Intrinsic cell rheology drives junction maturation.

A fundamental property of higher eukaryotes that underpins their evolutionary success is stable cell-cell cohesion. Yet, how intrinsic cell rheology and stiffness contributes to junction stabilization and maturation is poorly understood. We demonstrate that localized modulation of cell rheology governs the transition of a slack, undulated cell-cell contact (weak adhesion) to a mature, straight junction (optimal adhesion). Cell pairs confined on different geometries have heterogeneous elasticity maps and control their own intrinsic rheology co-ordinately. More compliant cell pairs grown on circles have slack contacts, while stiffer triangular cell pairs favour straight junctions with flanking contractile thin bundles. Counter-intuitively, straighter cell-cell contacts have reduced receptor density and less dynamic junctional actin, suggesting an unusual adaptive mechano-response to stabilize cell-cell adhesion. Our modelling informs that slack junctions arise from failure of circular cell pairs to increase their own intrinsic stiffness and resist the pressures from the neighbouring cell. The inability to form a straight junction can be reversed by increasing mechanical stress artificially on stiffer substrates. Our data inform on the minimal intrinsic rheology to generate a mature junction and provide a springboard towards understanding elements governing tissue-level mechanics.

Genes encoding bile acid, phospholipid and anion transporters are expressed in a human fetal cardiomyocyte culture.

OBJECTIVES: To establish a human fetal cardiomyocyte culture and to investigate whether the genes that encode transporters that may influence influx or efflux of bile acids are expressed in human fetal cardiomyocytes. DESIGN: Laboratory study. SETTING: Imperial College London. SAMPLE: Six fetal hearts were obtained at the time of termination of pregnancy at 12-13 weeks of gestation and used to generate primary human cardiomyocyte cultures. METHODS: To confirm the presence of cardiomyocytes, the cells were incubated with monoclonal antibodies to sarcomeric alpha-actinin and anticardiac myosin heavy chain. Real-time reverse transcription polymerase chain reaction was used to establish whether transcripts of genes that may influence bile acid transport are present in the culture (NTCP, BSEP, MDR3, FIC1, MRP2, MRP3, OATP-A, OATP-C, OATP-D, OATP-E) and whether taurocholate administration alters messenger RNA (mRNA) expression. MAIN OUTCOME MEASURES: Relative mRNA expression of genes of interest. RESULTS: Real-time polymerase chain reaction demonstrated the presence of mRNA for BSEP, MDR3, FIC1, OATP-C, OATP-D and OATP-E in fetal heart. Four transcripts remained in the cardiomyocyte culture (BSEP, MDR3, FIC1 and OATP-D), and we demonstrated the influence of taurocholate on gene expression. CONCLUSIONS: We have developed an in vitro model of the fetal heart that may be used for studies of the cardiac effect of endobiotics, e.g. bile acids, or of specific agents that may be used to treat the mother or fetus in pregnancy.

Comparison of the arrhythmogenic effects of tauro- and glycoconjugates of cholic acid in an in vitro study of rat cardiomyocytes.

Obstetric cholestasis is associated with intrauterine death. In obstetric cholestasis, primary bile acids are more commonly conjugated with taurine than glycine, while glycoconjugates predominate in normal pregnancy. Using an in vitro model of rat cardiomyocytes, we compared the effect of tauro- and glycoconjugated cholate on cardiomyocyte rhythm, contraction amplitude and network integrity. We demonstrated that taurocholate had a more marked effect on all of these parameters, and the effects of the glycoconjugates were fully reversible while those of tauroconjugates were not. The increased proportion of tauroconjugated bile acids in obstetric cholestasis may contribute to the aetiology of the intrauterine death associated with the condition.

Dexamethasone and ursodeoxycholic acid protect against the arrhythmogenic effect of taurocholate in an in vitro study of rat cardiomyocytes.

OBJECTIVE: To establish whether the therapeutic agents ursodeoxycholic acid and dexamethasone protect cardiomyocytes from taurocholate-induced arrhythmias in an in vitro model. DESIGN: Laboratory study. SETTING: Imperial College London, Hammersmith Campus. SAMPLE: Neonatal rat cardiomyocytes. METHODS: Using scanning ion conductance microscopy, we measured the rate, rhythm, amplitude of contraction and calcium dynamics of ventricular myocytes from one to two day old rats. Cells were pre-incubated for 16 hours in dexamethasone (80 or 800 nM) or 0.1 mM ursodeoxycholic acid before adding taurocholate at different concentrations (0.3-4.5 mM). MAIN OUTCOME MEASURES: Changes in rate and amplitude of contraction, calcium dynamics and rhythm. RESULTS: Taurocholate at concentrations of up to 3 mM induces abnormal changes including reductions in rate, amplitude of contraction, abnormal calcium dynamics and dysrhythmias. Although dexamethasone had no immediate protective effect on these changes, pre-incubation with dexamethasone was protective. Ursodeoxycholic acid pre-incubation was protective at taurocholate concentrations up to 1 mM. CONCLUSION: The therapeutic agents dexamethasone and ursodeoxycholic acid appear protective against the arrhythmogenic effect of taurocholate on cardiomyocytes.

A novel light source for SICM-SNOM of living cells.

We have developed a novel light source for use in a scanning near-field optical microscope (SNOM or NSOM) based on a nanopipette whose distance from the sample surface is controlled using scanning ion conductance microscopy. The light source is based on the general principle of the chemical reaction between a fluorophore in the pipette and ligand in the bath, to produce a highly fluorescent complex that is continually renewed at the pipette tip. In these experiments we used fluo-3 and calcium, respectively. This complex is then excited with an Ar+ laser, focused on the pipette tip, to produce the light source. This method overcomes the transmission problem of more traditional SNOM probes and has been used to acquire simultaneous high-resolution topographic and optical images of biological samples in physiological buffer. A resolution of approximately 220 nm topographic and approximately 190 nm optical was determined through imaging fixed sea-urchin sperm flagella. Live A6 cells were also imaged, demonstrating the potential of this system for SNOM imaging of living cells.

Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: application to single virus-like particle entry into a cell.

We have developed a method for simultaneous recording of high-resolution topography and cell surface fluorescence in a single scan which we call scanning surface confocal microscopy. The resolution of the system allows imaging of individual fluorescent particles in the nanometer range on fixed or live cells. We used this technique to record the interaction of single virus-like particles with the cell surface and demonstrated that single particles sink into the membrane in invaginations reminiscent of caveolae or pinocytic vesicles. This method provides a technique for elucidating the interaction of individual viruses and other nanoparticles, such as gene therapy vectors, with target cells. Furthermore, this technique should find widespread application for studying the relationship of fluorescently tagged molecules with components of the cell plasma membrane.

Simultaneous measurement of Ca2+ and cellular dynamics: combined scanning ion conductance and optical microscopy to study contracting cardiac myocytes.

We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca2+ near the cell surface is possible while retaining the cell functionality.

Laminin-2/4 from human placenta is a better adhesion agent for primary keratinocytes than laminin-1 from EHS sarcoma.

A comparison of the adhesion of human primary keratinocytes to laminin-1 from murine EHS sarcoma and laminin-2/4 from human placenta was carried out using two methods, cell adhesion to substrates covered with the laminin isoforms, and interaction of keratinocytes from suspension with latex beads coated with the proteins. Laminin-2/4 was considerably more potent as a promoter of attachment of primary human keratinocytes than laminin-1 (and fibronectin), with increased attachment of cells correlating well with the number of latex bead binding sites. Only small cells of diameter of less than 20 microm bound more than 5 beads. Staining of keratinocytes with involucrin antibodies confirmed the existence of an inverse relationship between laminin-2/4-coated bead binding and differentiation.

The bile acid taurocholate impairs rat cardiomyocyte function: a proposed mechanism for intra-uterine fetal death in obstetric cholestasis.

Obstetric cholestasis is a liver disease of pregnancy that can be complicated by sudden, hitherto unexplained, intra-uterine fetal death. Because intra-uterine death occurs suddenly, and because fetal heart rate abnormalities have been reported in obstetric cholestasis, we hypothesized that intra-uterine death is caused by impaired fetal cardiomyocyte function, resulting in fetal cardiac arrest. Obstetric cholestasis is associated with raised levels of maternal and fetal serum bile acids, and we propose that these may alter cardiomyocyte function. It was not possible to investigate the effects of bile acids on the intact human fetal heart at a cellular level. Therefore we used the closest available model of fetal myocardium at term: a primary culture of neonatal rat cardiomyocytes in which cells beat synchronously and develop pacemaker activity. The effect of the primary bile acid taurocholate (0.3 mM and 3 mM) on cultures of single cardiomyocytes, each with its own independent rate of contraction, was a reversible decrease in the rate of contraction and in the proportion of beating cells (P < 0.001). Addition of taurocholate to a network of synchronously beating cells caused a similar decrease in the rate of contraction. Furthermore, the integrity of the network was destroyed, and cells ceased to beat synchronously. Taurocholate also resulted in altered calcium dynamics and loss of synchronous beating. These data suggest that raised levels of the bile acid taurocholate in the fetal serum in obstetric cholestasis may result in the development of a fetal dysrhythmia and in sudden intra-uterine death.

Hybrid scanning ion conductance and scanning near-field optical microscopy for the study of living cells.

We have developed a hybrid scanning ion conductance and scanning near-field optical microscope for the study of living cells. The technique allows quantitative, high-resolution characterization of the cell surface and the simultaneous recording of topographic and optical images. A particular feature of the method is a reliable mechanism to control the distance between the probe and the sample in physiological buffer. We demonstrate this new method by recording near-field images of living cells (cardiac myocytes).

Cell volume measurement using scanning ion conductance microscopy.

We report a novel scanning ion conductance microscopy (SICM) technique for assessing the volume of living cells, which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The technique can measure a wide range of volumes from 10(-19) to 10(-9) liter. The cell volume, as well as the volume of small cellular structures such as lamelopodia, dendrites, processes, or microvilli, can be measured with the 2.5 x 10(-20) liter resolution. The sample does not require any preliminary preparation before cell volume measurement. Both cell volume and surface characteristics can be simultaneously and continuously assessed during relatively long experiments. The SICM method can also be used for rapid estimation of the changes in cell volume. These are important when monitoring the cell responses to different physiological stimuli.

Matrigel increases the rate of split wound healing and promotes keratinocyte ;take' in deep wounds in rats.

The influence of matrigel, a mixture of the components of thebasement membrane, on the wound healing was studied in a modelof experimental wounds in rats. Matrigel was found to increasethe rate of epithelization of split-thickness wounds. The modelof deep wound was developed in which the host animal could notprovide enough migrating and proliferating keratinocytes tocover the wound area. The model is relevant to severe burns andinjuries in humans. When rat keratinocyte suspension wastransplanted into deep wounds, cell retention in the wound bedwas only observed if matrigel was added together with the cells.Increasing matrigel concentration in the wound was seen toenhance the rate of wound area coverage by the cells. Althoughthe process of healing seemed macroscopically normal, afterhistological screening of the biopsies cell in the wouldappeared as amorphous aggregates and tubules rather thenstratified epidermis.

Role of feeder cells in spreading and cytoskeleton organization of newborn rat keratinocytes.

We studied the effect of feeder cells (fibroblasts) and a mixture of the extracellular matrix components, Matrigel, on spreading and cytoskeleton organization of newborn rat keratinocytes (REK). REK formed lamellipodia on being plated together with feeder cells and on the Matrigel as a substrate whereas the same REK plated alone on a plastic surface formed filopodia. REK lamellipodia formation in co-cultures depended on the fibroblast addition time. Although conditioned medium from fibroblast cultures was not enough to induce lamellipodia, the extracellular matrix left after fibroblast removal was as effective as Matrigel. Our results indicate that lamellipodia formation seems to depend on the factor(s) secreted by fibroblasts and associated with the extracellular matrix.