various objective criteria) in large volumes of essentially multivariate data.Within a sample, considered representative of acceptably artefact-free fibres, virtually the same four groups as before are identified. The only difference is that what were earlier grouped as the most oxidative FG fibres are now classed FOG; the acid-pretreated myosin ATPase reaction (previously little considered) contributes to this reclassification. At the five cluster level, a strong tendency exists for this small group, designated FG(O), to appear separately. At the two-cluster level classical distinctions in terms of high/low oxidative capacity, glycolytic capacity and myosin ATPase activity are each favoured by different similarity criteria.Surveying the whole sample of fibres, only criteria which favour non-rambling clusters produce similar results to those above. However, an artefact in one reaction, for which there is strong internal evidence, is able to explain almost all other effects. These results do not prove the biological rightness of a 4–5 cluster pattern, but they do demonstrate its mathematical strength and the reactions upon which it depends.The suggestion is made that cluster analysis and related multivariate statistical methods could profitably be applied to a wide range of further problems in cell taxonomy.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate     

Microtubules in pollen tube subprotoplasts: organization during protoplast formation and protoplast outgrowth

Summary Microtubules inNicotiana tabacum pollen tube subprotoplasts reassembled in wave-like to concentric cortical arrays. Crosslinks between microtubules were either 15 or 80 nm in length. Cortical actin filaments showed different distributions; no colocalization like that in pollen tubes was observed. Degradation of actin filaments by cytochalasin D had no influence on microtubule organization. Degradation of microtubules and/or actin filaments did not affect outgrowth of the subprotoplasts. Organization of the microtubules occurred independent of the presence of the generative cell and/or the vegetative nucleus. No relation of actin filament and microtubule organization with organelle distribution could be detected.Abbreviations AFs actin filaments - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol bis (2-amino ethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MTs microtubules - SPPs subprotoplasts - TRITC tetramethyl rhodamine B isothiocyanate     

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.     

Characterization of the translocator associated with pollen tube organelles

Summary In pollen tubes, the motive force of cytoplasmic streaming is assumed to be generated by the sliding of the translocator associated with cell organelles along actin filaments. In the present study, the characteristics of the translocator were studied by reconstituting the movement of pollen tube organelles along characean actin bundles. Movement of pollen tube organelles proceeded from the pointed end to the barbed end of the actin filaments of the characean cells. The reconstituted movement was not inhibited by vanadate. KCL at higher concentrations inhibited the movement. Furthermore, heavy meromyosin (HMM) prepared from rabbit skeletal muscle myosin partially inhibited the reconstituted movement and pCMB-modified HMM inhibited it completely. The present results strongly support our previous conclusion that the translocator which generates the motive force of cytoplasmic streaming in pollen tube is myosin.Abbreviations AMP-PNP adenylyl-imidodiphosphate - ATP adenosine-5-triphosphate - ATP--S adenosine-5-0-(3-thiotriphosphate) - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - HB homogenization buffer - HMM heavy meromyosin - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PPi pyrophosphate     

Molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in three Taiwan aboriginal tribes

We have investigated glucose-6-phosphate dehydrogenase (G6PD) deficiency in 220 unrelated aboriginal male subjects who belong to three different tribes (Saisiat, Ami, and Yami) in Taiwan. Our results show that the G6PD deficiency rates for Saisiat, Ami, and Yami people are 9.0% (6/67), 6.1% (6/99), and 0% (0/54), respectively. Among these deficiency cases, 4 of 6 (66.7%) Saisiat subjects have the 493 AG mutation and one carries the 1376 GT mutation, whereas, in Ami subjects, we found that four of six (66.7%) affected males have the 592 CT mutation and one carries the 493 AG mutation. These results contrast with our previous findings for Taiwan Chinese, in whom the 1376 GT mutation is the major mutant allele and accounts for 52.3% of the deficiency cases. This is the first report of G6PD deficiency characterized at the DNA level in Taiwan aboriginal populations.     

Myosin light chain patterns of individual fast and slow-twitch fibres of rabbit muscles

Summary Single muscle fibres were isolated by microdissection from freeze-dried samples of rabbit psoas and soleus muscles. The individual fibres were typed according to qualitative histochemical reactions for succinate dehydrogenase or NADH-tetrazolium reductase and for alkaline Ca²⁺-activated myofibrillar myosin ATPase after acid or alkaline preincubation. Methods are described for electrophoretic analysis by means of polyacrylamide disc electrophoresis in the presence of SDS of total myofibrillar proteins in single fibres after pre-extraction of soluble proteins. Fast-twitch white fibres revealed a myosin light chain pattern characteristic of fast-type myosin with three light chains of apparent molecular weights of 22,300 (LC₁), 18,400 (LC₂) and 16,000 (LC₃). Fast-twitch red fibres were indistinguishable in this respect from fast-twitch white fibres and showed an identical pattern of myosin light chains. Slow-twitch fibres could be characterized by a myosin light chain pattern typical of myosin of slow-twitch muscles with peptides of the apparent molecular weights of 23,500 (LC_1Sa), 23,000 (LC_1Sb) and 18,500 (LS_2S). Slow-twitch fibres isolated from soleus as well as from psoas muscle were indistinguishable with regard to their myosin light chain patterns, thus suggesting that fibres of the same histochemical type correspond in their myosin light chain patterns irrespective of their origin from different muscles.Dedicated to the memory of Ernest Gutmann who has contributed so much to our knowledge on differentiation of muscle and who died on August 6, 1977     

Histochemical and immunohistochemical properties of skeletal muscle fibres fromRana andXenopus

Summary Modern histochemical and immunohistochemical techniques have been used to type skeletal muscle fibres from threeRana species andXenopus laevis.Differing myosin properties and metabolic capacities (representing various contractile properties) define a minimum of four fibre types inRana and five inXenopus. TheRana andXenopus types are sufficiently similar so that a single nomencclature can be applied to them. This nomenclature uses an initial letter indicating the probable contractile performance (F=fast-twitch, S=slow-twitch and T=tonic), and a number indicating rank order of presumed shortening velocity.The largest, fastest fibres-F1-have low oxidative and, at best, moderate glycolytic capacities. Commonly adjacent to them are smaller, F2 fibres with variable but at least moderate metabolic capacities. F3 fibres are rarer and have on average the highest oxidative capacity, and at least moderate glycolytic capacity. They usually occur in the reddest parts of the muscle and, inRana, only in the vicinity of tonic fibres.Metabolically weak, classical amphibian tonic fibres (T5) occur in bothXenopus andRana, but onlyXenopus also has an S4 fibre type. This has moderate metabolic capacity and myosin properties suggesting it is probably capable of slow shortening as well as tonic hold. Immunohistochemically, S4 fibres are most similar to avian slow-twitch fibres.     

Supercontraction in crayfish muscle: Correlation with a peculiar actin localization

Summary 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.Abbreviations used in the text EGTA ethyleneglycol-bis (-aminoethyl ether)-N, N-tetraacetic acid - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis - TEMED N, N, N, N-tetramethylenediamine - TRIS Tris (hydroxymethyl) aminomethane A preliminary report on this work was presented at the meeting of the Union of Swiss Societies for Experimental Biology, Davos, 1978 (Benzonana et al., 1978)     

Thyroid hormone receptor-β-selective agonist GC-24 spares skeletal muscle type I to II fiber shift

Triiodothyronine (T3) is known to play a key role in the function of several tissues/organs via the thyroid hormone receptor isoforms alpha (TR) and beta (TR). We have investigated the effects of GC-24, a novel synthetic TR-selective compound, on skeletal muscle fiber-type determination, cross-sectional area, and gene expression in rat skeletal muscles. For fiber typing, cross sections of soleus and extensor digitorum longus (EDL) muscles were stained for myosin ATPase activity at various pHs. Serum T3, T4, and cholesterol levels were also determined. Analysis of highly T3-responsive genes, viz., myosin heavy chain IIa (MHCIIa) and sarcoendoplasmic reticulum adenosine triphosphatase (SERCA1), was performed by quantitative real-time polymerase chain reaction. Equimolar doses of T3 and GC-24 had a similar cholesterol-lowering effect. T3, but not GC-24, decreased fiber type I and increased fiber type II abundance in soleus and EDL muscles. Conversely, in EDL, both T3 and GC-24 decreased the mean cross-sectional area of type I fibers. MHCIIa gene expression was reduced (approximately 50%) by T3 and unchanged by GC-24. SERCA1 gene expression was strongly induced by T3 (approximately 20-fold) and mildly induced by GC-24 (approximately two-fold). These results show that GC-24 does not significantly alter the composition of skeletal muscle fiber type and further strengthens the putative use of GC compounds as therapeutic agents. This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil; 03/04886-1).     

Actin in Pumpkin Phloem Exudate

When analyzing cytoskeletal proteins in Cucurbita pepo phloem exudate by immunoblotting, we detected actin in an amount comparable to that in some plant tissues and a small amount of -tubulin. Electron-microscopic examination of the exudate permitted us to observe filaments that were capable of interacting with the myosin subfragment S1 from rabbit skeletal muscle and with phalloidin conjugated with colloidal gold. The addition of 0.5 mM phalloidin to the exudate in the medium containing 20 mM dithiothreitol (DTT) resulted in an increased number of filaments. Since high DTT concentrations induce a breakdown of filaments of the phloem protein PP1, it seems likely that the produced filaments were composed of actin. The addition of 50 mM MgCl₂ to the exudate resulted in the formation of dense bundles and paracrystals, which resembled those produced by muscle actin under similar conditions. Our results demonstrated that actin in phloem sap was capable of polymerization with filament formation.     

Differentiation of human skeletal muscle cells in culture: maturation as indicated by titin and desmin striation

Summary This report describes a phenotyping study of differentiating human skeletal muscle cells in tissue culture. Satellite cells (adult myoblasts), isolated from biopsy material, showed a proliferative behaviour in high-nutrition medium, but fused to form myotubes when grown in low-nutrition medium. The expression and structural organization of the intermediate filament proteins desmin and vimentin as well as the sarcomeric constituents -actin, -actinin, nebulin, myosin and especially titin during myofibrillogenesis in vitro, were studied by means of indirect immunofluorescence assays. The proliferating myoblasts contained both desmin and vimentin, -actinin and the filamentous form of actin. Shortly after the change of medium, expression of titin, sarcomeric myosin and skeletal muscle -actin was found in mononuclear cells in a diffuse, filamentous (titin, myosin, -actin) or punctate (titin, myosin) pattern. Four to 10 days after the medium change, mature myotubes showed desmin, titin, -actinin, nebulin, sarcomeric myosin and actin cross-striations, while vimentin was no longer detected. We conclude that human skeletal muscle cell cultures are an appropriate model system to study the molecular basis of myofibrillogenesis. Especially the presence of desmin in a striated fashion points to a high degree of maturation of the muscle cell cultures.