Culturing satellite cells from living single muscle fiber explants

Summary Conventional methods for isolating myogenic (satellite) cells are inadequate when only small quantities of muscle, the tissue in which satellite cells reside, are available. We have developed a tissue culture system that reliably permits isolation of intact, living, single muscle fibers with associated satellite cells from predominantly fast and slow muscles of rat and mouse; maintenance of the isolated fibers in vitro; dissociation, proliferation, and differentiation of satellite cells from each fiber; and removal of the fiber from culture for analysis.     

Variability in the shapes of satellite cells in normal and injured frog sartorius muscle

Surface topography of satellite cells is examined in the scanning electron microscope after application of a new method to eliminate connective tissue components. We describe the overall shape of satellite cells in normal frog muscle and show the range of variation in normal structure. Small increases in the width of tails and changes in the cell outline occur after muscle injury in vivo and after incubation in vitro. These changes may be signs of early mobilization of satellite cells related to their eventual role in muscle regeneration.     

Cross bridge slippage in skinned frog muscle fibres

Mechanically skinned single fibres of the semitendinosus muscles of Rana esculenta were investigated at ca. 4 degrees C. The fibres were activated by a Ca2+ jump technique, which allowed the development of a steady isometric tension within several seconds of entering a calcium rich solution at 4 degrees C. Sequences of length changes of different duration and amplitude were applied to the fibre. It could be demonstrated that the fibre behaved as a Hookean spring in the case of small amplitude length changes (up to 0.5% L0, ramp duration 0.5 ms) and that a sequence of length changes induced reversible changes in fibre state. In contrast, large stretches (greater than 1% L0) induced a muscle "give" if the stretch were not immediately preceded by a release. The data was interpreted on the basis of a strain induced detachment of cross bridges in combination with a rapid reattachment of presumably the same cross bridges in a discharged position. The rates of strain induced detachment and reattachment depended on the stretch amplitude. At amplitudes exceeding 2% L0 the rates were estimated to be at least several thousands per second.     

Cross bridge slippage in skinned frog muscle fibres

Mechanically skinned single fibres of the semitendinosus muscles of Rana esculenta were investigated at ca. 4 C. The fibres were activated by a Ca²⁺ jump technique, which allowed the development of a steady isometric tension within several seconds of entering a calcium rich solution at 4 C. Sequences of length changes of different duration and amplitude were applied to the fibre. It could be demonstrated that the fibre behaved as a Hookean spring in the case of small amplitude length changes (up to 0.5% L₀, ramp duration 0.5 ms) and that a sequence of length changes induced reversible changes in fibre state. In contrast, large stretches (> 1% L₀) induced a muscle give if the stretch were not immediately preceded by a release. The data was interpreted on the basis of a strain induced detachment of cross bridges in combination with a rapid reattachment of presumably the same cross bridges in a discharged position. The rates of strain induced detachment and reattachment depended on the stretch amplitude. At amplitudes exceeding 2% L₀ the rates were estimated to be at least several thousands per second.     

Visualization of myosin in living cells

Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasingly more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.     

Minimal latency of calcium release in frog twitch muscle fibres

Intracellular release of calcium in frog skeletal muscle fibres was monitored by the use of arsenazo III, in response to voltage clamped depolarizing pulses. A latency of a few milliseconds was evident between the onset of depolarization and the first detectable rise in the arsenazo-calcium signal, and this decreased logarithmically as the depolarization was increased. The minimal latency with strong depolarization (to +20 to +100 mV) was about 2 ms at 5 degrees C. This delay appears to be sufficiently long to be compatible with a chemically mediated coupling mechanism between depolarization and calcium release from the sarcoplasmic reticulum.     

The number of satellite cells in slow and fast fibres from human vastus lateralis muscle

The aim of this investigation was to study the distribution of satellite cells in slow (type I fibres) and fast (type II fibres) fibres from human vastus lateralis muscle. This muscle is characterised by a mixed fibre type composition and is considered as the site of choice for biopsies in research work and for clinical diagnosis. Biopsy samples were obtained from five healthy young volunteers and a total of 1,747 type I fibres and 1,760 type II fibres were assessed. Satellite cells and fibre type composition were studied on serial muscle cross-sections stained with specific monoclonal antibodies. From a total of 218 satellite cells, 116 satellite cells were found in contact with type I fibres (53.6±8% of the satellite cells associated to type I fibres) and 102 satellite cells in contact with type II fibres (46.4±8% of the satellite cells associated to type II fibres). There was no significant difference (P=0.4) between the percentages of satellite cells in contact with type I and with type II fibres. Additionally, there was no relationship between the mean number of satellite cells per fibre and the mean cross-sectional area of muscle fibres. In conclusion, our results show that there is no fibre type-specific distribution of satellite cells in a human skeletal muscle with mixed fibre type composition.     

Visualization of mRNA translation in living cells

The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for beta-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with beta-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. beta-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of beta-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions.     

Visualization of single fluorophores in living cells

Methods applicable to visualizing single fluorophores in living cells are described, namely, laser epifluorescence, confocal, near-field, two-photon, and total internal reflection microscopy. It is demonstrated that total internal reflection microscopy is the most appropriate for visualizing single fluorophores near the substrate-medium interface. This method can be used for studying receptors, ion channels, and numerous cytoskeletal and signal molecules located on or near the basal cell membrane. It is demonstrated that stringent criteria are necessary when identifying single molecules, as these objects emit a limited number of photons before irreversible photobleaching and their fluorescence is obscured by autofluorescence or out-of-focus fluorescence. The methods used for studying the lateral mobility of single molecules floating on the cell membrane are also described.     

Behaviour of chromosomal spindle fibres in living cells

Summary Mitosis in endosperm of Haemanthus katherinae was studied in vitro by the use of 16 mm time-lapse microcinematography. In several cells, chromosomal fibres were seen in phase contrast microscope. The fibres are more convergent in late metaphase and anaphase than in prometaphase. There exist two distinct fibre attachments at each daughter kinetochore, i.e. there are four in metaphase chromosome.Details of the division of a nearly cytoplasm-free mitotic apparatus are also reported as well as some data concerning the elongation of the spindle and activity of the phragmoplast.Dedicated to Professor H. Bauer on the occasion of his sixtieth birthday.     

Visualization of gap junction mobility in living cells

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.     

Caffeine and the myoplasmic calcium removal mechanisms in cut frog skeletal muscle fibres

Antipyrylazo III myoplasmic calcium transients were recorded in cut skeletal muscle fibres of the frog (Rana esculenta), using the double vaseline-gap voltage-clamp system. Intracellular calcium removal mechanisms were analysed, using a slightly modified model taken from the literature. Parameter values reported here are generally consistent with those obtained by the original model. Caffeine (0.5 mmol.l-1) moderately enhanced the overall myoplasmic calcium removal. In particular, the rate constant of the non-saturable uptake increased by 51% on the average, but there was a considerable fiber-to-fiber variation. The kinetic features of the binding sites representing the saturable uptake did not change significantly while the concentration of the available sites decreased by 36%. It is concluded that the caffeine-induced changes of the calcium removal components can be explained by supposing an increased resting myoplasmatic Ca2+ concentration in the presence of the drug.     

Visualization of the vesicular acetylcholine transporter in living cholinergic cells

We regret that we must retract the article Visualization of the Vesicular Acetylcholine Transporter in Living Cholinergic Cells by M. S. Santos, J. Barbosa Jr., C. Kushmerick, M. V. Gomez, V. F. Prado, and M. A. M. Prado ( J. Neurochem . 74 , 2425-2435 , 2000). We have made an unintentional mistake in the construction of the enhanced green fluorescent protein (EGFP) vectors, and consequently the vesicular acetylcholine transporter (VAChT) and its truncated forms are incorrectly expressed. We have repeated the key experiments with proper constructs and have found that the expression pattern is clearly different from that reported in the article. The truncated form of VAChT, without the C-terminal tail, presents a distinct pattern of expression when compared to VAChT, and we have found no evidence that the C-terminal tail of VAChT is able to drive EGFP to varicosity sites. As a consequence of this problem, our earlier conclusions were incorrect. We apologize for this mistake and for any problems that we may have caused.