Morphological characterization of actively fusing L6 myoblasts

We have characterized morphologically the surface of L6 myoblasts at the time of active cell fusion using transmission electron microscopy. Two subclones of the L6 line were used in these studies: the L6Cl55 line that fuses to form multinucleated syncytia and the NF44 non-fusing variant. Ultrastructural analysis revealed an electron-opaque material at localized points of cell-cell apposition in actively fusing L6Cl55 cells. This material may be transported by and secreted from smooth-surfaced cytoplasmic vesicles with an electron-dense core. In contrast to L6Cl55 cells, the electron-dense plaques were seen infrequently in cultures of the NF44 non-fusing variant. This previously unidentified substance may be associated with cell-cell recognition or adhesion, both necessary prerequisites for myoblast membrane fusion. Alternatively, the electron-dense plaques may be directly involved in the fusion event.     

Spinophilin stabilizes cell surface expression of alpha 2B-adrenergic receptors

The third intracellular (3i) loops of the alpha 2A- and alpha 2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the alpha 2A, alpha 2B, and alpha 2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three alpha 2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that alpha 2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the alpha 2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t(1/2) < or = 60 min) apical versus slower (t(1/2) = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the alpha 2-AR-interacting region (Sp151-483) by fusion with apically targeted p75NTR extended the half-life of alpha 2B-AR at the apical surface to approximately 3.6 h and eliminated the rapid phase (0-60 min) of alpha 2B-AR turnover on that surface. Furthermore, we examined alpha 2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp-/-) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-alpha 2B-AR was accelerated in mouse embryo fibroblasts derived from Sp-/- mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of alpha 2B-AR and presumably all three alpha2-AR subtypes at the surface of target cells and may act as a scaffold that could link alpha 2-ARs to proteins interacting with spinophilin via other domains.     

Identification of cytoskeletal components involved in attachment of L929 cells and macrophages to polystyrene

We have previously shown that lactoperoxidase (LPO) covalently coupled to polystyrene tissue culture flasks can be used to radioiodinate monolayer cell proteins that come into intimate contact with the LPO- polystyrene surface. These studies have now been extended to include a detailed examination of the class of iodinated polypeptides migrating with apparent molecular weights of 50,000 and 55,000 in SDS polyacrylamide gels. Whereas in cultured L929 cells the 55,000 band is predominantly iodinated, in thioglycollate-activated murine peritoneal macrophages the 55,000 and 50,000 bands are of equal intensity. It is possible that the marked degree of exposure of the 50,000 mol wt polypeptide to immobilized LPO is related to the unique strength of macrophages attachment. After labeling of both L929 cells and macrophages with immobilized LPO, all polypeptides in this molecular weight region were subjected to peptide mapping by simultaneous limited proteolysis and electrophoresis in a second SDS polyacrylamide slab gel. The results clearly show that the two major polypeptides in this region are identical within the limits of resolution of this technique. The 55,000 mol wt polypeptide can also be identified in Triton X-100 cytoskeletons from L929 cells after labeling with soluble LPO either before or after detergent lysis. We conclude that this cell surface polypeptide is in continuity with the cytoskeleton and is preferentially exposed to the substratum during attachment to polystyrene.     

Isolation of a monoclonal antibody viral polypeptide VP2 of simian virus 40

A stable hybridoma cell line, IIG8-203-2, that secretes a monoclonal antibody of the immunoglobulin subclass M was obtained by fusion of mouse myeloma cells with spleen cells of mice that had been immunized with the viral polypeptide VP2 of simian virus 40. The monoclonal antibody recognizes viral polypeptides that migrate with VP2 polypeptides in a sodium dodecyl sulfate-polyacrylamide gel. It also recognizes two intracellular polypeptides (29,000 and 37,000 daltons) in a detergent-insoluble fraction extracted 30 h after virus infection of TC7 cells.     

Role of Ca2+ and Ca2+-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca2+ transport into myoblasts. We have also demonstrated that a Ca2+-activated protease, CAF (mM), appears to relocate in response to the Ca2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

Rapid intracellular pathway gives rise to cell surface expression of the MHC class II-associated invariant chain (CD74)

In previous investigations, it had been shown that class II and associated invariant polypeptides are sorted to an endocytic route where transport is delayed. Invariant chain (Ii) is degraded in a post-Golgi compartment, presumably an endosomal vesicle, and only class II molecules emerge on the cell surface. By using a mAb against the extracytoplasmic domain of human Ii, we demonstrate, by electron microscopy and by cytofluorometry, surface expression of Ii on lymphoma cells and on human B lymphocytes. We examined surface expression of Ii upon inhibition by brefeldin A of intracellular transport from the endoplasmic reticulum to the Golgi stack. This treatment rapidly depletes the cell surface of Ii. In the subsequent absence of brefeldin A, Ii appears rapidly at the cell surface. Within 5 h, the previous level of surface Ii (sIi) is reconstituted. Chloroquine abrogates depletion of sIi by brefeldin A, apparently by inhibition of internalization of sIi. Because on its route to the cell surface Ii is not proteolytically digested, it was possible that Ii and associated class II molecules are not separated on this pathway. Immunochemical studies reveal that on the cell surface of a B lymphoma cell line a proportion of Ii and class II polypeptides are associated.     

Pattern of polypeptide synthesis in myoblast hybrids

We have examined cell hybrids derived from L6J1 rat myoblasts and A9 mouse fibroblastic cells for expression of the myogenic phenotype. Initial results showed that hybrid cells were no longer able to form myotubes and hence showed extinction of the myogenic phenotype. We then proceeded to characterize the pattern of protein synthesis in these cells using two-dimensional gel electrophoresis. Although we did detect extinction of synthesis of a small number of myoblast polypeptides in the hybrids these did not appear to be rat myoblast specific. Instead they correlated well with polypeptides lost upon viral transformation in another rat cell line. Analysis of the ability of parental cells and hybrids to grow in soft agar confirmed that both A9 cells and hybrids were more transformed than the parental L6J1 cells. The results are consistent with the interpretation that extinction of the ability to form myotubes is due to either transformation and/or a disrupted cell organization but is unlikely to be due to specific extinction of myoblast specific polypeptides, at least at the level detectable by 2D gel electrophoresis.     

Retinoic Acid Rapidly Decreases Phosphatidylinositol Turnover During Neuroblastoma Cell Differentiation

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line that can be induced to differentiate along the neuronal pathway by retinoic acid (RA). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H]inositol or [1(3)-3H]glycerol. No changes were observed in both [3H]inositol and [3H]glycerol uptake within 24 h of RA treatment. Decreased incorporation of the metabolic precursor into PI 4-monophosphate and PI 4,5-bisphosphate occurred within 1 h of RA treatment. No changes were seen in the specific radioactivity of the precursor pools up to 1 h of treatment with RA. Analysis of labeled PI metabolites from prelabeled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol content within 1 min of induction of LAN-1 cell differentiation. These findings constitute the earliest reported events in neuroblastoma cell differentiation.     

Differential turnover of methyl groups on methyl-accepting membrane proteins of irreversibly sickled erythrocytes

Methyl group turnover rates for specific methyl-accepting membrane proteins in intact irreversibly sickled cells (ISCs) have been determined. The turnover of methyl groups on all methyl acceptor membrane proteins carboxylmethylated in ISCs is not concerted but proceeds in an ordered sequence which is markedly different from that exhibited by unfractionated normal erythrocytes (AA). In ISCs methyl group turnover based on initial demethylation rate constants is most rapid for membrane polypeptides migrating in sodium dodecyl sulfate at 30,000-39,000 Da, 40,000-55,000 Da, polypeptides comigrating with cytoskeletal component band 4.1, and band 4.5. In contrast, initial methyl group turnover rates obtained after less than 20% of the total methyl groups are turned over in (AA) cells show most rapid demethylation rates for membrane polypeptides migrating at 40,000-55,000 Da, polypeptides comigrating with band 4.5, cytoskeletal components bands 2.1 and 4.1. Results also show significant differences between ISCs and most dense fractions from normal (AA) and nonsickle hemolytic anemias in the demethylation of cytoskeletal proteins, bands 2.1 and 4.1. These findings indicate qualitative differences in accessibility of methyl acceptor substrates to the methylating-demethylating enzyme activity in the cytosol of irreversibly sickled cells compared to discocytic control erythrocytes.     

An unexpectedly labile mitochondrially encoded protein is required for Mta expression

Maternally transmitted antigen (Mta) is a mouse major histocompatibility antigen recognized by cytotoxic T lymphocytes. A role for mitochondria in expression of this class I-like cell surface antigen has been previously established. We now show that a labile product of mitochondrial protein synthesis is required for Mta expression. Reexpression of Mta determinants after enzymatic removal occurred within 24 h, and the regeneration process was sensitive to chloramphenicol (CAP), a selective inhibitor of mitochondrial protein synthesis. Additionally, target cells treated with CAP for as little as 18 h showed diminished expression of Mta. The estimated half-life for Mtf products ranged from 6 to 15 h, less than the half-lives of known mitochondrial translation products. This suggests that the Mtf product is not generated by the normal turnover of stable mitochondrial respiratory proteins. Instead, these results indicate the existence of either labile unknown mitochondrially encoded peptides or a rapid turnover pathway for known mitochondrial products.     

Spontaneous fragmentation of several proteins in Drosophila pupae

Autoproteolysis is an essential activity in the expression of the entire genomes of a number of viruses. That is, new viruses can be produced only after large polyprotein products translated from the genome or from subgenomic mRNA degrade themselves to the polypeptides necessary for RNA replication or for the construction of new virus particles. We have recently shown that the major heat shock protein of Drosophila and a mouse cell line (70 kDa) also undergoes autoproteolysis with the production of specific patterns of smaller polypeptides. We show now that many other proteins in eucaryotic tissues also have a potential for self-degradation. We suggest that special coding regions in many genes may have important roles in both protein turnover and in the production of regulatory peptides.     

Immune reactive measles virus polypeptides on the cell's surface: turnover and relationship of the glycoproteins to each other and to HLA determinants

To better understand the mechanism(s) whereby antibody and complement and cytotoxic lymphocytes lyse infected cells, we studied the structure, interrelationship and turnover of measles virus polypeptides expressed on the cell's surface. Of the 6 major viral structural polypeptides, L, HA, P, NC, F, and M, found in purified virions or infected cells, only 2, the HA and F, resided on the surface of infected cells. The HA was present primarily in the form of a 160k dimer, and F was identified as a 64k polypeptide migrating distinct from other viral polypeptides. With reduction, the HA migrated as a 80k monomer, and F0, after cleavage, was found to be composed of a 42k nonglycosylated polypeptide, F1, and a 24k glycosylated protein, F2. The relationship between F0 and F1 and between the HA dimer and monomer was verified by tryptic peptide mapping. The turnover of HA and F from the cell's surface was 10 and 9 hr, respectively. However, in the presence of specific antibody after a marked loss of viral antigen from the surface, the turnover for HA and F was 15 and 12 hr, respectively. Despite being independent molecules, HA and F were closely linked, as they moved together (co-capped) over the plasma membrane when incubated with monospecific or monoclonal antibody. In contrast, neither HA nor F co-capped with the major histocompatibility antigens or with other host cell proteins, which indicates a separation between these host cell proteins and measles viral glycoproteins on the cell's surface.     

Cell surface proteins of rat myoblasts

During the course of cell fusion of a rat myoblast cell line, L6, the relative amount of a cell surface protein of molecular weight (MW) 200 000-250 000 (L200), detected on the two-dimensional gel electrophoretogram, increases to one of a few major cell surface proteins in the older culture in which myotubes are being formed. In the non-fusing variant (M3A) of L6, such a spot is not detectable. Instead, one protein of MW 90 000-100 000 (M100) is found in large quantities in M3A, but in a small amount in L6. These results suggest the possibility that these cell surface proteins, particularly L200, may play a key role in the development of muscle cells.     

Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions

In many epithelial tissues in the body (e.g. kidney distal nephron, colon, airways) the rate of Na(+) reabsorption is governed by the activity of the epithelial Na(+) channel (ENaC). ENaC activity in turn is regulated by a number of factors including hormones, physiological conditions, and other ion channels. To begin to understand the mechanisms by which ENaC is regulated, we have examined the trafficking and turnover of ENaC subunits in A6 cells, a polarized, hormonally responsive Xenopus kidney cell line. As previously observed by others, the half-life of newly synthesized ENaC subunits was universally short ( approximately 2 h). However, the half-lives of alpha- and gamma-ENaC subunits that reached the apical cell surface were considerably longer (t(12) > 24 h), whereas intriguingly, the half-life of cell surface beta-ENaC was only approximately 6 h. We then examined the effects of various modulators of sodium transport on cell surface levels of individual ENaC subunits. Up-regulation of ENaC-mediated sodium conductance by overnight treatment with aldosterone or by short term incubation with vasopressin dramatically increased cell surface levels of beta-ENaC without affecting alpha- or gamma-ENaC levels. Conversely, treatment with brefeldin A selectively decreased the amount of beta-ENaC at the apical membrane. Short term treatment with aldosterone or insulin had no effect on cell surface amounts of any subunits. Subcellular fractionation revealed a selective loss of beta-ENaC from early endosomal pools in response to vasopressin. Our data suggest the possibility that trafficking and turnover of individual ENaC subunits at the apical membrane of A6 cells is non-coordinately regulated. The selective trafficking of beta-ENaC may provide a mechanism for regulating sodium conductance in response to physiological stimuli.     

Borrelia Burgdorferi Upregulates the Adhesion Molecules E-selectin,P-selectin,ICAM-1 and VCAM-1 on Mouse Endothelioma Cells in vitro

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (≈50 h), bEnd3 cells were found to bind cells of a VLA-4⁺ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.     

A mutational hot-spot within an intron of the mouse beta 2-microglobulin gene.

beta 2-Microglobulin is the smaller, relatively non-polymorphic chain of class I major histocompatibility complex proteins. We have previously described a mutant mouse cell line which had been selected for loss of the class I thymus leukemia (TL) antigen and had concomitantly lost surface expression of H-2k antigens. Expression of class I antigens on the cell surface was restored by fusion to an antigenically distinct mouse lymphoma line, and the defect in the mutant was shown to be the loss of a functional beta 2-microglobulin gene. We now describe three additional mutants with the same phenotype, all selected for loss of TL but after different types of mutagenesis. All of these mutants have genomic rearrangements resulting in the absence of a functional beta 2-microglobulin gene. These data provide strong evidence for the requirement of beta 2-microglobulin for cell surface expression of the heavy chain of class I major histocompatibility complex proteins. We further show that the defects in at least one beta 2-microglobulin gene in each mutant cell line map to the same small DNA segment within the first intron. The breakpoints of these mutations define a hypermutable site within the mouse beta 2-microglobulin gene.     

Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.     

Preferential generation of human T cell hybrids with a tetraploid fusion partner

The factors determining successful derivation of human T lymphocyte hybrids are largely unknown. This report describes diploid and tetraploid clones of the T cell line CEM which were fused with either a human T cell line (Jurkat) or with peripheral blood lymphocytes (PBL). Fusions of all CEMR clones with the Jurkat cell line yielded hybrids at a very high frequency (1 X 10(-4)). Fusion of diploid clones of CEM with PBL yielded no hybrids, whereas fusion of tetraploid clones of CEM with PBL resulted in growth frequencies of 1 to 3 X 10(-6). Enumeration of hybrids immediately after fusion indicated that in all cases, fused cells represented 5 to 10% of the population. That the ability to yield viable hybrids after fusion was a characteristic of tetraploid cells was indicated by the finding that tetraploid variants of a diploid clone could also yield viable hybrids after fusion. Possible mechanisms for the difference in results generated with diploid and tetraploid cells, and characteristics of the hybrid cells generated, are also discussed.     

Generation of Cell Lines to Complement Adenovirus Vectors using Recombination-Mediated Cassette Exchange

Background

Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies.

Results

We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses.

Conclusions

RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression.