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PubMed1997

F-actin disruption attenuates agonist-induced [Ca2+], myosin phosphorylation, and force in smooth muscle.

Cytochalasins B and D (at 10 microM) inhibited stress development induced by 1 microM carbachol in bovine tracheal smooth muscle by 55% and 90%, respectively. Glucose depletion was ineffective in inhibiting carbachol-induced contraction, indicating that inhibition of glucose transport was not the cause. Cytochalasin D-treated smooth muscle cells appeared collapsed, with spiky protrusions from the cell membrane. Deconvolution of fluorescent images of fluorescein isothiocyanate-phalloidin-labeled smooth muscle cells revealed concentrations of actin filaments near the cell periphery, including near the spiky protrusions. Cytochalasin B attenuated carbachol-induced intracellular Ca2+ concentration ([Ca2+]), especially the initial peak intracellular [Ca2+]. Cytochalasin B also attenuated carbachol-induced myosin light chain phosphorylation. However, when the myosin phosphorylation data were plotted against time-matched intracellular [Ca2+] data, the two relationships in control and cytochalasin B-treated smooth muscle were similar, suggesting that the changes in myosin phosphorylation could be explained by the changes in intracellular [Ca2+]. These results suggest that actin filaments in smooth muscle cells are dynamic and may be an integral component of Ca2+ regulation and/or signal transduction in receptor-coupled mechanisms.

Actins↗
PubMed2006

Secretory granule biogenesis in sympathoadrenal cells: identification of a granulogenic determinant in the secretory prohormone chromogranin A.

Chromogranin A (CgA) may be critical for secretory granule biogenesis in sympathoadrenal cells. We found that silencing the expression of CgA reduced the number of secretory granules in normal sympathoadrenal cells (PC12), and we therefore questioned whether a discrete domain of CgA might promote the formation of a regulated secretory pathway in variant sympathoadrenal cells (A35C) devoid of such a phenotype. The secretory granule-forming activity of a series of human CgA domains labeled with a hemagglutinin epitope, green fluorescent protein, or embryonic alkaline phosphatase was assessed in A35C cells by deconvolution and electron microscopy and by secretagogue-stimulated release assays. Expression of CgA in A35C cells induced the formation of vesicular organelles throughout the cytoplasm, whereas two constitutive secretory pathway markers accumulated in the Golgi complex. The lysosome-associated membrane protein LGP110 did not co-localize with CgA, consistent with non-lysosomal targeting of the granin in A35C cells. Thus, CgA-expressing A35C cells showed electron-dense granules approximately 180-220 nm in diameter, and secretagogue-stimulated exocytosis of CgA from A35C cells suggested that expression of the granin may be sufficient to restore a regulated secretory pathway and thereby rescue the sorting of other secretory proteins. We show that the formation of vesicular structures destined for regulated exocytosis may be mediated by a determinant located within the CgA N-terminal region (CgA-(1-115), with a necessary contribution of CgA-(40-115)), but not the C-terminal region (CgA-(233-439)) of the protein. We propose that CgA promotes the biogenesis of secretory granules by a mechanism involving a granulogenic determinant located within CgA-(40-115) of the mature protein.

Adrenal Glands↗
PubMed2005

Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells by biarsenical labeling.

Human immunodeficiency virus type 1 (HIV-1) Gag is the primary structural protein of the virus and is sufficient for particle formation. We utilized the recently developed biarsenical-labeling method to dynamically observe HIV-1 Gag within live cells by adding a tetracysteine tag (C-C-P-G-C-C) to the C terminus of Gag in both Pr55Gag expression and full-length proviral constructs. Membrane-permeable biarsenical compounds FlAsH and ReAsH covalently bond to this tetracysteine sequence and specifically fluoresce, effectively labeling Gag in the cell. Biarsenical labeling readily and specifically detected a tetracysteine-tagged HIV-1 Gag protein (Gag-TC) in HeLa, Mel JuSo, and Jurkat T cells by deconvolution fluorescence microscopy. Gag-TC was localized primarily at or near the plasma membrane in all cell types examined. Fluorescent two-color analysis of Gag-TC in HeLa cells revealed that nascent Gag was present mostly at the plasma membrane in distinct regions. Intracellular imaging of a Gag-TC myristylation mutant observed a diffuse signal throughout the cell, consistent with the role of myristylation in Gag localization to the plasma membrane. In contrast, mutation of the L-domain core sequence did not appreciably alter the localization of Gag, suggesting that the PTAP L domain functions at the site of budding rather than as a targeting signal. Taken together, our results show that Gag concentrates in specific plasma membrane areas rapidly after translation and demonstrate the utility of biarsenical labeling for visualizing the dynamic localization of Gag.

Arsenicals↗
PubMed2026

Cell cycle-dependent protein dynamics in budding yeast resolved by deconvolution of bulk proteomics.

The cell division cycle is characterised by oscillatory dynamics in regulatory mechanisms and biosynthesis, coordinated with genome replication and segregation. To understand these dynamics, quantitative cell cycle-dependent protein concentration data are essential. Unfortunately, accurately resolving cell cycle-dependent protein dynamics is challenging because single-cell proteomics is currently infeasible and bulk proteomics requires - inherently imperfect - cell synchronisation. Here, we developed a computational method to deconvolve cell cycle-dependent protein concentration dynamics and applied it to new budding yeast bulk proteome data. Key to this method was a yeast population model, parameterised with experimental cell cycle progression and volume growth data, for quantifying the desynchronisation in sampled populations. We performed deconvolution on 3272 proteins, using cross-validation to determine regularisation parameters, and identified 539 proteins with cell cycle-dependent dynamics. Many of these dynamics were consistent with known yeast biology and dynamic proteins were enriched for several metabolic process, extending previous observations and supporting the emerging picture of metabolic activity as varying substantially over cell cycle phases. We consider the generated cell cycle-resolved budding yeast proteome data a key resource.

Journal Article↗
PubMed2026

Endothelial cell-specific DNA methylation alterations in breast cancer.

DNA methylation alterations are well-established contributors to carcinogenesis, yet, in the tumor microenvironment (TME), patterns of lineage and cell-specific methylation alterations are not well understood. Single-cell DNA methylation profiling in the TME is limited by technical challenges and high costs. Here, we use bulk DNA methylation, cell type deconvolution (HiTIMED), and an interaction testing framework (CellDMC) to identify reproducible, computationally inferred lineage-specific epigenetic alterations in the TME supported by orthogonal data sources. Tumor endothelial cells (TECs), critical regulators of angiogenesis, vascular permeability, and immune cell trafficking, acquire structural and functional abnormalities that promote tumor growth. We hypothesize that TECs have altered DNA methylation compared with endothelial cells in non-tumor tissues. In genome-scale methylation data from discovery and validation datasets (tumor n = 1071; non-tumor n = 415), we identify and validate >4500 TEC-specific CpGs with altered methylation, many mapping to genes involved in angiogenesis and endothelial function. Integration with gene expression data indicates that TEC-specific methylation alterations may reprogram transcriptional networks controlling angiogenesis. High-resolution, cell lineage-specific epigenetic landscapes can be inferred from bulk methylation data, implicating TEC-specific DNA methylation alterations as potential drivers of cancer angiogenesis and vascular dysfunction and providing a framework for future mechanistic and translational studies of the tumor vasculature.

DNA Methylation↗
PubMed2001

Improved spatial discrimination of protein reaction states in cells by global analysis and deconvolution of fluorescence lifetime imaging microscopy data.

The deconvolution of fluorescence lifetime imaging microscopy (FLIM) data that were processed with global analysis techniques is described. Global analysis of FLIM data enables the determination of relative numbers of molecules in different protein reaction states on a pixel-by-pixel basis in cells. The three-dimensional fluorescence distributions of each protein state can then be calculated and deconvolved. High-resolution maps of the relative concentrations of each state are then obtained from the deconvolved images. We applied these techniques to quantitatively image the phosphorylation state of ErbB1 receptors tagged with green fluorescent protein in MCF7 cells.

Cell Line↗
PubMed2004

Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a D-octapeptide derivative.

Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5(+)) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 microg ml(-1)), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 microg ml(-1)). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer D-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted D-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC(50)], 4 microM) which chemosensitized AD/PDR5(+) to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC(50), 30 microM) and Cdr2p (IC(50), 2 microM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter Ben(R)p, or the FLC drug target lanosterol 14 alpha-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC(50), 1 microM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.

ATP-Binding Cassette Transporters↗
PubMed2025

Penalised regression improves imputation of cell-type specific expression using RNA-seq data from mixed cell populations compared to domain-specific methods.

Gene expression studies often use bulk RNA sequencing of mixed cell populations because single cell or sorted cell sequencing may be prohibitively expensive. However, mixed cell studies may miss expression patterns that are restricted to specific cell populations. Computational deconvolution can be used to estimate cell fractions from bulk expression data and infer average cell-type expression in a set of samples (e.g., cases or controls), but imputing sample-level cell-type expression is required for more detailed analyses, such as relating expression to quantitative traits, and is less commonly addressed. Here, we assessed the accuracy of imputing sample-level cell-type expression using a real dataset where mixed peripheral blood mononuclear cells (PBMC) and sorted (CD4, CD8, CD14, CD19) RNA sequencing data were generated from the same subjects (N=158), and pseudobulk datasets synthesised from eQTLgen single cell RNA-seq data. We compared three domain-specific methods, CIBERSORTx, bMIND and debCAM/swCAM, and two cross-domain machine learning methods, multiple response LASSO and ridge, that had not been used for this task before. We also assessed the methods according to their ability to recover differential gene expression (DGE) results. LASSO/ridge showed higher sensitivity but lower specificity for recovering DGE signals seen in observed data compared to deconvolution methods, although LASSO/ridge had higher area under curves than deconvolution methods. Machine learning methods have the potential to outperform domain-specific methods when suitable training data are available.

Humans↗
PubMed2005

Deconvolution of concentration recordings at live cell preparations via shape error optimization.

In many fields of science and engineering including several areas in analytical chemistry, deconvolution needs to be performed on measured data to extract meaningful information. This situation arises when a variable of interest has to be indirectly estimated from a measurable quantity that depends on this variable in some known manner. This dependence, called the "forward problem", has to be computationally undone to obtain the information sought from the experimental results. Solving this "inverse problem" requires deconvolution whenever the forward problem involves convolution. Despite its ubiquitous importance, however, performance of the methodologies used for deconvolution remains often unsatisfactory. An example is in bioanalytical applications where microsensing at live preparations is performed to obtain information on biological transport. It is in this context that a novel approach to solve inverse problems, shape error optimization, is proposed and tested in this work. The experimental paradigm addressed is in the area of multidrug resistance (MDR) in cancer that gives rise to passive and active drug efflux from cells. Doxorubicin (DOX) concentration is monitored with a carbon fiber microelectrode in vitro at close proximity to a monolayer of cells expressing MDR. The measured local concentration is the result of convolution of cellular efflux with the impulse response of diffusion in the extracellular medium. Hence, estimating DOX efflux, which is the biologically meaningful information, leads to a deconvolution problem. Performance of deconvolution via shape error optimization is compared with that of two conventional techniques: discrete Fourier transform and square error optimization. The results obtained are also applicable to other areas of science and engineering where deconvolution is commonly used for processing experimental data.

Algorithms↗
PubMed2006

Improved cell typing by charge-state deconvolution of matrix-assisted laser desorption/ionization mass spectra.

Robust, specific, and rapid identification of toxic strains of bacteria and viruses, to guide the mitigation of their adverse health effects and optimum implementation of other response actions, remains a major analytical challenge. This need has driven the development of methods for classification of microorganisms using mass spectrometry, particularly matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), that allows high-throughput analyses with minimum sample preparation. We describe a novel approach to cell typing based on pattern recognition of MALDI mass spectra, which involves charge-state deconvolution in conjunction with a new correlation analysis procedure. The method is applicable to both prokaryotic and eukaryotic cells. Charge-state deconvolution improves the quantitative reproducibility of spectra because multiply charged ions resulting from the same biomarker attaching a different number of protons are recognized and their abundances are combined. This allows a clearer distinction of bacterial strains or of cancerous and normal liver cells. Improved class distinction provided by charge-state deconvolution was demonstrated by cluster spacing on canonical variate score charts and by correlation analyses. Deconvolution may enhance detection of early disease state or therapy progress markers in various tissues analyzed by MALDI-MS.

Algorithms↗
PubMed1992

Evidence that androgen negative feedback regulates hypothalamic gonadotropin-releasing hormone impulse strength and the burst-like secretion of biologically active luteinizing hormone in men.

Testosterone injections or pharmacological amounts of dihydrotestosterone infused in men, and androgen-secreting tumors in women, can suppress plasma LH bioactivity assessed in an in vitro rat Leydig-cell bioassay. However, such observations do not define the physiological nature of endogenous androgen feedback actions on the hypothalamo-pituitary axis. To explore the feedback role of endogenous androgen on the male gonadotropic axis, we used a potent, selective, nonsteroidal competitive antagonist of the androgen receptor, flutamide HCl. Eight young men (ages 21-30) each received flutamide (750 mg orally daily x 3 days) and placebo and underwent blood sampling at 10-min intervals for 28 h, the last 2 h of which included two consecutive iv pulses of GnRH (10 micrograms). Plasma bioactive LH concentrations were measured in the rat Leydig cell bioassay. Deconvolution analysis was used to evaluate the number, amplitude, mass, and duration of bioactive LH secretory bursts and simultaneously estimate the half-life of endogenous LH. Flutamide treatment increased mean plasma bioactive LH concentrations from 27 +/- 2.3 to 54 +/- 9.1 IU/L (P = 0.018). Increased LH concentrations were achieved by a significantly amplified mass of LH secreted per burst, which rose from 14 +/- 1.8 (control) to 24 +/- 2.8 (flutamide) IU/L distribution volume. The amplitude (maximal secretion rate) of bioactive LH release episodes also increased from 1.3 +/- 0.21 (control) to 2.2 +/- 0.29 (flutamide) IU/L/min. These responses were specific, since flutamide did not influence bioactive LH half-life [49 +/- 6.5 (control) vs 52 +/- 4.1 (flutamide) min], LH secretory burst duration, frequency, or interburst interval. The total 24-h production rate of bioactive LH rose significantly from 310 +/- 35 (control) to 570 +/- 82 (flutamide) IU/L.day. In contrast, no features of LH secretory bursts evoked by exogenous GnRH pulses were altered significantly by antiandrogen. In summary, in vivo blockade of endogenous androgen negative feedback actions in normal men selectively amplifies the mass and amplitude of bioactive LH secretory bursts without altering their number or duration, or the half-life of LH, or the amount of LH released in response to exogenous GnRH. Therefore, we infer that in the steroid milieu of normal men endogenous androgen acting via the androgen receptor can negatively regulate hypothalamic GnRH stimulus strength, and hence the rate and mass of biologically active LH secretion in vivo.

Activity Cycles↗
PubMed1993

Evidence for attenuation of hypothalamic gonadotropin-releasing hormone (GnRH) impulse strength with preservation of GnRH pulse frequency in men with chronic renal failure.

To investigate the nature of putative disturbances in pulsatile gonadotropin (LH) secretion in men with chronic renal failure, we undertook blood sampling at 10-min intervals for 24 h in 9 hemodialysis-dependent uremic men and 16 community- and age-matched controls. Serum LH concentrations were measured in a 2-site immunoradiometric assay, which does not cross-react with free alpha or LH beta-subunit and correlates well with an in vitro Leydig cell bioassay. Deconvolution analysis was applied to calculate the number, amplitude, mass, and duration of spontaneous LH release episodes and simultaneously estimate the half-life of endogenous LH in each subject. We observed that: 1) the estimated half-life of immunoradiometric LH removal from plasma averaged 103 +/- 11 min in normal and 207 +/- 29 min in uremic men (P < 0.01); 2) the number of LH secretory bursts was slightly higher in uremic than healthy men (e.g. 20 +/- 2.2 vs. 15 +/- 1.0 secretory bursts/24 h, respectively; P = 0.05); 3) the mass of LH secreted per burst was approximately 50% lower in chronic renal failure than in health, namely 1.4 +/- 0.18 vs. 2.8 +/- 0.42 IU/L (P < 0.01); 4) the decrease in the mass of LH secreted per burst was not due to a decline in LH secretory burst amplitude, but rather an attenuation of LH secretory burst duration (4.8 +/- 0.35 min in uremic vs. 11 +/- 1.3 min in normal men; P < 0.001); 5) the mean 24-h serum immunoradiometric LH concentration was significantly higher in uremia at 5.7 +/- 0.68 vs. 3.6 +/- 0.41 IU/L in controls (P = 0.017); and 6) serum estradiol concentrations were increased in uremia, but total and free testosterone concentrations did not differ significantly between the two subject groups. In response to synthetic GnRH (10 micrograms, administered iv after the 24-h basal sampling period), the mean mass of immunoradiometric LH released within each calculated LH secretory burst was similar in uremic (n = 8) and normal (n = 21) individuals. We conclude that uremia is accompanied by a specific defect in the pulsatile mode of LH secretion, which is marked by an abbreviation of LH secretory burst duration and a consequent fall in the mass of LH secreted per spontaneous release episode. There is no overall decline in LH secretory pulse frequency or gonadotroph responsiveness to a submaximally effective dose of exogenous GnRH. Such findings are consistent with diminished hypothalamic GnRH impulse strength.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed2003

Expression deconvolution: a reinterpretation of DNA microarray data reveals dynamic changes in cell populations.

Cells grow in dynamically evolving populations, yet this aspect of experiments often goes unmeasured. A method is proposed for measuring the population dynamics of cells on the basis of their mRNA expression patterns. The population's expression pattern is modeled as the linear combination of mRNA expression from pure samples of cells, allowing reconstruction of the relative proportions of pure cell types in the population. Application of the method, termed expression deconvolution, to yeast grown under varying conditions reveals the population dynamics of the cells during the cell cycle, during the arrest of cells induced by DNA damage and the release of arrest in a cell cycle checkpoint mutant, during sporulation, and following environmental stress. Using expression deconvolution, cell cycle defects are detected and temporally ordered in 146 yeast deletion mutants; six of these defects are independently experimentally validated. Expression deconvolution allows a reinterpretation of the cell cycle dynamics underlying all previous microarray experiments and can be more generally applied to study most forms of cell population dynamics.

Cell Cycle↗
PubMed2003

Accessibility of nuclear chromatin by DNA binding polyamides.

Pyrrole-imidazole polyamides bind DNA with affinities comparable to those of transcriptional regulatory proteins and inhibit the DNA binding activities of components of the transcription apparatus. If polyamides are to be useful for the regulation of gene expression in cell culture experiments, one pivotal issue is accessibility of specific sites in nuclear chromatin. We first determined the kinetics of uptake and subcellular distribution of polyamides in lymphoid and myeloid cells using fluorescent polyamide-bodipy conjugates and deconvolution microscopy. Then cells were incubated with a polyamide-chlorambucil conjugate, and the sites of specific DNA cleavage in the nuclear chromatin were assayed by ligation-mediated PCR. In addition, DNA microarray analysis revealed that two different polyamides generated distinct transcription profiles. Remarkably, the polyamides affected only a limited number of genes.

Alkylation↗
PubMed1999

A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens.

Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.

Antigens↗
PubMed1998

Stealth ryanodine-sensitive Ca2+ release contributes to activity of capacitative Ca2+ entry and nitric oxide synthase in bovine endothelial cells.

1. The involvement of ryanodine-sensitive Ca2+ release (RsCR) in bradykinin (Bk)-induced Ca2+ release, capacitative Ca2+ entry (CCE) and nitric oxide synthase (NOS) activation was assessed in freshly isolated bovine coronary artery endothelial cells. 2. Using deconvolution microscopy fura-2 was found throughout the whole cytosol, while the cell membrane impermeable dye FFP-18 was exclusively in the cell membrane. Thus, perinuclear ([Ca2+]pn) and subplasmalemmal Ca2+ concentration ([Ca2+]sp) were monitored using fura-2 and FFP-18. 3. Inhibition of Na+-Ca2+ exchange by lowering extracellular Na+ concentration augmented the Bk-induced [Ca2+]pn signal in Ca2+-free solution. This effect was abolished when RsCR was prevented with 25 micromol l-1 ryanodine, while inhibition of RsCR had no effect on Bk-induced increase in [Ca2+]pn without inhibition of Na+-Ca2+ exchange. 4. Initiating RsCR by 200 nmol l-1 ryanodine increased [Ca2+]sp, while [Ca2+]pn remained constant. However, when Na+-Ca2+ exchange was prevented, ryanodine was also able to elevate [Ca2+]pn. 5. Blockage of RsCR diminished Ca2+ extrusion in response to stimulation with Bk in normal Na+-containing solution. 6. Inhibition of RsCR blunted Bk-activated CCE, while inhibition of Na+-Ca2+ exchange during stimulation enhanced CCE. 7. Although direct activation of RsCR failed to activate NOS, inhibition of RsCR diminished the effect of ATP and Bk on NOS, while the effect of thapsigargin remained unchanged. 8. These data suggest that during stimulation subplasmalemmal RsCR occurs, which contributes to the activities of CCE and NOS. Thus, the function of the subplasmalemmal Ca2+ control unit must be extended as a regulator for CCE and NOS.

Animals↗
PubMed2001

High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells.

High-resolution, fluorescence deconvolution (DV) microscopy was implemented to obtain a detailed view of the organization and structural composition of gap junctions assembled from one or two different connexin isotypes in live and fixed cells. To visualize gap junctions, the structural protein components of gap junction channels, the connexin polypeptides alpha1(Cx43), beta1(Cx32), and beta2(Cx26), were tagged on their C-termini with the autofluorescent tracers green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants. Tagged connexins were expressed in transiently transfected HeLa cells. Comprehensive analysis including dye-transfer analysis demonstrated that the tagged connexins trafficked, assembled, and packed normally into functional gap junction channel plaques. Such gap junction plaques were examined by single, dual, and triple-color DV microscopy. High-resolution images and three-dimensional volume reconstructions of gap junction plaques were obtained by this technique, which revealed several new aspects of gap junction structure. Specifically, the studies demonstrated that the mode of channel distribution strictly depends on the connexin isotypes. Here we present such images, and volume reconstructions in context with images obtained by other light, and electron microscopic techniques, such as laser scanning confocal, conventional wide-field fluorescence, thin section, and freeze-fracture electron microscopy. In addition, we give a simple description of the principal mechanisms of DV microscopy, name advantages and disadvantages, and discuss issues such as dual-color imaging using CFP and YFP, spatial resolution, colocalization, and avoiding imaging artifacts.

Connexin 26↗
PubMed2003

[Use of three-dimensional fluorescence deconvolution microscopy for study of spatial distribution of secretory vesicles in living cells].

A three-dimensional image of a living cell is helpful for cell secretion study. In this report, the three-dimensional fluorescence deconvolution microscopy for observing living cells was studied, because this technique can obtain a quick three-dimensional imaging with minimal fluorescence quenching and cytotoxicity for living cell observation. The property of three-dimensional point spread function (PSF) of imaging system was analyzed. The relationship between experimental and theoretical PSF was illustrated, and the theoretical PSF was proved that it could reflect the principle of imaging system with NA 1.65 objective in use. Three-dimensional deconvolution algorithm in this report was proved effective by well-defined three-dimensional specimens. Furthermore, the rat pancreatic beta cell secretory vesicles labeled by acridine orange was observed by using this technique. Results showed that the blurring induced by out-of-focus light was removed by the deconvolution algorithm effectively, under current experiment conditions (with NA 1.65 objective) the experimental PSF approximated the theoretical PSF very well, and deconvolved living cell images exhibited the spatial distribution of the secretory vesicles clearly.

Algorithms↗