A chemical approach for the localization of membrane sites involved in lymphocyte activation.

The aldehyde groups formed on periodate oxidation of cell surface sialyl residues were used to insert a mitogenic site onto the lymphocyte membrane by attachment of biotin hydrazide or 2,4-dinitrophenyl hydrazine. The biotin- or 2,4-dinitrophenyl-conjugated cells were both agglutinated and stimulated when cultured with avidin or anti-2,4-dinitrophenyl antibody respectively. On the other hand, biotin or DNP-conjugated cells, modified via functional groups on the membrane proteins, were agglutinated but not stimulated when cultured with avidin or anti-DNP antibody respectively. Our results show that the specific interaction of a protein at the periodate oxidation site leads to blastogenesis.     

Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast

Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.     

Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting.

Human cells incubated with N-levulinoylmannosamine (ManLev) process this unnatural metabolic precursor into N-levulinoyl sialic acid (SiaLev), which is incorporated into cell surface glycoconjugates. A key feature of SiaLev is the presence of a ketone group that can be exploited in chemoselective ligation reactions to deliver small-molecule probes to the cell surface. A mathematical model was developed and tested experimentally to evaluate the prospects of using cell surface ketones as targets for covalent small-molecule drug delivery. We quantified the absolute number of ketone groups displayed on cell surfaces as a function of the concentration of ManLev in the medium. The apparent rate constants for the hydrolysis and disappearance of the cell surface conjugates were determined, as well as the apparent rate constant for the formation of covalent bonds with cell surface ketones. These values and the mathematical model confirm that chemoselective reactions on the cell surface can deliver to cells similar numbers of molecules as antibodies. Thus, cell surface ketones are a potential vehicle for a metabolically controlled small-molecule drug delivery system.     

Remodeling of the myoblast membrane accompanies development

We have prepared a library of cloned hybridomas that produce monoclonal antibodies reactive with the surface of E63 rat myoblasts. Using immunofluorescence analysis of antigens on single cells we have studied the expression of determinants at distinct stages of development. Conditions were established for quantitative photometry and were used to confirm the diversity in stage-specific expression that accompanies development. The remodeling of the myoblast membrane also involves stage-specific and transient changes in topography and aggregation of many antigens, and the period surrounding fusion is one of particular activity. The localization of antigens on the upper and attached surfaces of myogenic cells was often distinct, and quantitative and spatial stage-specific reorganizations of antigens differed with respect to these two surfaces. This polarity represents an additional level of complexity in the continuous remodeling of the muscle cell membrane. Comparisons of quantitative and topographic analyses of antigens on E63 cells with Rat-1 fibroblasts and developmentally defective (fu-) myoblasts indicate that the outer membranes of these nonmyogenic cells are distinct from differentiating myoblasts. One determinant, H36, is absent on Rat-1 cells and on all fu- lines tested, and undergoes interesting stage-specific changes in expression and topography.     

Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting

Human cells incubated with N-levulinoylmannosamine (ManLev) process this unnatural metabolic precursor into N-levulinoyl sialic acid (SiaLev), which is incorporated into cell surface glycoconjugates. A key feature of SiaLev is the presence of a ketone group that can be exploited in chemoselective ligation reactions to deliver small-molecule probes to the cell surface. A mathematical model was developed and tested experimentally to evaluate the prospects of using cell surface ketones as targets for covalent small-molecule drug delivery. We quantified the absolute number of ketone groups displayed on cell surfaces as a function of the concentration of ManLev in the medium. The apparent rate constants for the hydrolysis and disappearance of the cell surface conjugates were determined, as well as the apparent rate constant for the formation of covalent bonds with cell surface ketones. These values and the mathematical model confirm that chemoselective reactions on the cell surface can deliver to cells similar numbers of molecules as antibodies. Thus, cell surface ketones are a potential vehicle for a metabolically controlled small-molecule drug delivery system.     

Fusion Competence of Myoblasts Rendered Genetically Null for N-Cadherin in Culture

Myoblast fusion is essential to muscle tissue development yet remains poorly understood. N-cadherin, like other cell surface adhesion molecules, has been implicated by others in muscle formation based on its pattern of expression and on inhibition of myoblast aggregation and fusion by antibodies or peptide mimics. Mice rendered homozygous null for N-cadherin revealed the general importance of the molecule in early development, but did not test a role in skeletal myogenesis, since the embryos died before muscle formation. To test genetically the proposed role of N-cadherin in myoblast fusion, we successfully obtained N-cadherin null primary myoblasts in culture. Fusion of myoblasts expressing or lacking N-cadherin was found to be equivalent, both in vitro by intracistronic complementation of lacZ and in vivo by injection into the muscles of adult mice. An essential role for N-cadherin in mediating the effects of basic fibroblast growth factor was also excluded. These methods for obtaining genetically homozygous null somatic cells from adult tissues should have broad applications. Here, they demonstrate clearly that the putative fusion molecule, N-cadherin, is not essential for myoblast fusion.     

Rodent myoblast interactions with laminin require cell surface glycoconjugates but not laminin glycosyl groups

Laminin glycosyl groups are necessary for the spreading of murine melanoma cells which become attached to this glycoprotein. Laminin has been implicated in myogenesis but the potential role of its glycosyl groups in this process has not been examined. In this study we report the effects of the carbohydrate moieties of laminin on myoblast adhesion, spreading, and differentiation. Unglycosylated laminin from tunicamycin-treated cultures of a mouse cell line, M1536 B3, was used in the experiments. Glycosylated laminin from a murine tumor and from cultures of M1563 B3 cells served as controls. Cell binding experiments with C2C12 mouse myoblasts showed that the cells preferred a laminin-coated surface, compared to the uncoated plastic surface (nontissue culture wells). Myoblasts did not distinguish between glycosylated and unglycosylated laminin substrates. Both glycosylated and unglycosylated forms of laminin promoted myoblast growth and differentiation. In contrast, cells on uncoated plastic surfaces grew very slowly and did not further differentiate. The L6 rat myoblast response to glycosylated and unglycosylated laminin was the same. These results indicate that although rodent myoblasts in culture require a laminin substratum for spreading, growth, and differentiation on a proprietary plastic surface, laminin carbohydrates are not implicated in those cellular responses. In contrast, parallel studies using the lectin, Con A, indicate that cell surface glycoconjugates of myoblasts are implicated in the response of these cells to a laminin substratum.     

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti-genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.     

Changes in oleic acid oxidation and incorporation into lipids of differentiating L6 myoblasts cultured in normal or fatty acid-supplemented growth medium.

L6 myoblasts accumulate large stores of neutral lipid (predominantly triacylglycerol) when cultured in fatty acid-supplemented growth medium. No accumulation of neutral lipid was evident in myotubes (differentiated myoblasts) when treated similarly. Triacylglycerol accumulation was rapid and dependent on exogenous fatty acid concentration. Triacylglycerol content in myoblasts cultured in fatty acid-supplemented growth medium was approx. 3-fold higher than that in myotubes treated similarly and 2-3-fold higher than that in myoblasts cultured in normal growth medium. Incorporation studies using [I-14C]oleic acid showed that myoblasts and myotubes take up exogenous fatty acid at similar rates. However, cells cultured in fatty acid-supplemented growth medium remove more exogenous fatty acid than do cells cultured in normal growth medium. Over 90% of the incorporated label was found in phospholipid and triacylglycerol fractions in all situations studied. Myoblasts incorporated a more significant proportion (P less than 0.001) of label into triacylglycerol compared with that of myotubes. No differences in fatty acid oxidation rates were detected when differentiating L6 cells cultured in normal growth medium were compared with those cultured in fatty acid-supplemented growth medium. However, fatty acid oxidation rates were observed to increase 3-5-fold upon myoblast differentiation. We conclude that there is a marked change in the pattern of lipid metabolism when myoblasts (primarily triacylglycerol-synthesizing cells) differentiate into myotubes (primarily phospholipid-synthesizing cells). Understanding these changes, which coincide with normal muscle development, may be important, since a defect in this natural switch could explain the observed accumulation of lipid in muscle characteristic of some of the muscular dystrophies and other lipid-storage myopathies.     

Prostaglandin binding activity and myoblast fusion in aggregates of avian myoblasts

Myoblast aggregates provide a system for studying cell interactions which have several advantages over standard, stationary cultures. In gyrotory rotation, aggregate size can be controlled and is independent of cell migration. In muscle aggregates, fibroblasts are excluded, yet myoblast differentiation and fusion occur in a highly synchronous fashion. Specific PG binding occurs in chick or quail myoblast aggregates: in chick the peak of binding is at 35-36 hr. Aggregation is complete 16 hr before PG binding activity appears. This suggests either that gyrotory aggregation is not identical to myoblast recognition, or that PG binding activity occurs subsequent to myoblast recognition. Myoblast aggregates begin to release PG before 18 hr. The amount detected remains constant until binding begins at 34 hr when PG binding to the aggregates begins. Thus, both the release of PG and PG receptor activity are characteristics of the myoblasts and release of prostaglandin precedes appearance of the binding activity. As a first step in identifying the PG receptor and determining its appearance on the myoblast cell surface, we have prepared antisera against myoblast surfaces which blocks receptor-ligand interaction and have absorbed it against both peripheral and intrinsic membrane fractions. The results indicate that the PG receptor is a myoblast peripheral membrane macromolecule.     

Isolation and preliminary characterisation of DNA-protein complexes from the mitochondria of Saccharomyces cerevisiae

Four independent rat L6 myoblast cell lines have been selected in a single step for resistance to the cytotoxic effects of the lectin concanavalin A (conA). In contrast to parental wild-type myoblast lines, all of the variant clones are unable to undergo normal cellular differentiation to form multinucleated myotubes or biochemical differentiation to produce an increase in the specific activity of the muscle-specific enzyme, creatine phosphokinase (CPK). The correlation between lectin resistance and loss of fusion potential is very tight; clonal variation studies show that there is less than a 2.8×10^?8 chance that the two are not directly related. Membrane preparations from the conA-resistant myoblast lines incorporate significantly less GDP-[¹⁴C]mannose into the lipid intermediates of protein glycosylation than preparations from parental wild-type cells. Also, conversion of mannose label to fucose occurs in myoblasts and this pathway is more active in conA-resistant cells than wild-type cells. Reduced binding of labelled conA to the cell surfaces of variant myoblasts was observed which may result from alterations to membrane glycoprotein receptors. These studies suggest that mannosylated glycoproteins of the cell surface play a role in the development of the myotubes from myoblasts. Lectin-resistant myoblasts should be useful model systems for investigating what appears to be a pleiotropic mutation affecting the myogenesis process through membrane modifications.     

Fluorescence properties of labeled proteins near silver colloid surfaces

The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver island films. We first adsorbed a monolayer of biotinylated-BSA as a base that was used to capture labeled avidin molecules. For labeled avidin on silver island films, we observed an increase of the fluorescence intensity of between 18 and 80 with one-photon excitation and up to several hundredfold or larger with two-photon excitation. The probes were moderately more photostable in the presence of silver islands. There was also a dramatic decrease in the lifetimes with the amplitude-weighted values decreasing from 7- to 35-fold. The data suggest that these spectral changes are due to both increased rates of excitation near the metallic particles and increases in the rates of radiative decay. Because these silver island surfaces are very heterogeneous, we are hopeful that larger increases in intensity and photostability can be obtained for probes situated at an optimal distance from the ideal island surfaces.     

Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin

Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts induced to fuse. Additionally, we have compared the localization of syncytin with the localization of caveolin-3 and of myogenin, which are also involved in myoblast fusion and maturation. Syncytin was localized to areas of the cell membrane and to filopodial structures connecting myoblasts to each other and to myotubes. Weaker staining was present over intracellular vesicles and tubules. Caveolin-3 was detected in the sarcolemma and in vesicles and tubules in a subset of myoblasts and myotubes. The strongest staining occurred in multinucleated myotubes. Wide-field fluorescence microscopy indicated a partial colocalization of syncytin and caveolin-3 in a subset of myoblasts. Super-resolution microscopy showed such colocalization to occur in the sarcolemma. Myogenin was restricted to nuclei of myoblasts and myotubes and the strongest staining occurred in multinucleated myotubes. Syncytin staining was observed in both myogenin-positive and myogenin-negative cells. Antisense treatment downmodulated syncytin-1 expression and inhibited myoblast cell fusions. Importantly, syncytin-1 antisense significantly decreased the frequency of multinucleated myotubes demonstrating that the treatment inhibited secondary myoblast fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma.     

Cell surface proteins of Drosophila. II. A comparison of embryonic and ecdysone-induced proteins

The cell-surface proteins of Drosophila embryos at gastrula and myoblast fusion stages were characterized by radioiodination and two-dimensional gel electrophoresis. Over 13% of the cell surface proteins detected in gastrula embryos were not found in myoblast fusion stage embryos or in Drosophila embryonic cell line EH34A3 cells. Nearly 18% of the cell-surface proteins detected in myoblast fusion stage embryos were evident only at that stage. Embryonic cell-surface proteins were compared with cell-surface proteins from untreated EH34A3 cells and EH34A3 cells treated with 20-hydroxyecdysone, which induces cell aggregation and the expression of "new" proteins at the cell surface (D. F. Woods and C. A. Poodry, 1983, Dev. Biol. 96, 23-31). Only one of the proteins induced by ecdysone in EH34A3 cells was detected in the NP-40 soluble fraction of radioiodinated cell lysates, even after fractionation by lectin affinity chromatography and immunoprecipitation to enrich for putative ecdysone induced proteins. However, extraction of the NP-40 insoluble pellet of embryo cells revealed one additional protein that was present both in myoblast fusion stage embryos and hormone-treated culture cells. It was concluded that except for these two proteins, the cell-surface proteins induced in cultured cell lines by treatment with 20-hydroxyecdysone are not present in significant amounts in gastrula or myoblast fusion stage embryos.     

A role for the Ca2(+)-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis

The formation of multinucleate skeletal muscle cells (myotubes) is a Ca2(+)-dependent process involving the interaction and fusion of mononucleate muscle cells (myoblasts). Specific cell-cell adhesion precedes lipid bilayer union during myoblast fusion and has been shown to involve both Ca2(+)-independent (CI)2 and Ca2(+)-dependent (CD) mechanisms. In this paper we present evidence that CD myoblast adhesion involves a molecule similar or identical to two known CD adhesion glycoproteins, N-cadherin and A-CAM. These molecules were previously identified by other laboratories in brain and cardiac muscle, respectively, and are postulated to be the same molecule. Antibodies to N-cadherin and A-CAM immunoblotted a similar band with a molecular weight of approximately 125,000 in extracts of brain, heart, and pectoral muscle isolated from chick embryos and in extracts of muscle cells grown in vitro at Ca2+ concentrations that either promoted or inhibited myotube formation. In assays designed to measure the interaction of fusion-competent myoblasts in suspension, both polyclonal and monoclonal anti-N-cadherin antibodies inhibited CD myoblast aggregation, suggesting that N-cadherin mediates the CD aspect of myoblast adhesion. Anti-N-cadherin also had a partial inhibitory effect on myotube formation likely due to the effect on myoblast-myoblast adhesion. The results indicate that N-cadherin/A-CAM plays a role in myoblast recognition and adhesion during skeletal myogenesis.     

Transient involvement of gap junctional communication before fusion of newborn rat myoblasts

Heptanol-sensitive gap junction communication was characterized by the gap-FRAP method (fluorescence recovery after photobleaching) in confluent rat myoblasts developing in primary culture. Cell to cell dye diffusion was mainly restricted to a short period of the prefusion lag period and disappeared duringfusion promotion except between some myoblasts and myotubes. This short period of occurrence of gap junction communication might be transiently and partially involved during the first steps preparing the subsequent fusion, since treatment with an uncoupler (heptanol) reduced the formation of multinucleated myotubes. During subsequent steps, functional gap junctions are not involved between myoblasts in the process of fusing, but a possible secondary involvement for fusion of remaining myoblasts to newly-formed myotubes is discussed. These data, together with results from other authors, suggest a regulatory role of gap junction communication in development and fusion of skeletal muscle cells, by providing a pathway for exchanging small molecules from one myoblast to another.     

Use of biotinylated chitosan for substrate-mediated gene delivery

To improve transfection efficiency of nonviral vectors, biotinylated chitosan was applied to complex with DNA in different N/P ratios. The morphologies and the sizes of formed nanoparticles were suitable for cell uptake. The biotinylation decreased the surface charges of nanoparticles and hence reduced the cytotoxicity. The loading capacities of chitosan were slightly decreased with the increase of biotinylation, but most of the DNA molecules were still complexed. Using different avidin-coated surfaces, the interaction between biotinylated nanoparticles to the substrate may be manipulated. The in vitro transfection results demonstrated that biotinylated nanoparticles may be bound to avidin coated surfaces, and the transfection efficiencies were thus increased. Through regulating the N/P ratio, biotinylation levels, and surface avidin, the gene delivery can be optimized. Compared to the nonmodified chitosan, biotinylated nanoparticles on biomaterial surfaces can increase their chances to contact adhered cells. This spatially controlled gene delivery improved the gene transfer efficiency of nonviral vectors and could be broadly applied to different biomaterial scaffolds for tissue engineering applications.