Functional domains are specified to single-cell resolution in a Drosophila epithelium

Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P{GAL4} enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary (“stellate”) cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an “identified cell” system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.

Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution

We have developed an extremely sensitive technique, termed immuno-detection amplified by T7 RNA polymerase (IDAT) that is capable of monitoring proteins, lipids, and metabolites and their modifications at the single-cell level. A double-stranded oligonucleotide containing the T7 promoter is conjugated to an antibody (Ab), and then T7 RNA polymerase is used to amplify RNA from the double-stranded oligonucleotides coupled to the Ab in the Ab-antigen complex. By using this technique, we are able to detect the p185her2/neu receptor from the crude lysate of T6–17 cells at 10−13 dilution, which is 109-fold more sensitive than the conventional ELISA method. Single-chain Fv fragments or complementarity determining region peptides of the Ab also can be substituted for the Ab in IDAT. In a modified protocol, the oligonucleotide has been coupled to an Ab against a common epitope to create a universal detector species. With the linear amplification ability of T7 RNA polymerase, IDAT represents a significant improvement over immuno-PCR in terms of sensitivity and has the potential to provide a robotic platform for proteomics.

Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins.

Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes. However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry. Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells. We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1. Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP. Each mutant retains only one of the two excitation peaks of the wild-type protein. GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines. Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.

Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging.

The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2+, were simultaneously monitored in living cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isoproterenol- or forskolin-evoked cAMP accumulation (measured in vivo as an increased FlCRhR emission ratio) was reduced when cytosolic free Ca2+ concentration was elevated before, simultaneously with, or after cAMP activation. However, in REF-52 fibroblasts, Ca2+ neither prevented nor reduced forskolin-stimulated cAMP production. These results provide novel in vivo evidence for the Ca2+ modulation of the cAMP transduction pathway in C6-2B cells. The simultaneous microscopic measurement of cAMP and Ca2+ kinetics in single cells makes it now possible to study the regulatory interactions between these second messengers at the cellular and even the subcellular level.

Sex and the single cell: meiosis in yeast.

Recent studies of Saccharomyces cerevisiae have significantly advanced our understanding of the molecular mechanisms of meiotic chromosome behavior. Structural components of the synaptonemal complex have been identified and studies of mutants defective in synapsis have provided insight into the role of the synaptonemal complex in homolog pairing, genetic recombination, crossover interference, and meiotic chromosome segregation. There is compelling evidence that most or all meiotic recombination events initiate with double-strand breaks. Several intermediates in the double-strand break repair pathway have been characterized and mutants blocked at different steps in the pathway have been identified. With the application of genetic, molecular, cytological, and biochemical methods in a single organism, we can expect an increasingly comprehensive and unified view of the meiotic process.

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

Single cell analysis of cytokine gene coexpression during CD4+ T-cell phenotype development.

CD4+ T cells from alpha beta-T-cell receptor transgenic mice were analyzed for coexpression of cytokine mRNAs during phenotype development using a double-label in situ hybridization technique. T cells that produced cytokines in the primary response were a fraction of the activated population, and only a minority of the cytokine-positive cells coexpressed two cytokines. In secondary responses, frequencies of double-positive cells increased, although they remained a minority of the total. Of the cytokine pairs examined, interleukin (IL)-4 and IL-5 were the most frequently coexpressed. IL-4 and interferon gamma showed the greatest tendency toward segregation of expression, being rarely coexpressed after the primary stimulation. These data indicate that there is significant heterogeneity of cytokine gene expression by individual CD4+ T cells during early antigenic responses. Coexpression of any pairs of cytokines, much less Th1 and Th2 cytokines, is generally the exception. The Th0 phenotype is a population phenotype rather than an individual cell phenotype.

Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor

In this report, we have analyzed the human T cell repertoire derived in vivo from a single T cell precursor. A unique case of X-linked severe combined immunodeficiency in which a reverse mutation occurred in an early T cell precursor was analyzed to this end. It was determined that at least 1,000 T cell clones with unique T cell receptor-β sequences were generated from this precursor. This diversity seems to be stable over time and provides protection from infections in vivo. A similar estimation was obtained in an in vitro murine model of T cell generation from a single cell precursor. Overall, our results document the large diversity potential of T cell precursors and provide a rationale for gene therapy of the block of T cell development.

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

The yeast Cac1 protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres

Cac1p is a subunit of yeast chromatin assembly factor I (yCAF-I) that is thought to assemble nucleosomes containing diacetylated histones onto newly replicated DNA [Kaufman, P. D., Kobayashi, R. & Stillman, B. (1997) Genes Dev. 11, 345–357]. Although cac1Δ cells could establish and maintain transcriptional repression at telomeres, they displayed a reduced heritability of the repressed state. Single-cell analysis revealed that individual cac1Δ cells switch from transcriptionally “off” to transcriptionally “on” more often per cell cycle than wild-type cells. In addition, cac1Δ cells were defective for transcriptional silencing near internal tracts of C1–3A sequence, but they showed no defect in silencing at the silent mating type loci when analyzed by a reverse transcription–PCR assay. Despite the loss of transcriptional silencing at telomeres and internal C1–3A tracts, subtelomeric DNA was organized into nucleosomes that had all of the features characteristic of silent chromatin, such as hypoacetylation of histone H4 and protection from methylation by the Escherichia coli dam methylase. Thus, these features of silent chromatin are not sufficient for stable maintenance of a silent chromatin state. We propose that the inheritance of the transcriptionally repressed state requires the specific pattern of histone acetylation conferred by yCAF-I-mediated nucleosome assembly.

Graded transcriptional response to different concentrations of a single transactivator

Threshold mechanisms of transcriptional activation are thought to be critical for translating continuous gradients of extracellular signals into discrete all-or-none cellular responses, such as mitogenesis and differentiation. Indeed, unequivocal evidence for a graded transcriptional response in which the concentration of inducer directly correlates with the level of gene expression in individual eukaryotic cells is lacking. By using a novel binary tetracycline regulatable retroviral vector system, we observed a graded rather than a threshold mechanism of transcriptional activation in two different model systems. When polyclonal populations of cells were analyzed at the single cell level, a dose-dependent, stepwise increase in expression of the reporter gene, green fluorescent protein (GFP), was observed by fluorescence-activated cell sorting. These data provide evidence that, in addition to the generally observed all-or-none switch, the basal transcription machinery also can respond proportionally to changes in concentration of extracellular inducers and trancriptional activators.

Comparative T cell receptor repertoire selection by antigen after adoptive transfer: A glimpse at an antigen-specific preimmune repertoire

The low frequency of precursor cells specific for any particular antigen (Ag) makes it difficult to characterize preimmune T cell receptor (TCR) repertoires and to understand repertoire selection during an immune response. We have undertaken a combined adoptive transfer single-cell PCR approach to probe the Ag-specific preimmune repertoires of individual mice. Our strategy was to inject paired irradiated recipient mice with normal spleen cells prepared from individual donors and to compare the TCR repertoires subsequently selected during a CD8 response to a defined model Ag. We found that although some TCRs were shared, the TCR repertoires selected by mice receiving splenocytes from the same donor were not identical in terms of the TCRs selected and their relative frequencies. Our results together with computer simulations imply that individual mice express distinct Ag-specific preimmune TCR repertoires composed of expanded clones and that selection by Ag is a random process.

Real-time visualization of intracellular hydrodynamics in single living cells

Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements, CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high spatial resolution. Studies of the hydrodynamics in the microorganism Dictyostelium discoideum indicated the presence of a microscopic region near the plasma membrane where the mobility of water molecules is severely restricted. Modeling the transient hydrodynamics eventuated in the determination of cell-specific cytosolic diffusion and plasma membrane permeability constants. Our experiments demonstrate that CARS microscopy offers an invaluable tool for probing single-cell water dynamics.

Insulin Action on GLUT4 Traffic Visualized in Single 3T3-L1 Adipocytes by Using Ultra-fast MicroscopyV⃞

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20–30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5–7 min at 37°C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.