The cesium-induced delay in myoblast membrane fusion is accompanied by changes in cellular subfraction lipid composition.

We have recently demonstrated that the delay in myoblast membrane fusion induced by cesium is accompanied by changes in isolated membrane lipids (Santini, M.T., Indovina, P.L., Cantafora, A. and Blotta, I. (1990) Biochim. Biophys. Acta 1023, 298-304). In the present study, we have investigated changes in the lipid profile of total cell homogenates and microsomal membrane fractions during myoblast membrane fusion as well as the effects that addition of cesium may have on these lipid variations in order to try to understand the production and translocation of lipids during this myogenic process. The data presented here indicate that the lipid composition of cell homogenates and microsomes varies in a different manner from isolated plasma membranes during myogenic fusion. In addition, cesium affects, in a different manner, the normally-occurring lipid production and distribution which takes place in each subcellular fraction.     

Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes

Steady-state and time-resolved fluorescence spectroscopy has been used to obtain information on oxidation processes and associated dynamical and structural changes in model membrane bilayers made from single unilamellar vesicles (SUV's) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) mixed with increasing amounts of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC). The highly unsaturated arachidonoyl chain containing four double bonds is prone to oxidation. Lipid oxidation was initiated chemically by a proper oxidant and could be followed on line via the fluorescence changes of an incorporated fluorescent lipophilic fatty acid: 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BP-C11). The oxidation rate increases with an increasing amount of SAPC. Size measurements of different SUV's incorporated with a trace amount of a phosphatidylcholine analogue of BP-C11 using fluorescence correlation spectroscopy have demonstrated that an increase of lipid unsaturation results in smaller sized SUV's and therefore to a larger curvature of the outer bilayer leaflet. This suggests that the lipid-lipid spacing has increased and that the unsaturated fatty acyl chains are better accessible for the oxidant. Oxidation results in some characteristic physical changes in membrane dynamics and structure, as indicated by the use of specific fluorescence probes. Fluorescence measurements of both dipyrenyl- and diphenylhexatriene-labelled PC introduced in non-oxidised and oxidised DOPC-SAPC membranes clearly show that the microfluidity (local fluidity at the very site of the probes) significantly decreases when the oxidised SAPC content increases in the lipid mixture. A similar effect is observed from the lateral diffusion experiments using monopyrenyl PC in the same membrane systems: the lateral diffusion is distinctly slower in oxidised membranes.     

The calcium-dependent myoblast adhesion that precedes cell fusion is mediated by glycoproteins

Presumptive myoblasts from explants of chick embryo pectoral muscle proliferate, differentiate, and fuse to form multinucleate myotubes. One event critical to multinucleate cell formation is the specific adhesion of myoblasts before union of their membranes. In the studies reported here five known inhibitors of myotube formation--trifluoperazine, sodium butyrate, chloroquine, 1,10 phenanthroline, and tunicamycin--were tested for their effect on the Ca++-dependent myoblast adhesion step. The first four inhibitors of myotube formation do not perturb myoblast adhesion but rather block fusion of aggregated cells, which suggests that these agents perturb molecular events required for the union of the lipid bilayers. By contrast, tunicamycin exerts its effect by inhibiting the myoblast adhesion step, thereby blocking myotube formation. The effect of tunicamycin can be blocked by a protease inhibitor, however, which implies that the carbohydrate residues protect the glycoproteins from proteolytic degradation rather than participate directly in cell-cell adhesion. Whereas trypsin treatment of myoblasts in the absence of Ca++ destroys the cells' ability to exhibit Ca++-dependent adhesion, the presence of Ca++ during trypsin treatment inhibits the enzyme's effect, which suggests that myoblast adhesion is mediated by a glycoprotein(s) that has a conformation affected by Ca++. Finally, myoblast adhesion is inhibited by an antiserum raised against fusion-competent myoblasts. The effect of the antiserum is blocked by a fraction from the detergent extract of pectoral muscle that binds to immobilized wheat germ agglutinin, which again suggests that glycoproteins mediate Ca++-dependent myoblast adhesion.     

Phase behavior of isolated photoreceptor membrane lipids is modulated by bivalent cations

The phase behavior of isolated photoreceptor membrane lipids is further investigated by ³¹P-NMR, in view of earlier discrepant results [(1979) Biochim. Biophys. Acta 558, 330–337; (1982) FEBS Lett. 124, 93–99]. We present evidence that the discrepancy is due to bivalent cations. When resuspended in aqueous media at neutral pH in the absence of bivalent cations, the isolated photoreceptor membrane lipids largely adopt the bilayer configuration. However, upon addition of such cations (Ca²⁺ Mg²⁺) or when resuspended in their presence, the formation of other phases (hexagonal H_II, lipidic particles) results. The rate of this transition depends on cation concentration and temperature. The transition is not easily reversed by addition of EDTA. Implications with regard to photoreceptor membrane structure and function need further study.     

Changes in the surface membrane during myoblast fusion.

1. During fusion of chick-embryo myoblasts in culture, the surface membrane is affected as follows. Uptake of 2-aminoisobutyrate and 2-deoxyglucose, each of which is concentrated 20-fold relative to its concentration in the medium, is unaltered; uptake of alpha-methyl glucoside and choline (15 mM), each of which equilibrates relative to its concentration in the medium, approximately doubles. An approximate doubling also occurs in iodinatable surface protein (and in total protein) and in cell surface area as judged by light-microscopy. Adenylate cyclase (in the absence or the presence of fluoride) increases by more than 2-fold. 2. It is concluded that, during myoblast fusion cells increase in size, and this is reflected in an increased rate of simple diffusion; the rate of facilitated processes such as the uptake of amino acids and sugars, on the other hand, remains unaltered, though the activity of certain enzymes is increased. These results indicate that specific changes in the function of surface membrane occur during myoblast fusion in vitro.     

The diet-induced metabolic syndrome is accompanied by whole-genome epigenetic changes

Consuming a high-fat/high-fructose diet (HFD) starting at a young age leads to the development of obesity and to the progression of metabolic syndrome (MS). We are interested in the relationship between MS and DNA methylation as a mediator of the metabolic memory and the early appearance of these diseases in the progeny. To this end, Wistar rats were fed a HFD for 1 year, and every 12 weeks, biochemical analyses were performed. After 24 weeks, animals fed the HFD showed alterations related to MS such as elevated blood levels of fasting glucose, triglycerides, and insulin compared with their littermate controls. During the experimental period, the control females exhibited a 40 % lower 5-methylcytosine (5-mC) level compared to the control males. The HFD affected the 5-mC levels in males and females differently. The HFD induced a 20 % decrease in the 5-mC levels in males and a 15 % increase in females. We found that the HFD induces an early presentation of MS in the progeny of treated animals and that the DNA methylation was altered in the F₁ generation. The presentation of MS is positively associated with changes in the global percentage of 5-mC in the DNA.     

The kinetics of myoblast fusion

The kinetics of myoblast fusion were estimated using two complementary assays. Both utilized suspensions of fusion-competent cells, i.e. 48-52-h cultures of chick pectoral muscle grown in a low-calcium medium, thus minimizing contributions arising from cellular migration. One assay, designed to measure the onset of membrane contiguity, relies on the transfer of a lipid dye, diI-C18-[3], from labelled to unlabelled cells. The other assay, designed to estimate the kinetics of appearance of morphologically distinct multinucleate cells in suspension (myoballs), relies on enzymic dissociation of cellular aggregates followed by nuclear staining. The assays demonstrate significant membrane contiguity within 20-30 min after initiating the fusion process; however, the multinucleate myoball morphology does not appear for at least one additional hour.     

Effect of membrane curvature-modifying lipids on membrane fusion by tick-borne encephalitis virus

Enveloped viruses enter cells by fusion of their own membrane with a cellular membrane. Incorporation of inverted-cone-shaped lipids such as lysophosphatidylcholine (LPC) into the outer leaflet of target membranes has been shown previously to impair fusion mediated by class I viral fusion proteins, e.g., the influenza virus hemagglutinin. It has been suggested that these results provide evidence for the stalk-pore model of fusion, which involves a hemifusion intermediate (stalk) with highly bent outer membrane leaflets. Here, we investigated the effect of inverted-cone-shaped LPCs and the cone-shaped oleic acid (OA) on the membrane fusion activity of a virus with a class II fusion protein, the flavivirus tick-borne encephalitis virus (TBEV). This study included an analysis of lipid mixing, as well as of the steps preceding or accompanying fusion, i.e., binding to the target membrane and lipid-induced conformational changes in the fusion protein E. We show that the presence of LPC in the outer leaflet of target liposomes strongly inhibited TBEV-mediated fusion, whereas OA caused a very slight enhancement, consistent with a fusion mechanism involving a lipid stalk. However, LPC also impaired the low-pH-induced binding of a soluble form of the E protein to liposomes and its conversion into a trimeric postfusion structure that requires membrane binding at low pH. Because inhibition is already observed before the lipid-mixing step, it cannot be determined whether impairment of stalk formation is a contributing factor in the inhibition of fusion by LPC. These data emphasize, however, the importance of the composition of the target membrane in its interactions with the fusion peptide that are crucial for the initiation of fusion.     

Inhibition of myoblast fusion by bromoconduritol

It has recently been reported that the glucosidase I inhibitor, N-methyl-1-deoxynojirimycin (MDJN), inhibits myoblast fusion whereas the mannosidase inhibitor, 1-deoxymannojirimycin (ManDJN), has no effect on fusion. We now report that bromoconduritol, which is an active-site-directed covalent inhibitor of glucosidase II, also inhibits fusion at concentrations that have no effect on the plating efficiency or growth of rat L6 myoblasts. Significant inhibition of fusion was obtained at concentrations as low as 50 micrograms of bromoconduritol/mL, whereas inhibition of cell growth did not occur until concentrations of 250 micrograms/mL were reached. Rat L6 myoblasts were grown in the presence and absence of processing inhibitors and were surface labelled with 125I. Analysis of the iodinated proteins by two-dimensional gel electrophoresis demonstrated that a number of high-molecular-weight proteins (greater than 90,000) detected at the surface of control cells were absent from the surface of cells treated with MDJN or bromoconduritol. It is suggested that MDJN and bromoconduritol prevent the translocation of these proteins to the cell surface. The high-molecular-weight proteins detected at the surface of control cells were also detectable in ManDJN-treated cells, indicating that inhibition of N-linked complex oligosaccharide formation does not affect the translocation of these proteins to the myoblast cell surface.     

Characterisation of myogenic cell membrane: II. Dynamic changes in membrane lipids during the differentiation of mouse C2 myoblast cells

Proliferating mouse C2 myoblast cells resist haemagglutinating virus of Japan, Sendai virus (HVJ) mediated cell fusion. However, differentiating C2 cells can be induced to fuse by HVJ, suggesting that the rigid membrane of C2 cells changes during the differentiation. To investigate this phenomenon, changes in membrane lipids which affect fluidity were examined. Membrane cholesterol gradually decreased with the differentiation of C2 cells. However, spontaneous fusion to form myotubes and artificial fusion induced by HVJ were both inhibited when the level of cholesterol was prevented from falling in the cell membrane. The membranes of differentiating C2 cells contained more unsaturated fatty acids than those of proliferating cells. Thus, when differentiating C2 cells were treated with stearate (a saturated fatty acid), they failed to form myotubes and were insensitive to HVJ-mediated fusion. Whereas, if proliferating C2 cells were given linolenate (an unsaturated fatty acid), they became capable of HVJ-induced fusion. These results indicate that differentiating C2 cells change their fusion sensitivity by decreasing cholesterol, probably at the same time as they increase the unsaturated fatty acid content of the cell membrane.     

Localized and transient changes in plasma membrane fluidity during in vitro myoblast fusion: an 1H-NMR study.

High resolution proton NMR was used to study the cell surface molecular events which take place during in vitro myoblast differentiation and fusion. The CH3 and (CH2)n spectral signals were followed throughout in vitro myogenic development. The results show that although both the T1 and T2 relaxation times of the CH3 and (CH2)n groups are sensitive to the fusion process, T1 is the most sensitive. Both T1 of CH3 and (CH2)n increased before fusion indicating a higher degree of molecular motion and then returned to their original values. These results demonstrate how mobile lipid domains observed with proton NMR can be used to study the changes taking place during myoblast differentiation, particularly myoblast membrane fusion.     

Inhibition of myoblast fusion by lysosomotropic amines

Fusion of cultured chick embryo myoblasts was inhibited by treatment with several lysosomotropic amines. The concentrations required for half-maximal inhibition of fusion were approximately 2 μM for chloroquine, 30 μM for tributylamine, 3.2 mM for ammonium chloride, and 3.3 mM for methylamine. All the amines inhibited fusion appreciably at concentrations lower than those that reduced cell density. Both the rate and extent of fusion were affected by the amines, which had to be present for about 20 hr before the usual onset of fusion. Inhibition of fusion was reversible by transfer of inhibited cells to fresh medium. The amines did not cause accumulation of a nondialyzable inhibitor in the culture medium. Levels of creatine kinase increased by eight-fold or more between 18 and 65 hr in cultures treated with tributylamine or chloroquine, although this increase was not as pronounced as in control cultures. The increased creatine kinase activity in amine-treated cultures was due mainly to the BB and MB isozymes, with relatively little increase in the MM isozyme.     

Tandem events in myoblast fusion

Myoblasts, derived from primary chick pectoral muscle explants and grown on collagencoated culture dishes in a low calcium medium, are harvested with EDTA and are gently agitated in suspension. In the presence of calcium, the cells rapidly form easily dissociable aggregates which exclude fibroblasts. The apparent strength of adhesion increases with time until, under appropriate conditions, the myoblasts fuse in suspension to form multinucleate cells. The calcium-dependent dissociable aggregation shows optima for pH, temperature, calcium concentration, and culture age that closely parallel those observed for myotube formation measured with cells attached to tissue culture plates. We conclude from this marked correlation between the effects of these variables on myoblast aggregation and myotube formation that cell-cell adhesion is an integral part of myoblast fusion. Furthermore, we suggest that the formation of multinucleate cells is the result of a sequence of steps beginning with cell-cell recognition and adhesion, progressing to membrane union, and ultimately ending after subsequent morphologic changes producing the morphologies characteristic of multinucleate cells both in suspension and on tissue culture plates.     

The Wiskott-Aldrich syndrome protein (WASP) is essential for myoblast fusion in Drosophila

In higher organisms, mononucleated myoblasts fuse to form multinucleated myotubes. During this process, myoblasts undergo specific changes in cell morphology and cytoarchitecture. Previously, we have shown that the actin regulator Kette (Hem-2/Nap-1) is essential for myoblast fusion. In this study, we describe the role of the evolutionary conserved Wiskott-Aldrich syndrome protein that serves as a regulator for the Arp2/3 complex for myoblast fusion. By screening an EMS mutagenesis collection, we discovered a new wasp allele that does not complete fusion during myogenesis. Interestingly, this new wasp3D3-035 allele is characterized by a disruption of fusion after precursor formation. The molecular lesion in this wasp allele leads to a stop codon preventing translation of the CA domain. Usually, the WASP protein exerts its function through the Arp2/3-interacting CA domain. Accordingly, a waspDeltaCA that is expressed in a wild-type background acts as dominant-negative during the fusion process. Furthermore, we show that the myoblast fusion phenotype of kette mutant embryos can be suppressed by reducing the gene dose of wasp3D3-035. Thus, Kette antagonizes WASP function during myoblast fusion.     

Phagocytosis by human macrophages is accompanied by changes in ionic channel currents

The present study has shown that changes in ionic channel currents accompany the phagocytosis of particles by mononuclear phagocytes. The patch-clamp technique in the cell-attached configuration was applied to human monocyte-derived macrophages to measure the activity of single transmembrane ionic channels in intact cells. During such measurements, IgG-opsonized and non-opsonized latex particles were offered for phagocytosis under continuous video-microscopical observation. Single particles were presented to the phagocytes at a membrane location some distance from that of the patch electrode. After a lag period following particle attachment, enhanced inward and outward time-variant single channel currents coinciding with particle engulfment were observed. On the basis of current-voltage characteristics and membrane potential measurements, the outward-directed channels were identified as K+ channels. Phagocytosis was also accompanied by slow transient changes in background membrane currents, probably due to changes in the membrane potential of the phagocytosing cell. Phagocytosis of IgG-coated latex particles differed from phagocytosis of uncoated or albumin-coated particles by a shorter lag time between particle attachment and the onset of enhanced ionic channel activity.     

Changes in myoblast membrane electrical properties during cell-cell adhesion and fusion in vitro

Characteristic of the process of myogenesis are the changes in the composition and organization of the cell membrane. While poorly understood, these changes have biochemical and biophysical relevance. Recently, changes in molecular order of the myoblast membrane which accompany differentiation in vitro have been observed (Santini, M.T., Indovina, P.L. and Hausman, R.E. (1987) Biochim. Biophys. Acta 896, 19–25). To further investigate these cell fusion processes we have examined additional physical parameters: conductivity and permittivity of the myoblast membrane during differentiation which reflect the molecular arrangement of the membrane. The determination of these parameters is possible because in the radio frequency range suspensions of cells in an electrolyte buffer show a characteristic conductivity dispersion due to the interfacial polarization. An analysis of our experimental data based on a ‘single-shell’ model showed that conductivity and permittivity of the membrane of pre- and post-fusion myoblasts varied significantly and abruptly. The conductivity of the cell interior (cytosol) remained constant. We discuss the significance of the observed changes in these membrane parameters for myogenesis.     

The actin nucleator WASp is required for myoblast fusion during adult Drosophila myogenesis

Myoblast fusion provides a fundamental, conserved mechanism for muscle fiber growth. We demonstrate here that the functional contribution of Wsp, the Drosophila homolog of the conserved actin nucleation-promoting factor (NPF) WASp, is essential for myoblast fusion during the formation of muscles of the adult fly. Disruption of Wsp function results in complete arrest of myoblast fusion in all muscles examined. Wsp activity during adult Drosophila myogenesis is specifically required for muscle cell fusion and is crucial both for the formation of new muscle fibers and for the growth of muscles derived from persistent larval templates. Although Wsp is expressed both in fibers and individual myoblasts, its activity in either one of these cell types is sufficient. SCAR, a second major Arp2/3 NPF, is also required during adult myoblast fusion. Formation of fusion-associated actin 'foci' is dependent on Arp2/3 complex function, but appears to rely on a distinct, unknown nucleator. The comprehensive nature of these requirements identifies Arp2/3-based branched actin polymerization as a universal mechanism underlying myoblast fusion.     

Spontaneous myoblast fusion is mediated by cell surface Ca-dependent protein kinase(s)

Mononucleated myoblasts divide in vitro until they attain confluency and fuse, forming multinucleated myotubes. Fusion is an extracellular Ca2+-dependent process. We used for our studies an established line of skeletal myoblasts (L6) as well as a non-fusing Myo- alpha-amanitin-resistant mutant of this line (Ama102). Our results show that extracellular calcium at concentrations which elicit myoblast fusion activates the phosphorylation of a protein species of 48 kD, present at the surface of mononucleated myoblasts of the fusing wild type (L6). At fusion, as the cells become independent of the extracellular calcium concentration for their further differentiation, this activation can no longer be observed. In fusion inhibition experiments, where we used lowered calcium levels, the phosphorylation of the 48 kD protein band is clearly decreased. When the myoblasts are fed with standard medium, they fuse rapidly and the phosphorylation of the 48 kD species is markedly increased. The above-described phenomenon takes place at the cell surface and is completed in a short time. The use of Myo- mutant showed that it is developmentally regulated. In view of our results, it is reasonable to postulate that Ca2+-activated phosphorylation of the cell surface could be on the basis of spontaneous myoblast fusion.     

Inhibition of myoblast fusion by tunicamycin and pantomycin

The formation of myotubes by a continuous rat myoblast line, L6, can be inhibited by non-toxic concentrations of tunicamycin and pantomycin. The effect of tunicamycin, an inhibitor of UDP-N-acetyl-glucosamine: dolichol phosphate N-acetylglucosaminyltransferase, could be reversed by N-acetylglucosamine but not by mannose, glucose or UDP-N-acetylglucosamine.