Single-cell Events Research Articles

Multiparametric evaluation of apoptosis: Effects of standard cytotoxic agents and the cyanoguanidine CHS 828

A multiparametric high-content screening assay for measurement of apoptosis was developed. HeLa cells and lymphoma U-937 cells were exposed to cytotoxic drugs in flat-bottomed optical microtiter plates. After incubation, the DNA-binding dye Hoechst 33342, fluorescein-tagged probes that covalently bind active caspases and chloromethyl-X-rosamine to detect mitochondrial membrane potential (MMP) were added. Image acquisition and quantitative measurement of fluorescence in a defined number of cells per well was performed using the automated image capture and analysis instrument ArrayScan. The usefulness of the assay was tested in cells exposed to standard cytotoxic drugs as well as in experimental cytotoxic cyanoguanidine CHS 828. A time- and dose-dependent activation of caspase-3, decrease in MMP, and increase in nuclear fragmentation and condensation were observed for the standard drugs, with the ability to correlate the parameters on a single cell basis. CHS 828 induced caspase-3 activation and reduction in MMP with modest changes in nuclear morphology. The method described was considered to be a rapid and information-rich apoptosis assay suitable both for correlating morphological and biochemical apoptotic events in single cells as well as for screening and evaluation of novel substances with apoptosis-inducing capabilities.

An Emerging Impedance Sensor Based on Cell-Protein Interactions: Applications in Cell Biology and Analytical Biochemistry

Electric cell-substrate impedance sensing (ECIS) is described for real time monitoring of cell behavior including attachment, motility, and cytotoxicity in a continuous fashion. This emerging technology subjects cells growing on microelectrodes to small ac electric fields. Cells interact differently with various proteins used to precoat the gold electrode. Initial intact extracellular matrix protein binding to cells is often mediated through adhesion receptors in the integrin family with fibronectin as the commonest coating protein. Upon the attachment and spreading of cells, the impedance increases because the cells act as insulating particles to restrict the current flow. An intriguing feature is the fluctuation in the measured impedance, which is always associated with living cells and persists even if the cell layer becomes fully confluent. This behavior is attributed to vertical motions or micromotions of the cells, an indication of cell viability and morphology change. ECIS presents a novel way to assess wound healing and serves as an alternative to animal testing for drug screening and toxicology studies. The size of the electrode can be reduced to 25–50 µm to open up an exciting possibility for monitoring single cell events. Microfabrication will allow up to several hundred different individual culture wells containing the detecting electrodes to be fabricated on a single chip. In this context, the methodology becomes a truly powerful and high throughput analytical device.

On the scents of smell in the salamander.

Our sense of smell is based on a remarkable chemical-detection system that possesses high sensitivity, broad discriminability and plastic, yet stable, function. Understanding how olfactory stimuli translate into perception is a problem of daunting complexity. How do odour-coding events in single cells correlate with emergent properties from the ensemble, and with behaviour? For comprehensive descriptions of neural function, analysis must extend from examination of how elemental principles relate to the function of the whole. The tiger salamander has long been used as an experimental model in studies of olfaction, enabling general questions about olfactory function to be approached.

Digital imaging microscopy of firefly luciferase activity to directly monitor differences in cell transduction efficiencies between AdCMVLuc and Ad5LucRGD vectors having different cell binding properties

The luciferase reporter gene incorporated into adenoviral vectors is very useful for monitoring viral transduction of different cell types or for comparing the transduction efficiency of different viral constructs of one cell type. Luciferase protein expression can be detected and quantified with very high sensitivity from whole cells or organ extracts. However, its disadvantages become obvious when aiming at evaluation of transduction events at the single cell level. The results obtained from whole cell extracts cannot be directly correlated to single cell events. In this paper direct cellular luciferase imaging using cell permeable luciferin substrates is applied for comparative analysis of cellular transduction events by two adenoviral vectors with different cell binding properties. Using digital imaging microscopy we show a more than ten-fold increase in transduction efficiency by Ad5LucRGD vectors versus AdCMVLuc vectors on human A549 cells.

Synchronized Spontaneous Ca2+ Transients in Acute Anterior Pituitary Slices

We investigated the organization of spontaneous rises in cytosolic free Ca2+ concentration ([Ca2+]i) due to electrical activity in acute pituitary slices. Real time confocal imaging revealed that 73% of the cells generated fast peaking spontaneous [Ca2+]i transients. Strikingly, groups of apposing cells enhanced their [Ca2+]i in synchrony with a speed of coactivation >1,000 microm/s. Single-cell injection of Neurobiotin or Lucifer yellow labeled clusters of cells, which corresponded to coactive cells. Halothane, a gap junction blocker, markedly reduced the spread of tracers. Coupling between excitable cells was mainly homologous in nature, with a prevalence of growth hormone-containing cells. We conclude that spontaneously active endocrine cells are either single units or arranged in synchronized gap junction-coupled assemblies scattered throughout the anterior pituitary gland. Synchrony between spontaneously excitable cells may help shape the patterns of basal secretion.

Transgenic maize plants by tissue electroporation.

In this paper, we describe the transformation of regenerable maize tissues by electroporation. In many maize lines, immature zygotic embryos can give rise to embryogenic callus cultures from which plants can be regenerated. Immature zygotic embryos or embryogenic type I calli were wounded either enzymatically or mechanically and subsequently electroporated with a chimeric gene encoding neomycin phosphotransferase (neo). Transformed embryogenic calli were selected from electroporated tissues on kanamycin-containing media and fertile transgenic maize plants were regenerated. The neo gene was transmitted to the progeny of kanamycin-resistant transformants in a Mendelian fashion. This showed that all transformants were nonchimeric, suggesting that transformation and regeneration are a single-cell event. The maize transformation procedure presented here does not require the establishment of genotype-dependent embryogenic type II callus or cell suspension cultures and facilitates the engineering of new traits into agronomically relevant maize inbred lines.

Observation of neuronal activity using real-time voltage-sensitive dye imaging

Sensory systems are confronted with the problem of taking “information” in the outside world and encoding and manipulating it in forms that can be used in the neuronal world. A major challenge is to document how the transition between these worlds takes place (transduction) and, once it has taken place, how the data are manipulated by neural circuits (integration). Since the brain is an intrinsically parallel device, carrying out many functions simultaneously, it would appear as important to record brain activity in a similarly parallel manner as to record events in single cells and membranes. Optical recording of neuronal events offers a first step toward thing to observe events that are distributed among the cells and processes of a neuronal network.In the sense of smell odors appear to be encoded by activity distributed across many neurons at each level of the system studied so far, from the sensory cells in the nose to the pyramidal cells in prepyriform cortex (for review see). Thus, to elucidate how the molecular properties of odorants are represented by neurons it is probably necessary to observe the patterns of distributed activation. The distribution of activity across many neuronal elements, in contrast to representing odor molecules by dedicated “labelled lines”, confers redundancy and fault tolerance on a system that is crucial for complex behaviors that underly survival for many animals species, as well as providing flexibility for being sensitive to large numbers of compounds.

Transient expression from microprojectile-mediated DNA transfer in pinus taeda

Transfer of plasmid DNA to Pinus taeda L. (loblolly pine) cotyledon cells by microprojectile bombardment has been demonstrated using beta-glucuronidase (GUS). GUS histochemical staining indicated active enzyme in localized centers (blue spots) 24 hours after bombardment. GUS expression declined during subsequent culture, but remained detectable in meristematic tissue 62 days post-bombardment, however, transgenic shoots were not recovered. Localized GUS expression events resulted predominantly from single-cell events containing one microprojectile. The staining pattern was complex, with indigo found both in the central target cell and in adjacent cells. Cellular damage sustained by GUS-positive cells ranged from undetectable to sufficiently extensive to cause cell death. Microprojectile bombardment provides a useful method to assay transient gene expression in loblolly pine and has potential for the production of transgenic plants in pine.

DNA Replication and differentiation in rat myoblasts studied with monoclonal antibodies against 5‐bromodeoxyuridine, actin, and α2‐macroglobulin

During the differentiation of skeletal muscle, mononucleate myoblasts proliferate, then stop replicating, spontaneously fuse, and express a large number of genes which encode the muscle phenotype. We have used monoclonal antibodies specific for 5-bromodeoxyuridine, myoactin, and equine alpha 2-macroglobulin to follow and establish the sequence of events that surround the transition from a replicating to a differentiating population. Triple-label immunofluorescence microscopy was used to visualize the changes in DNA synthesis, formation of myoactin fibers, and the cessation of endocytosis of alpha 2-macroglobulin that accompany myogenesis. Our results indicate that myoblasts cease actively endocytosing alpha 2-macroglobulin after stopping DNA synthesis but prior to fusion. Formation of myoactin fibers rarely occurs in mononucleate myoblasts and only in post-mitotic cells, but they are common in multinucleate myotubes. We suggest that the regulation of DNA synthesis is critical to normal myogenesis and that detection of incorporated BrdUrd by immunofluorescence, in conjunction with other antibodies and nucleic acid probes, is a convenient method with which to study and sequence the molecular events in single cells as they relate to the transition in DNA synthesis that accompanies differentiation.

Homogeneous immunoassay based on chemiluminescence energy transfer.

The chemiluminescent compound aminobutylethyl-isoluminol (ABEI) and its isothiocyanate derivative have been coupled to a range of haptens (progesterone, cyclic AMP, cyclic GMP) and protein antigens (IgG, C9). All the derivatives were chemiluminescent, immunologically active, and stable for more than six months. When the ABEI-labeled antigens bind to their respective fluorescein-labeled antibodies, there is a shift in the ratio of chemiluminescence at 460 nm (blue) to that at 525 nm (green). This nonradiative energy transfer was used to establish homogeneous immunoassays, which require no separation step. These assays were at least as sensitive as the conventional radioimmunoassays and could accurately measure substances in serum (i.e., for IgG, results correlated by r = 0.96 relative to those by 125I assay) and tissue extracts (cyclic AMP, r = 0.91 relative to 3H assay), and also were used to evaluate the kinetics of antibody-antigen binding. Chemiluminescence energy transfer provides a new method for quantifying ligand-ligand interactions in the 10(-15) to 10(-18) mol range without first separating bound and free ligand. This provides a unique opportunity to investigate chemical events in single cells and intact cells.