Localization of the actin-binding protein fesselin in chicken smooth muscle

This report compares cellular localization of fesselin in chicken smooth, skeletal and cardiac muscle tissues using affinity purified polyclonal fesselin antibodies. Western blot analyses revealed large amounts of fesselin in gizzard smooth muscle with lower amounts in skeletal and cardiac muscle. In gizzard, fesselin was detected by immunofluorescence as discrete cytoplasmic structures. Fesselin did not co-localize with talin, vinculin or caveolin indicating that fesselin is not associated with dense plaques or caveolar regions of the cell membrane. Immunoelectron microscopy established localization of fesselin within dense bodies. Since dense bodies function as anchorage points for actin and desmin in smooth muscle cells, fesselin may be involved in establishing cytoskeletal structure in this tissue. In skeletal muscle, fesselin was associated with desmin in regularly spaced bands distributed along the length of muscle fibers suggesting localization to the Z-line. Infrequently, this banding pattern was observed in heart tissue as well. Localization at the Z-line of skeletal and cardiac muscle suggests a role in contraction of these tissues.     

Proteasomes are tightly associated to myofibrils in mature skeletal muscle

Proteasomes are the major actors of nonlysosomal cytoplasmic protein degradation. In particular, these large protein complexes (about 2500 kDa) are considered to be responsible for muscular degradation during skeletal muscle atrophy. Despite their unusual and important size, they are widely described as soluble and mobile in the cytoplasm. In mature skeletal muscle, we have previously observed a sarcomeric distribution of proteasomes, as revealed by the distribution of α1/p27K, a subunit of the 20S core-particle (prosome) of proteasome. Here, we extend these observations at the electron microscopic level in vivo. We also show that this sarcomeric pattern is dependent of the extension of the sarcomere. Using isolated myofibrils, we demonstrate that proteasomes are still attached to the myofibrils after the isolation procedure, and reproduce the observations made in vivo. In addition, the extraction of actin by gelsolin largely removes proteasomes from isolated myofibrils, but some of them are held in place after this extraction, showing a sarcomeric disposition in the absence of any detectable actin, and suggesting the existence of another molecular partner for these interactions. From these results, we conclude that most of detectable 20S proteasomes in skeletal muscle cells is tightly attached to the myofibrils.     

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.     

Effects of long term denervation on smooth muscle of the chicken expansor secundariorum

Summary Denervation of the expansor secundariorum muscle of the adult and 2 week chicken, by sectioning the brachial plexus, resulted in an approximate twofold increase in dry weight over 8 weeks. Unlike skeletal muscle, no ultrastructural changes were exhibited by the smooth muscle cells for a period of up to 5 months post denervation. No evidence of hypertrophy of the individual muscle cells was observed, but following colchicine treatment a definite increase in the number of mitotic figures was noted within muscle bundles indicating that the increase in dry weight of the expansor muscle is due to hyperplasia of the smooth muscle cells. The results are discussed in relation to in vitro studies of the interaction of sympathetic nerves with smooth muscle.     

Chicken cardiac myofibrillogenesis studied with antibodies specific for titin and the muscle and nonmuscle isoforms of actin and tropomyosin

Summary Myofibrillogenesis was studied in cultured chick cardiomyocytes using indirect immunofluorescence microscopy and antibodies against - and -actin, muscle and nonmuscle tropomyosin, muscle myosin, and titin. Initially, cardiomyocytes, devoid of myofibrils, developed variable numbers of stress fiber-like structures with uniform staining for anti-muscle and nonmuscle actin and tropomyosin, and diffuse, weak staining with anti-titin. Anti-myosin labeled bundles of filaments that exhibited variable degrees of association with the stress fiber-like structures. Myofibrillogenesis occurred with a progressive, and generally simultaneous, longitudinal reorganization of stress fiber-like structures to form primitive sarcomeric units. Titin appeared to attain its mature pattern before the other major contractile proteins. Changes in the staining patterns of actin, tropomyosin, and myosin as myofibrils matured were interpreted as due to longitudinal filament alignment occurring before ordering in the axial direction. Non-muscle actin and tropomyosin were found with sarcomeric periodicity in the initial stages of sarcomere myofibrillogenesis, although their staining patterns were not identical. The localization of the sarcomeric proteins -actin and muscle tropomyosin in stress fiber-like structures and the incorporation of non-muscle proteins in the initial stages of sarcomere organization bring into question the meaning of sarcomeric proteins in regard to myofibrillogenesis.     

Changes in myofibrils and cytoskeleton of neonatal hamster myocardial cells in culture: an immunofluorescence study

Myocardial cells in culture offer many possibilities for studying cellular and molecular biology of cardiac muscles. However, it is important to know how long these cells can be maintained in vitro without significant structural and biochemical changes. In this study, we have investigated the morphological changes of myofibril proteins and cytoskeletons by using immunofluorescent techniques in cultured neonatal hamster myocardial cells at different culture durations. Our results have demonstrated that these cultured cells still contain intact myofibrils and cytoskeletal proteins after 6 days in vitro incubation, however, the organization of some of these proteins is altered. The proteins most sensitive to these in vitro conditions are: myosin heavy chain, actin and desmin. The data indicate that the duration of the culture and the contractile activity of the myocardial cells in culture can influence organization of their contractile apparatus and cytoskeleton.     

Structural diversity in muscle fibres of chicken breast

Summary Chicken breast muscle is usually considered to be a relatively homogeneous white muscle and has therefore been widely used for studies of muscle proteins. In a previous study, however, we have found different M-region structures in different fibres from this muscle. Because of this result, we have now carried out a combined histochemical and ultrastructural survey of this muscle. In particular, we have made use of large transverse cryo-sections that include most of the muscle cross-section.Although the white region is fairly homogeneous in fibre content according to normal histochemical criteria (mAT-Pase), we have found that there is a gradation of fibre structure across the muscle. The bulk of the muscle stains conventionally for Type-II fibres according to mATPase tests (the white part) but, in the small red part of the muscle, there are also Type-I fibres together with the Type-II fibres. Superimposed on this division into Type-I and Type-II fibres are variations in fibre size, oxidative and glycolytic staining properties, and variations of Z-band width and M-band structure; there is no strict correlation among any of these parameters. The apparently uniform staining across most of the muscle when tested for myofibrillar ATPase may be a misleading indicator of fibre properties.     

Expression of immunoreactive myosin and myoglobin in the developing chicken gizzard

Summary Antibodies to adult-type myosin and myoglobin from chicken gizzard were used to study the expression of these proteins during chicken embryogenesis. Using the indirect immunofluorescent technique, myosin was detected as discrete fluorescent foci in the central part of the presumptive chicken gizzard as early as day 5 of development. During the following days, immunoreactive myosin extends both craniocaudally as well as laterally and reaches the serosal and luminal borders by day 13/14. On day 16, the adult fascicular pattern is achieved. As judged by enzymelinked immunoassay and spectroscopic methods, myoglobin did not appear until day 18.Dedicated to Mrs. C.F. Schoenberg, Department of Anatomy, Cambridge, Great Britain     

Immunoreactive substance P in the chicken gut: Distribution,development and possible functional significance

Summary The distribution and cellular localization of substance P in the chicken gut was studied by immunocytochemistry and immunochemistry. Substance P-containing nerve fibers are numerous in the gut wall. They occur in the smooth muscle layer as well as in the mucosa, where they are associated with blood vessels or surround the intestinal crypts. The fibers are particularly numerous in the myenteric and submucosal plexuses, where substance P-containing nerve-cell perikarya are also encountered. Substance P was found also in scattered endocrine cells of the small intestine, caeca and colon. Previously, bombesin-containing cells, which are numerous in the proventriculus, have been mistakenly identified as substance P cells due to crossreactivity of certain antisera against substance P. Immunochemistry revealed the highest concentration of substance P in the duodenum. The gel chromatographic behavior of chicken substance P differs slightly from that of synthetic bovine substance P, suggesting that chicken substance P differs structurally from mammalian substance P. Substance P-containing nerve fibers in the chicken gut develop slowly after hatching, apparently beginning in the duodenum; at approximately 20 weeks after hatching the distribution pattern is fully developed.A functional investigation was performed on the isolated chicken caecum to clarify the role of substance P in the contractile behavior of smooth muscle. Substance P contracted the caecum over a wide dose range; the contractile response was greater in 20 week-old chickens than in 4 and 10 week-old animals. Electrical field stimulation caused a relaxation of the caecum and a contraction upon cessation of stimulation. Neither of these responses, both of which are neurally mediated, were inhibited by adrenergic and cholinergic blockade. It is conceivable that the contractile response following electrical stimulation is caused by substance P released from nerve fibers in the smooth muscle.     

Trypanosoma cruzi: reversal of inhibition of host muscle differentiation after exposure to elevated temperatures

The L6E9 myoblast cell line can be grown as individual cells in "growth medium," or can be induced to fuse and differentiate to form multinucleated myotubes either at 37 C or at 40.5 C in "differentiation medium." It has previously been shown that myoblasts with infected Trypanosoma cruzi (Brazil strain) cannot differentiate to form myotubes. Moreover, the mRNAs for contractile proteins are not induced in these infected cells. Infected myoblasts grown in "differentiation medium" at 37C were unable to differentiate by 7 days. The infection was maintained at 100%, and the number of trypomastigotes in the supernatant increased with time (peak greater than 10(6)/ml). At 40.5C, however, infected myoblasts gradually eliminated their infection. The percentage of parasitized cells was reduced to less than 1% by the 7th day of observation. There was also a decrease in the number of trypomastigotes in the supernatant. Moreover, significant fusion was observed in these cultures by morphological criteria. Using 32P-labeled recombinant DNA probes, it was shown that, at 37C, there was an inhibition of mRNAs for muscle-specific contractile proteins (myosin heavy chain and alpha-actin), whereas nonspecific mRNAs were not inhibited. Furthermore, infected myoblasts exposed to 40.5C exhibited no inhibition of mRNAs for myosin heavy chain and alpha-actin. Myoblasts cleared of their infection could readily be reinfected. This study demonstrates that the inhibition of muscle differentiation induced by T. cruzi is reversible when cultures are exposed to elevated temperatures.     

Projection of statocyst sensory neurons associated with crescent hairs in the crayfish Procambarus clarkii Girard

Summary Both smooth muscle and striated muscle are present in the iris of the chick embryo. The two types of musculature form mixed clusters which include undifferentiated cells and many nerve fibres, but they are structurally quite distinct and have different origins. The smooth musculature originates around the 10th day from a laminar invagination (iridial lamella) of the posterior epithelium, and is therefore an ectodermal derivative. The striated musculature appears slightly later than the smooth musculature and originates from undifferentiated cells which are regarded as mesenchymal. After the 15th day in ovo the smooth musculature stops growing; its cells become confined to an area very near the pupillary margin and many develop pigment granules in the sarcoplasm. Many smooth muscle cells seem to undergo regressive changes; however, cells with the typical appearance of visceral muscle cells are still present in the iris of 3-month-old chickens. High density of innervation and vasculari/ation, wide range of striated muscle fibre diameters, presence of lipid vacuoles and of large clusters of mitochondria in the striated fibres, occurrence of peripheral couplings of the sarcoplasmic reticulum, and presence of numerous fibroblast processes in the interstices between fibres, characterize the sphincter pupillae of the mature iris.This work was supported by grants from the Medical Research Council and the Central Research Fund of the University of London     

Embryonic chicken gizzard: immunolocalization of collagen and smooth muscle myosin

Summary Antibodies to chicken gizzard myosin and to chicken skin collagen type I allow the myofibrillar and connective tissue development in the embryonic chicken gizzard to be followed. Fibroblasts are assumed to synthesize collagen prior to the onset of smooth muscle cell development in the muscle primordium (day 5); they are presumably also responsible for collagen synthesis close to the presumptive lamina propria and in the developing tubular glands (day 14 to 17). From day 6 to 8, myosin and collagen are colocalized intracellularly, and from day 9 onward collagen fibers start to appear extracellularly, eventually forming the trellis-like connective tissue septa that give the rhomboid profile found in the adult muscle. The close association of collagen and myosin in early development suggests that the muscle cells themselves produce and export collagen.     

Acetylcholinesterase molecular forms in the fast or slow muscles of the chicken and the pigeon

Molecular forms of acetylcholinesterase (AChE) were examined in various skeletal muscles of the chicken and the pigeon. In chicken pectoralis m., AChE was found to be restricted to endplate containing segments, and no asymmetric form could be detected in aneural samples. In the chicken muscles studied, a relation has been established between globular (G1,G2,G4) forms or asymmetric (A8,A12) forms, and muscle fibre types. Asymmetric forms are preponderant in fast-twitch muscles, whereas in slow tonic muscles 80% of the AChE activity is due to globular forms. However, comparison with pigeon muscles shows that AChE chicken muscle patterns may not be generalized.     

Temperature acclimation and the expression of contractile protein isoforms in the skeletal muscles of the common carp (Cyprinus carpio L.)

Summary Common carp (Cyprinus carpio L.) were acclimated to either 2, 5, 8, 11, 15, 20, or 23°C for 12 weeks (12 h light: 12 h dark). Fish did not feed after 6 weeks at temperatures below 8°C. Skinned fibres were prepared from fast myotomal muscle by freeze-drying. Measured at 0°C unloaded contraction velocity (Vmax) and maximum isometric tension generation (Po) were 2–3 times higher in the 11°C-than 23°C-acclimated groups, and had intermediate values in 15 °C-acclimated fish. Po and Vmax at 0°C were not significantly different for carp maintained at 2, 5, 8, or 11°C. Measured at the acclimation temperature of each group Vmax and Po were 51% and 71% lower for fibres from 2°C- than 23°C-acclimated fish. The results indicate a partial capacity adaptation of muscle power output in fish acclimated between 11°C and 23°C. At 8°C the ATPase activity of myofibrils was 2 times higher in fish acclimated to 8°C than to 20°C. The effects of temperature acclimation on the protein composition of myofibrils was investigated using one- and two-dimensional electrophoresis. Peptide maps of purified myosin heavy chains and actin prepared by proteolytic digestion with either Staphylococcus aureus V8 protease or chymotrypsin were similar for both acclimation groups. The molecular weights and isoelectric points of the major isoforms of tropomyosin, troponin C, troponin I, troponin T, and myosin light chains (MLC1, MLC2 and MLC3) were also similar in 8°C- and 20°C-acclimated carp. A 20 kDa molecular weight protein with a pI intermediate between that for MLC2 and MLC3 was found in myofibrils and single fibres from carp acclimated to 8°C but was not present in carp acclimated to 20°C. It is suggested that this band corresponds to a myosin light chain isoform unique to cold-acclimated fish. Evidence was also obtained that myofibrils from warm-acclimated fish contained a second minor isoform of troponin I.     

Human smooth muscle cell cultures of the stomach morphologic and biochemical studies

Summary Smooth muscle cell cultures were prepared from stomach explants obtained surgically from 10 patients with duodenal ulcer. The cultured cells grew in either overlapping layers in “hills and valleys” or in parallel arrays. The ultrastructure studies showed plasmalemmal vesicles, bundles of myofilaments associated with dense bodies, and gap junctions. The synthesis of contractile proteins illustrated the preponderance of actin on myosin and tropomyosin. The synthesis of contractile proteins in stomach smooth muscle cell cultures is significantly higher than in skin fibroblast cultures, i.e. 20 x higher for myosin, 10 x higher for actin, and 30 x higher for tropomyosin.     

Molecular cloning and expression of two chicken invariant chain isoforms produced by alternative splicing

The biosynthesis of distinct forms of the invariant chain (Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice. However, there have been no reports on the existence of Ii isoforms in avian species. In the present study, we identified two chicken Ii cDNAs by RT-PCR and RACE, and examined the Ii gene copy number, mRNA expression and protein expression by Southern blotting, Northern blotting and immunofluorescence confocal microscopy, respectively. One of the Ii cDNAs, named Ii-1, was 1,151 bp in length, and had an open reading frame (ORF) of 672 nucleotides, in agreement with a previously identified chicken Ii sequence; the other, named Ii-2, was 1,337 bp long and had an ORF of 861 nucleotides. Southern blotting confirmed that these cDNAs were derived from a single copy gene. Northern blotting performed with total RNA from various tissues of 6-week-old chickens revealed high levels of Ii-1 and Ii-2 mRNA expression in the spleen and bursa of Fabricius, and low levels of Ii-1 expression in the thymus, heart and liver, while Ii-2 was not expressed in these tissues. High levels of expression of both Ii isoforms were detected in the spleen and bursa of Fabricius during late embryogenesis. Immunofluorescence staining showed that Ii proteins were expressed in the cell membranes of the splenocytes. These data suggest that chicken Ii exists in two isoforms resulting from alternative splicing, and is strongly expressed in the major immune organs.     

Comparison of vascular smooth muscle cells from adult human,monkey and rabbit in primary culture and in subculture

Summary A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or dedifferentiated after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or dedifferentiation process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of dedifferentiated cells at all times.The advantages of differentiated rather than dedifferentiated smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.The authors wish to thank Professor H.H. Bentall of the Royal Postgraduate Medical School, Hammersmith Hospital, London, for making available human material, and Dr. S. Zeki of Department of Anatomy, University College London for material from monkeys. We are also extremely grateful to Professor G. Burnstock for the use of his laboratory facilitiesHolder of a John Halliday Travelling Fellowship from the Life Insurance Medical Research Fund of Australia and New ZealandResearch Fellow with the National Heart Foundation of AustraliaSupported by the Deutsche Forschungsgemeinschaft     

Autonomic innervation of the ocular choroid membrane in the chicken

Summary The distribution pattern of adrenergic fibres innervating the ocular choroid membrane of the chicken was studied by means of fluorescence and electron microscopy. In addition, the origin of these fibres was investigated after superior cervical ganglionectomy. Adrenergic axons reach the choroid, partly forming the perivascular plexuses and partly running in the choroid nerves and the choroidal branches of the ciliary nerves. The axon terminals distribute to the smooth muscle cells of the arterial wall and to the extensive system of smooth muscle cells of the intervascular stroma. After unilateral ganglionectomy, fluorescent fibres almost completely disappeared, and degenerative changes could be observed in the terminal varicosities on both smooth muscle cell populations. These findings suggest that the adrenergic axons either originate from neurones within the ipsilateral superior cervical ganglion, or pass through this ganglion. The persistence of normal terminals in short- and long-term ganglionectomised animals shows that the vasal and intervascular muscle cells of the choroid membrane are provided with both an adrenergic and a cholinergic innervation.This work was supported by grant No 80.00442.04 from the Italian National Research Council (CNR)     

A novel striated tropomyosin incorporated into organized myofibrils of cardiomyocytes in cell and organ culture

Striated muscle tropomyosin is classically described as consisting of 10 exons, 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b, in both skeletal and cardiac muscle. A novel isoform found in embryonic axolotl heart maintains exon 9a/b of striated muscle but also has a smooth muscle exon 2a instead of exon 2b. Translation and subsequent incorporation into organized myofibrils, with both isoforms, was demonstrated with green fluorescent protein fusion protein construct. Mutant axolotl hearts lack sufficient tropomyosin in the ventricle and this smooth/straited chimeric tropomyosin was sufficient to replace the missing tropomyosin and form organized myofibrils.     

Clathrin and clathrin-accessory proteins in rat kidney cortex epithelia

Several vectorial transport routes in mammalian cells involve clathrin and associated proteins. In kidney epithelia urine production requires numerous transport processes. However, only little is known about the distribution of clathrin and its associated proteins in this organ in situ. We now report on the presence and distribution of clathrin and its accessory proteins AP1, AP2, Eps15, Epsin, CALM and Clint/EpsinR in the epithelia of the rat kidney cortex using immunoblotting, immunofluorescence and immuno-electron microscopy. Our data show that all investigated proteins are ubiquitously present in rat kidney cortex epithelia, however, with distinct distribution patterns. In the renal corpuscle, podocytes showed the most conspicuous labelling. Clathrin, AP2 and CALM were highly expressed in foot processes, while AP1 was primarily localized in the cell body. In the proximal tubule all proteins were present in dots along the plasma membrane and most conspicuous below the brush border. However, clathrin and AP2 co-localized in vesicle subtypes distinct from those containing clathrin and AP1. In the distal tubule and in the cortical collecting duct all proteins were found in the apex of the cells; however, AP1 and Clint/EpsinR showed additional staining in perinuclear dots. The occurrence and distribution of the investigated proteins in kidney epithelia are discussed with respect to their possible involvement in the functions of the specific nephron segment.Preliminary results were presented at the 28th Annual Meeting of the German Society for Cell Biology, March 16th–19th 2005, Heidelberg, Germany.