The movement protein BC1 promotes redirection of the nuclear shuttle protein BV1 of Abutilon mosaic geminivirus to the plasma membrane in fission yeast.

In order to monitor their interaction and cellular localisation, the movement protein (MP; syn. BC1) and the nuclear shuttle protein (NSP; syn. BV1) of the geminivirus Abutilon mosaic virus (AbMV) were ectopically expressed in Schizosaccharomyces pombe cells, either alone or together under the control of an inducible promoter. For highest resolution, electron microscopy using freeze-fracture immunolabelling served to detect these proteins in situ. As expected from previous in planta and yeast experiments, NSP accumulated within the nuclei, whereas MP was targetted to the protoplasmic face of plasma membranes when expressed alone. Upon coexpression, NSP was localised at the plasma membranes, where it was strongly attached. These results support a model in which NSP transports viral DNA to the cell periphery to facilitate cell-to-cell movement of viral DNA within plants. In contrast to AbMV MP, no plant-specific protein seems to be necessary for the translocation of NSP to the plasma membrane.

Yeast two-hybrid systems confirm the membrane- association and oligomerization of BC1 but do not detect an interaction of the movement proteins BC1 and BV1 of Abutilon mosaic geminivirus.

Most of the plant begomoviruses use two proteins to transport their DNA from cell to cell, BV1 to shuttle it between nucleus and cytoplasm and BC1 to facilitate movement across plasmodesmata. In order to analyse their interaction for Abutilon mosaic geminivirus (AbMV) in yeast ( Saccharomyces cerevisiae), BC1 and BV1 genes were cloned into various plasmid vectors suitable for two-hybrid analysis. BC1 was fused to the binding domain (GBD) and BV1 to the activation domain (GAD) of the GAL4 transcription factor to check for interactions in the nucleus. Additionally, BC1 as well as BV1 were integrated into pMyr or pSos vectors to analyze protein binding at the plasma membrane using the CytoTraptrade mark system. Using freeze-fracture immuno-labelling (FreeFI), singly-expressed GBD:BC1 was localized at the plasma membrane although it was fused to a nuclear localization signal provided by the construct. GAD:BV1 was found in the nucleus of transformed cells as expected. Upon co-transformation of both constructs, cells grew poorly and exhibited symptoms of autolysis without any detectable level of GBD:BC1 or GAD:BV1, as revealed by FreeFI. In conclusion, both fusion proteins did not meet in the same compartment and appeared to be harmful to yeast if constitutively co-expressed. When expressed from pSos vector, BC1 induced the CytoTrap detection signal in the absence of pMyr indicating that BC1 protein alone is able to target the effector protein to the inner face of the plasma membrane. A mutated form of BC1 (DeltaBC1) lacking the previously identified membrane-binding domain was no longer able to auto-induce the CytoTrap signal cascade. Using DeltaBC1, an N-terminal, or a C-terminal third of BC1 revealed a homo-oligomerization of the C-terminal region of BC1 in two-hybrid analysis, but no interaction of BC1 with BV1.

Localizing the movement proteins of Abutilon mosaic geminivirus in yeast by subcellular fractionation and freeze-fracture immuno-labelling.

The movement proteins BC1 and BV1 of Abutilon mosaic geminivirus fused to glutathion-S-transferase (GST) and Flag-peptide were expressed in fission yeast (Schizosaccharomyces pombe) cells to analyse the fundamental intracellular distribution of these proteins in an eukaryotic cell in the absence of plant-specific factors. Most of BC1 protein sedimented rapidly after cell lysis and differential centrifugation. Using freeze-fracture immuno-labelling, the protein was detected in situ predominantly at plasma membranes and to a lower extent at cytoplasmic vesicles but not in the cytoplasm, the nuclei, or the mitochondria. Anti-BC1, anti-GST, and anti-Flag antibodies tagged smooth flecks only at the protoplasmic faces of the plasma membrane. The consequences of the BC1 behaviour for its use in two-hybrid analysis in yeast are discussed. In contrast, BV1 was detected mainly in the nucleus and partially in the cytoplasm but never associated with membranes.

Functional rescue of defective mutant connexons by pairing with wild-type connexons.

We have compared the functional and structural integrity of gap junction channels assembled from a Cx45 truncation mutant with those of gap junction channels assembled from wild-type (wt) Cx45 and Cx43. These channel-forming proteins are constitutively expressed in HeLa cells. The truncation mutant lacks the last 26 amino acids of the COOH-terminus, including nine serine phosphorylation sites that are associated with regulatory processes of these channels. We determined the presence of gap junction plaques in these cells with the immunogold freeze fracture technique, which showed that plaque formation is similar in all the clones investigated. Junctional permeability was probed with calcein transfer and flow cytometry analyses and junctional conductance was measured in cell pairs with double whole-cell patch-clamp techniques. For homotypic pairing only the truncated mutant did not form permeable channels. However, coupling was restored for heterotypic channels (pairing wtCx45- or wtCx43- with mutant-connexons), whose junctional communication was not different from that of the homotypic channels. Our results indicate that the presence of gap junction plaques does not warrant functional coupling and that heterotypic trCx45/wtCx45 channels can be regulated by the intact wtCx45 connexons. This dominant-positive effect is also operative when wtCx43 are paired with trCx45 connexons.

Supramolecular dynamics of gap junctions.

During the life cycle of a membrane protein its molecular structure may change and for aggregated proteins this process may be observed on the supramolecular level. Here we demonstrate that this is the case for gap junction channels which maintain cell-cell communication. Freshly synthesized connexins are integrated as hexamers (connexons) into the plasma membrane where they form plaques after pairing with connexons of an attached cell. We inhibited protein trafficking by applying the fungal metabolite brefeldin A (BFA), quantified cell-cell coupling by calcein transfer and fluorescence-activated flow cytometry, and examined the degradation and formation of gap junction plaques by indirect immunofluorescence and immunogold labeling. Under control conditions 50% of the detected plaques were ubiquitylated and less than 10% showed a two-dimensional crystalline packing. One hour after BFA reversal about 60% of the plaques were crystalline and ubiquitylation dropped to 14%. Label for ubiquitin was predominantly found on non-crystalline plaques. We, therefore, conclude that newly formed gap junction plaques are of crystalline morphology which changes to a pleomorphic structure when individual channels are modified during their aging process. This dynamic in plaque morphology correlates with channel inactivation and plaque ubiquitylation.

Gap-junctional coupling measured by flow cytometry.

A method is described which reliably quantifies the degree of intercellular communication via gap junctions by combining a dye-loading technique with fluorescence-activated flow cytometry. Our experiments expand former measurements of other groups by analyzing the time- and density-dependent onset of coupling with a fixed ratio of donor to recipient cells. The high sensitivity of this technique provides a better resolution than the microelectrode technique and allows the detection of small changes in gap-junctional coupling by examining a large number of cells in a single experiment. Suspended cells were loaded with the membrane-permeable dye calcein AM, which is intracellularly hydrolyzed by nonspecific esterases, and the resulting polyanionic calcein is thus trapped inside these donor cells. Gap junctions, however, are permeable for this fluorescent dye, as can be observed when suspended donor cells are added to recipient cells (i.e., monolayer cultures) in which case cell-cell contact is established within less than 60 min. In addition, one of these two cell populations can also be stained with a membrane-resident dye (e.g., DiI), which facilitates the identification of different cell populations (donors, recipients, and noncoupled cells) not only by epifluorescence microscopy but also by flow cytometry. Our analyses reveal that junctional coupling depends not only on the connexin type (homo- or heterotypic junction) but also on the origin (species) of the contacting cells (homo- or heterospecific contact). We confirm earlier reports in which homotypic-homospecific coupling was demonstrated with different techniques in connexin-transfected HeLa and RIN cells as well as in BICR/M1R(k) and 3T3/SV40 cells. In contrast to other publications, we show that a significant heterotypic-homospecific coupling between Cx40- and Cx43-HeLa transfectants can be resolved, whereas no coupling was detected for heterotypic-heterospecific contacts between Cx40-HeLa transfectants and the Cx43-expressing cell lines BICR/M1R(k), 3T3/SV40, and RIN.

Connexin transfection induces invasive properties in HeLa cells.

Gap-junctional coupling of tumor cells facilitates their invasion into normal tissue as was further investigated with connexin-transfected HeLa cells by an in vitro assay. Wild-type HeLa cells are coupling deficient and did not show invasive properties when multicell spheroids were confronted with precultured embryonic chicken heart fragments. After transfection with cDNA of Cx31, Cx40, or Cx43, these cells were homotypically coupled; Cx40- and Cx43-transfected HeLa cells also were heterotypically coupled with embryonic chicken heart cells in monolayer coculture. Transfected clones revealed invasive properties in the chicken heart in vitro assay. However, the pattern of invasion differed between these transfectants: After 4 days Cx43-transfected HeLa cells were found in the central part of heart spheroids; Cx40- and Cx31-transfected HeLa cells, however, did not invade the central core but were detected in the outer part of heart spheroids. We conclude that connexin expression supports invasion of tumor cells into normal tissue, but heterotypic gap-junctional coupling is not required for this process.

Characterization of the gap junction protein connexin37 in murine endothelium, respiratory epithelium, and after transfection in human HeLa cells.

Affinity-purified antibodies to oligopeptides derived from two different regions of the carboxyterminus and cytoplasmic loop or to the last 103 C-terminal amino acids of mouse connexin37 (Cx37) were used to characterize expression of this gap junctional protein in endothelium of several murine tissues. Cx37 was expressed in endothelium of large blood vessels in brain, liver, kidney, spleen, heart, and lung, but not in capillaries. In addition, weak Cx37 immuno-signals were observed in lung respiratory epithelium of small bronchi and in alveolar epithelial cells of bronchioli. The ratios of Cx37 protein to Cx37 mRNA in adult and embryonic kidney as well as skin were 29-303-fold larger than in lung, suggesting that Cx37 mRNA was translated at different efficiencies in kidney and skin versus lung. Cx37 protein was more abundant in embryonic kidney and lung than in the corresponding adult tissues. After differential centrifugation of plasma membrane fractions in sucrose gradients, we found that Cx37-containing gap junctions in lung were much smaller than Cx32 and Cx26 aggregates from liver. HeLa cells were transfected with mouse Cx37 cDNA. In these cells, mouse Cx37 protein was phosphorylated mainly at serine, less at tyrosine, and very little at threonine residues. Three conductance states were resolved at 110, 240, and 315 pS.

Dispersed and aggregated gap junction channels identified by immunogold labeling of freeze-fractured membranes.

An indirect immunogold labeling technique was applied to replicas of freeze-fractured membranes of rapidly frozen unfixed cells. The endogenous gap junction protein Cx43 of BICR/M1Rk rat mammary tumor cells was preferentially identified in quasi-crystalline gap junction plaques as were the transfected connexins Cx40, Cx43, and Cx45 in HeLa (human cervical carcinoma) cells. With this method we also detected contact areas with dispersed gap junction channels which are the only structural correlation for endogenous Cx45 in HeLa wild-type cells where no gap junction plaques exist. In double-transfected HeLa cells a colocalization of Cx40 and Cx43 was occasionally detected in quasi-crystalline gap junction plaques, whereas in contact areas with dispersed particles only one Cx type was present. Our results indicate that functional gap junction channels exist outside the quasi-crystalline plaques.

Production of tissue plasminogen activator (tPA) in two and three dimensionally growing cultures of Bowes melanoma cells.

Bowes melanoma cells synthesize more tissue plasminogen activator (tPA) in monolayer cultures than in multicell spheroids. Cellular production of tPA in these cells was measured during a cultivation period of 800 h. Without changing the cell culture assay, we were able to obtain monolayers, multilayers, and multicell spheroids (cell aggregates) by stirring microcarrier beads in 500-mL spinner flasks operated at 50 rpm. Thus, the medium conditions in the liquid were similar for cells in monolayers and in multicell spheroids. Probes for measurements of intracellular and extracellular parameters were taken from the same culture at distinct times; therefore, their variations during cultivation can directly be compared. Because cells were cultured in an unregulated (with regard to pH, glucose, etc.) spinner flask, their concentration was kept below 10(6) cells/mL, thus avoiding too fast and too severe depletion of oxygen and other medium factors. Nevertheless, the tPA productivity decreased from 8 ng/h/10(6) cells (monolayer) to 4 ng/h/10(6) cells (multicell spheroids with microcarrier nucleus, 800 mum diameter), matching the decrease of total cellular protein. Due to medium depletion, the cell cycle distribution changed from 45% to 68% G(1) cells in a characteristic way during growth of multicell spheroids. This is accompanied by changes in amino acids, glucose, lactate, and pH, which may account for the reduction of tPA productivity. (c) 1996 John Wiley & Sons, Inc.

Bioeffects of diagnostic ultrasound in vitro.

Biological effects induced by ultrasound were frequently reported for continuous wave (cw) mode. Thresholds for the onset of bioeffects of pulsed ultrasound, starting from diagnostic conditions, have not yet been defined by standardized in vitro models. We therefore investigated the effects of pulsed ultrasound on cultured cells using diagnostic ultrasound devices, a selfmade transducer and a sonochemical laboratory reactor tunable from pulsed diagnostic conditions to cw ultrasound. Additionally, we determined physical parameters of the ultrasonic field by different types of hydrophones. Sonochemical reactions and the effects induced by the ultrasonic fields in cultured cells indicated a threshold for bioeffects.

Disturbance of cellular calcium homeostasis by in vitro application of shock waves.

Extracorporeally generated shock waves used in lithotripsy of urinary and biliary stones exhibit tissue lesions in vivo and destroy or damage cells in vitro. The involvement of cavitation-generated free radicals in these harmful effects is discussed controversially. We investigated changes in cytoplasmic calcium concentration and intracellular calcium localization after shock-wave treatment of suspended cell cultures using flow cytometry and electron microscopy and present evidence for the disturbance of mitochondrial Ca2+ a sequestration and, therefore, for a chemically induced cell injury.

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha-type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

TNF receptors TR60 and TR80 can mediate apoptosis via induction of distinct signal pathways.

TNF membrane receptors are usually co-expressed in many tissues but their relative contribution to cellular TNF responses is for most situations unknown. In a TNF cytotoxicity model of KYM-1, a human rhabdomyosarcoma cell line, we recently demonstrated that each of the two TNFRs is on its own capable of inducing cell death. Here we show that both receptors are able to induce apoptosis, as revealed from a similar onset of DNA fragmentation and typical morphologic criteria. To obtain additional information about the signaling pathways involved in TR60- and TR80-induced programmed cell death, we have used a series of selective inhibitors of intracellular signaling molecules. The overall pattern emerging from these experiments provides strong evidence for distinct signal pathway usage of TR60 and TR80, indicating protein kinase(s)-mediated control of TR60 signaling and a tight linkage of TR80 to arachidonate metabolism. The subsequent establishment of KYM-1-derived cell lines that display TNFR selective resistance further supports a segregation of TR60 and TR80 signaling pathways for induction of apoptotic cell death. Moreover, these results demonstrate an independent control of the distinct signaling cascades used by TR60 and TR80. This allows a highly flexible regulation of a cellular TNF response in those cases in which both receptors contribute to overall TNF responsiveness.

Reduced cavitation-induced cellular damage by the antioxidative effect of vitamin E.

Fragmentation of human urinary and biliary stones by shock waves in extracorporeal lithotripsy is accompanied by tissue damage. Both the fragmentation as well as the side effects are often attributed to cavitation. The hazardous potential of cavitation is not only of a physical nature but also of a chemical nature, because of the generation of free radicals, e.g. .OH, .H and .O2. After the application of shock waves, we have demonstrated cavitation-generated free radicals in cell-free solutions and also in the surviving and intact suspended MGH-U1 cells by hydroethidine measurements. Under electron microscopical inspection, the same cells exhibited perinuclear cisternae, damaged mitochondria and numerous intracellular vacuoles. The contribution of free radicals to cell damage was investigated by reducing the vitamin E level in rats by a tocopherol free diet and by incubating L1210 cells in a tocopherol enriched medium. After 250 shock waves, ex vivo erythrocytes revealed a 75% increase in total cell disruption over cells from non-depleted rats. The in vitro experiments with L1210 cells exhibited a moderate protection by the addition of this scavenger of free radicals.

Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells.

Human HeLa or SkHep1 cells, defective in intercellular communication through gap junctions, were transfected with coding sequences of murine connexin40 (Cx40) and -43. The transfected cells were restored in gap junctional coupling as shown by 100-fold increased electrical conductance. When studied by the double whole-cell patch-clamp technique, Cx40 HeLa transfectants exhibited single channel conductances of gamma = 121 +/- 7 pS and gamma = 153 +/- 5 pS. They were voltage gated with an equivalent gating charge of z = 4.0 +/- 0.5 for a voltage of half-maximal inactivation U0 = 44 +/- 7 mV. The corresponding values of connexin43 (Cx43) HeLa transfectants are: gamma = 60 +/- 4 pS and gamma = 40 +/- 2 pS as well as z = 3.7 +/- 0.8 and U0 = 73 +/- 7 mV. Transfer of the dye Lucifer Yellow was always considerably lower in Cx40- than in Cx43-transfectants though their total junctional conductance was similar or even higher than for Cx43-transfectants. In order to characterize cell and tissue distribution as well as phosphorylation of connexin40 and -43 proteins, antibodies to C-terminal oligopeptides of these proteins were prepared and used for immunoblotting, immunoprecipitation, and immunofluorescence analysis of transfected cells where they exhibited the punctate pattern characteristic of gap junctions on contacting membranes. Phosphorylation of connexin40 was shown by immunoprecipitation from 32P-labeled, transfected SkHep1 cells. Analyses of protein distribution in tissues revealed that the amount of connexin40 detected in heart was higher than in lung which is the inverse of the level of connexin40 mRNA in these tissues, suggesting posttranscriptional control of expression. Connexin40 protein in adult mouse heart and skin is about 20-fold more abundant than in the corresponding embryonic tissue. Connexin43 in adult mouse heart appears to be more highly phosphorylated than in embryonic heart or in transfected human cells.

Biophysical characterization of gap-junction channels in HeLa cells.

HeLa cells seem not to be junctionally coupled when probed with techniques such as Lucifer yellow spreading and/or ionic coupling measured with three inserted microelectrodes. When investigated with double whole-cell patch-clamp measurements, HeLa cells in monolayer cultures were electrically coupled in 39% of the cases with very low transjunctional conductances (average one to five open channels). These gap-junction channels had a single-channel conductance gamma = 26 +/- 6 pS and were voltage-gated with an equivalent gating charge z = 3.1 +/- 1.5 for a voltage of half-maximal inactivation Uo = 49 +/- 10 mV. The voltage-dependent component represents only 31 +/- 8% of the total junctional conductance. The voltage-insensitive conductance is characterized by a residual open probability po (infinity) = 0.34 +/- 0.12, which corresponds to a ratio Gmin/Gmax = 0.50 +/- 0.12. Dissociation of monolayer cells into cell pairs yielded about 58% coupled cell pairs with no notably altered single-channel properties.

Beta-galactosidase activity in transfected Ltk- cells is differentially regulated in monolayer and in spheroid cultures.

We have investigated whether three-dimensional cultivation of cells to multicell spheroids influences the expression of a transfected gene. Ltk- cells (mouse fibroblasts, thymidine kinase negative) have been transfected with a bacterial lacZ gene which was coupled to a beta-actin promoter. The transfected cells synthesize beta-galactosidase, a cytoplasmic enzyme which can easily be stained for histology with 5-bromo-4-chloro-3-indoxyl beta-D-galactoside and for cytometry with fluorescein di-(beta-D-galactopyranoside). As we have shown with monolayer cells, beta-galactosidase is produced independently of cell density, medium condition, and cell cycle. In multicell spheroids, however, the portion of producing cells was reduced from approximately 98% to approximately 2% within a week. This reduction is also independent of cell density, medium condition, and cell cycle. Nonproducing multicell spheroid cells, however, regained their ability to synthesize beta-galactosidase within a few days when the cells were recultivated as monolayers. Since the lacZ gene was not lost, its expression might have been regulated by its beta-actin promoter. We, therefore, investigated whether the endogenous synthesis of beta-actin was similarly regulated. A correlation between the distinct reduction in beta-galactosidase-producing cells and filamentous or total actin concentration was not unequivocally observed.

Sensitivity of normal and malignant cells to shock waves.

We examined the cytotoxic effect of shock waves for primary (embryonic chick kidney and thigh muscle) and permanently growing normal and malignant cells (human, rat, and mouse) in suspension. To avoid the influence of different media, the cells were suspended in phosphate buffered saline and shock wave treated. In all cases the acute cytotoxic effect (measured by flow cytometry) was a function of the applied shock waves. The investigated cells differed in their LD 50 values which, however, do not reveal a general difference in sensitivity to shock waves for normal and malignant cells.