Thomas R. Podleski

Fibronectin delays the fusion of L6 myoblasts

Abstract The effect of fibronectin on myogenesis has been studied in vitro. The addition of purified fibronectin to the myogenic cell line L 6 blocks fusion and causes an increase in cell number. The effects of fibronectin could be prevented by the immunoprecipitation of fibronectin from solutions using affinity-purified antifibronectin antibodies. Mild trypsinization of the cells (10 μ/ml trypsin for 20 min) which removes surface fibronectin, causes the rate of fusion to increase when the trypsinization is done just before the cells begin to fuse, day 4 (after the plating of the cells), an inhibition when done on one day earlier, day 3, and has no effect when done after the cells have begun to fuse, day 5. By measuring the binding of rhodamine-labeled antifibronectin antibodies to intact cells, it was found that surface fibronectin increased from day 3 to day 4 and then decreased on day 5. The stimulating effect of trypsin on fusion, therefore, corresponds to the day surface fibronectin reaches a peak. Affinity-purified antifibronectin antibodies were also shown to be capable of enhancing fusion. It is concluded from these results that high levels of fibronectin stimulate events which reduce fusion, whereas the removal of surface fibronectin during critical times either stimulates or reduces fusion.

Acetylcholine receptor distribution on myotubes in culture correlated to acetylcholine sensitivity.

1. A linear relation, with a slope of 0‐9 +/‐ 0‐2 on a log‐log plot, was obtained between acetylcholine (ACh) sensitivity and alpha‐bungarotoxin (alpha‐BTX) binding site density in developing L6 and rat primary myotubes. ACh sensitivity was defined as g/Qn where g is conductance, Q is ACh charge and n is the Hill coefficient. Experimentally we found n approximately 1‐7 for our myotubes, which is similar in value to that reported for adult systems. 2. The linear relationship is compatible with an organization whereby each ion channel is always complexed with a fixed number of ACh receptors such that the dose‐response characteristics of each such complex are independent of average ACh receptor density. 3. Light microscope autoradiography showed that the alpha‐bungarotoxin binding sites on L6 myotubes are uniformly distributed over the surface, while primary rat myotubes exhibit gradients and hot spots. Electron microscope autoradiography indicated that about 70% of the [125I]alpha‐bungarotoxin label was on the surface of the myotubes. The alpha‐bungarotoxin site density, after subtracting myoblast background, varied from 5 to 400 sites/micrometer2 on different L6 myotubes, and from 54 to 900 sites/micrometer2 on primary rat myotubes, with occasional hot spots of 3000‐4000 sites/micrometer2. The conductance sensitivities varied from 10(‐4) to 2 X 10(‐2) Momega‐1/nC1‐7.

Evidence that the types and specific activity of lectins control fusion of L6 myoblasts

Abstract The specific activity of both the non-thiodigalactoside blockable and thiodigalactoside blockable lectin activities increase as a prelude to fusion. The peak of the non-thiodigalactoside blockable lectin activity is coincident with early fusion. The specific activity of both types of lectin activities decrease with the progression of fusion. The non-thiodigalactoside blockable lectin activity appears to be absent from myotubes.

Control of acetylcholine receptor mobility and distribution in cultured muscle membranes. A fluorescence study

Abstract The molecular control of the distribution and motion of acetylcholine receptors in the plasma membrane of developing rat myotubes in primary cell culture was investigated by fluorescence techniques. Acetylcholine receptors were marked with tetramethylrhodamine-labeled α-bungarotoxin and lateral molecular motion in the membrane was measured by the fluorescence photobleaching recovery technique. Three types of experiments are discussed: (I) The effect of enzymatic cleavages, drugs, cross-linkers, and physiological alterations on the lateral motion of acetylcholine receptors and on the characteristic distribution of acetylcholine receptors into patch and diffuse areas. (II) Observation of the distribution and/or motion of fluorescence-labeled concanavalin A receptors, lipid probes, cell surface protein, and stained cholinesterase in acetylcholine receptor patch and diffuse areas. (III) The effect of a protein synthesis inhibitor and electrical stimulation on membrane incorporation of new acetylcholine receptors. Some of the main conclusions are: (a) acetylcholine receptor lateral motion is inhibited by concanavalin A plant lectin and by anti-α-bungarotoxin antibody, but marginally enhanced by treatment with a local anesthetic; (b) patches are stabilized by an immobile cellular structure consisting of molecules other than the acetylcholine receptors themselves; (c) this structure is highly selective for acetylcholine receptors and not for other cell membrane components; (d) acetylcholine receptor patch integrity and diffuse area motion are independent of direct metabolic energy requirements and are sensitive to electrical excitation of myotube; (e) lipid molecules can move laterally in both acetylcholine receptor patches and diffuse areas; and (f) acetylcholine receptor lateral motion in diffuse areas and immobility in patch areas are not altered by specific agents which are known to affect extrinsic cell surface proteins, or cytoplasmic microfilaments and microtubules.

Studies on lectin activity during myogenesis.

Abstract The molecular properties of two hemagglutinating proteins, one a lectin called electrolectin and a second protein called myonectin, are described in the L 6 cells, a myogenic cell line. The activities of these two proteins change during myogenesis. Electrolectin is found in two forms: (1) s -electrolectin is in the supernatant fraction of a 100000 g centrifugation; (2) p -electrolectin is in the pellet. Myonectin is found almost exclusively in the supernatant fraction. Also present in the supernatant fraction is a protein which blocks s -electrolectin activity. The blocking effects of this protein can be removed by a variety of techniques including gel filtration, heating, trypsinization and extensive dialysis. All of these procedures result in the inactivation of myonectin. Since the blocking protein also chromatographs with myonectin, these observations suggest that myonectin and the blocking protein may be the same. Based on gel chromatography, s -electrolectin and p -electrolectin have similar filtration properties whereas myonectin is very different, and can be easily separated from electrolectin. Since trypsinization of intact cells leads to the loss of myonectin, it is concluded that myonectin is largely limited to the surface of the cells. Several procedures were used to alter the rate and extent of the differentiation of the cells in order to determine the relationship between the agglutinating proteins and cellular differentiation. Plating cells at different densities or blocking fusion did not alter the activity of p -electrolectin. Changes in s -electrolectin and myonectin activities were observed, but the decrease in their activities which normally accompanies fusion occurred even when fusion was blocked. It is concluded, therefore, that fusion alone is not responsible for the decrease in the activities of these proteins.

Differential responses of L5 and rat primary muscle cells to factors in rat brain extract

Crude brain extract (100,000 g supernate from newborn or fetal rat brain homogenate) was studied for its effects on the number and distribution of acetylcholine receptors (AChRs) on myotubes of the L5 cloned myogenic cell line and compared to that of rat primary cultures. Gamma counting, light autoradiography and scanning electron microscopic autoradiography were used. We found that the L5 cells responded to the brain extract with an increase in the average AChR site density (2-5-fold) and with an increase in AChR clustering. Clustering was manifested by both an increase in the number of AChR clusters and in the ratio of receptor site density within clusters relative to that between clusters. The increase in average AChR site density was shown to be due to an increase in the rate of AChR insertion into the surface membrane with little change in the rate of receptor degradation. As also previously reported, the rat myotubes had a similar clustering response but only a very slight (approximately 1.2-fold) increase in average AChR site density. The surface area of myotubes was also increased slightly (approximately 1.2-1.3-fold) by the brain extract. Autoradiography viewed by scanning EM was found to be very useful in illustrating the shape and distribution of the receptor clusters. After the brain extract was fractionated on Sephadex G-200, the fractions with greatest clustering activity could be separated from those causing predominantly an increase in receptor site density. Increased receptor site density was primarily produced by the low molecular weight fractions (less than 12 kD), whereas the strongest (but not exclusive) effect on clustering was produced by the high molecular weight fractions (greater than 140 kD). Furthermore, the two cell types assayed had different sensitivity to the different factors. L5 cells responded to both the high and low molecular weight factors while rat primary cells are sensitive primarily to the high molecular weight factors.

Physical and chemical characterization of the major lactose-blockable lectin activity from fetal calf skeletal muscle

The lactose-blockable lectin activity from fetal calf skeletal muscle has been purified to apparent homogeneity. The purification entails differential centrifugation, ammonium sulfate precipitation, asialofetuin affinity chromatography with a lactose gradient and ion-exchange chromatography on DEAE-cellulose. In the last step, the activity is resolved into a major and minor species, designated ion-exchange-purified lectins I and II, respectively. Both lectin activities are reversibly inhibited by lactose and appear as single bands with identical mobilities on SDS-polyacrylamide gel electrophoresis. Lectin II was not obtained in sufficient quantities for further characterization. Lectin I is characterized by a functional requirement for reducing agents and sensitivity to N-ethylmaleimide, which suggests a role for an essential thiol in its activity. Subunit molecular weight determinations by SDS-polyacrylamide gel electrophoresis (12 000 +/- 1 000) and by gel filtration in 6 M guanidine . HCl (13 000 +/- 1 000), when compared with that obtained under native conditions on Bio-Rad P-60 gels (27 000 +/- 2 000), suggest a true Mr of 25 000 +/- 3 000 for the dimeric molecule. Amino acid composition data, when fitted to this molecular weight, lead to the tentative conclusion that the intact dimer is composed of two very similar but compositionally non-identical chains, designated by alpha and beta. While the only detectable N-terminal amino acid is tryptophan, the isoelectric focusing pattern of lectin I supports this heterodimeric structure. In addition, a lactose-sensitive hemagglutinating activity which can be separated from the lactose-blockade activity by affinity chromatography was also observed.

A specific decrease of the fluorescence depolarization of perylene in muscle membranes from mice with muscular dystrophy

The microviscosity of erythrocyte membranes and muscle microsomes from age matched 6-week old control mice REJ 129 Dy/Dy, and mice with muscular dystrophy REJ 129 DY/DY has been estimated by measuring the fluorescence depolarization of perylene. There was no difference between the erythrocyte membranes. The muscle microsomes from dystrophic animals had about 20% lower values than the controls. The temperature dependence indicated that a transition occurs in both sets of muscle microsomes, but the transition temperature was lower in the dystrophic microsomes. Cholesterol, phospholipid and triglyceride analyses of the membranes showed no difference between the erythrocyte membranes. The largest difference in the muscle microsomes was a two-fold increase in cholesterol level found in the dystrophic microsomes. No simple correlation could be made between the lipid analysis and the microviscosity measurements. Since the change in microviscosity is found in membranes isolated from the tissue primarily affected by the dy gene, we suggest that the change in microviscosity may be important in the development of the disease.

Correlation of membrane potential and potassium flux in the electroplax of electrophorus

Abstract 1. 1. Exposure of the innervated membrane to high external potassium concentrations or to carbamylcholine results in an initial rapid depolarization with a further gradual decline to a steady level which varies with the concentration of the depolarizing agent. 2. 2. The time course for repolarization following withdrawal of the depolarizing agent is slower than for depolarization. 3. 3. Depolarization of the innervated membrane by high external potassium concentrations is followed by a transitory increase in potassium efflux but a sustained influx. The influx and efflux increase to the same level when both membranes are simultaneously depolarized by high external potassium concentrations. 4. 4. Depolarization of the innervated membrane by the action of carbomylcholine at the synapses is followed by a transitory increase in potassium efflux and little change in the influx. 5. 5. Repolarization is followed by a less marked but more prolonged transitory decrease in the efflux.

Acetylcholine receptor regulation in L5 muscle cells is independent of increases in collagen secretion induced by ascorbic acid

Ascorbic acid is the active component of fetal brain extract that induces increased acetylcholine receptor (AChR) expression in L5 rat clonal muscle cell cultures. The induction of AChR expression, as determined by 125I-alpha-bungarotoxin binding, occurs with a delay of 20-25 h. We report that the delayed increase in AChR can be triggered by a 5-h exposure to ascorbic acid. These studies suggest that intermediary processes may be involved. Ascorbic acid treatment also causes a threefold increase in collagen secretion in L5 cultures by 3 h. The rapid increase in collagen secretion and the delayed induction of surface AChR suggested that there may be a link between these two responses. However, although bacterial collagenase eliminates secreted collagen, it had no effect on the increase in surface AChR. Thus, the ascorbic acid effect on elevating AChR expression is independent of its effect on collagen secretion.