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Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

Several techniques rely on detection of fluorescence signals to identify or study phenomena or to elucidate functions. Separation of these fluorescence ...

University of South Alabama

Chemokines responsible for recruiting lymphocytes such as activated T cells into the cornea have not been clearly defined. IP-10, I-TAC, and MIG are chemoattractants for these lymphocytes. The goal of this study was to determine whether human corneal keratocyte (HCKs) in culture synthesize these chemokines in response to proinflammatory mediators.

Synthesis of alpha-chemokines IP-10, I-TAC, and MIG are differentially regulated in human corneal keratocytes.

The purpose of this study was to characterize the synthesis of alpha-chemokines IP-10, MIG, and I-TAC by human corneal epithelial cells (HCE) following exposure to proinflammatory mediators. Supernatants were collected from HCE cultures stimulated with individual or combinations of TNF-alpha, IL-1alpha, and IFN-gamma, and assayed for alpha-chemokines by ELISA. RT-PCR was used to detect IFN-gamma receptor mRNA. Activation of STAT 1 was determined by Western blotting. Stimulation of HCE with either IL-1alpha or TNF-alpha increased IP-10 protein synthesis up to 6-fold, whereas insignificant levels of MIG and I-TAC were induced. The epithelial cells were found to express IFN-gamma receptors constitutively. Exposure to the ligand resulted in STAT 1 phosphorylation and production of nanogram amounts of IP-10, I-TAC, and MIG. When HCE were stimulated with combinations of TNF-alpha and IFN-gamma, or IL-1alpha and IFN-gamma, the levels of IP-10 and I-TAC secreted were > 150-fold higher than that produced following exposure to a single cytokine. In contrast, MIG protein synthesis was not enhanced upon stimulation with cytokine combinations. The abundant production of ELR(-)alpha -chemokines following appropriate stimulation suggests that HCE may play an important role in the recruitment of effector cells such as activated T-lymphocytes to inflamed corneal tissue. The data also indicate that the synthesis of IP-10, I-TAC, and MIG are differentially regulated in HCE.

Human corneal epithelial cells synthesize ELR(-)alpha-chemokines in response to proinflammatory mediators.

Receptor desensitization is a ubiquitous regulatory mechanism that defines the activatable pool of receptors, and thus, the ability of cells to respond to environmental stimuli. In recent years, the molecular mechanisms controlling the desensitization of a variety of receptors have been established. However, little is known about the molecular mechanisms that underlie desensitization of natriuretic peptide receptors, including natriuretic peptide receptor-A (NPR-A). Here we report that calcineurin (protein phosphatase 2B, PP2B, PPP3C) regulates homologous desensitization of NPR-A in murine Leydig tumor (MA-10) cells. We demonstrate that both pharmacological inhibition of calcineurin activity and siRNA-mediated suppression of calcineurin expression potentiate atrial natriuretic peptide (ANP)-induced cGMP synthesis. Treatment of MA-10 cells with inhibitors of other phosphoprotein phosphatases had little or no effect on ANP-induced cGMP accumulation. In addition, overexpression of calcineurin blunts ANP-induced cGMP synthesis. We also present data indicating that the inhibition of calcineurin potentiates ANP-induced testosterone production. To better understand the contribution of calcineurin in the regulation of NPR-A activity, we examined the kinetics of ANP-induced cGMP signals. We observed transient ANP-induced cGMP signals, even in the presence of phosphodiesterase inhibitors. Inhibition of both calcineurin and phosphodiesterase dramatically slowed the decay in the response. These observations are consistent with a model in which calcineurin mediated dephosphorylation and desensitization of NPR-A is associated with significant inhibition of cGMP synthesis. PDE activity hydrolyzes cGMP, thus lowering intracellular cGMP toward the basal level. Taken together, these data suggest that calcineurin plays a previously unrecognized role in the desensitization of NPR-A and, thereby, inhibits ANP-mediated increases in testosterone production.

Calcineurin regulates homologous desensitization of natriuretic peptide receptor-A and inhibits ANP-induced testosterone production in MA-10 cells.

In recent years a variety of fluorescent probes for measurement of cGMP signals have been developed (Nikolaev et al., Nat. Methods 3:23-25, 2006; Honda et al., Proc Natl Acad Sci USA 98:2437-42, 2001; Nausch et al., Proc Natl Acad Sci USA 105:365-70, 2008). The probes are comprised of known cGMP binding sites-e.g., from phosphodiesterase type 5 (PDE5) or protein kinase G (PKG)-attached to fluorescent proteins. Binding of cGMP triggers conformational changes that alter the emitted fluorescence. In the case of Förster resonance energy transfer (FRET)-based probes, binding of cGMP alters the distance between the donor and acceptor fluorophores and thus alters FRET. However, FRET-based probes inherently have low signal-to-noise ratios, limiting the utility of these probes. Here we describe the use of hyperspectral imaging and analysis approaches to increase the signal-to-noise ratio of FRET-based cGMP measurements. These approaches are appropriate for monitoring changes in cGMP signals either in cell populations using a spectrofluorimeter or in single cells using spectral microscope systems with appropriate spectral filtering capabilities.

Hyperspectral imaging of FRET-based cGMP probes.

Förster resonance energy transfer (FRET) techniques have proven invaluable for probing the complex nature of protein-protein interactions, protein folding, and intracellular signaling events. These techniques have traditionally been implemented with the use of one or more fluorescence band-pass filters, either as fluorescence microscopy filter cubes, or as dichroic mirrors and band-pass filters in flow cytometry. In addition, new approaches for measuring FRET, such as fluorescence lifetime and acceptor photobleaching, have been developed. Hyperspectral techniques for imaging and flow cytometry have also shown to be promising for performing FRET measurements. In this study, we have compared traditional (filter-based) FRET approaches to three spectral-based approaches: the ratio of acceptor-to-donor peak emission, linear spectral unmixing, and linear spectral unmixing with a correction for direct acceptor excitation. All methods are estimates of FRET efficiency, except for one-filter set and three-filter set FRET indices, which are included for consistency with prior literature. In the first part of this study, spectrofluorimetric data were collected from a CFP-Epac-YFP FRET probe that has been used for intracellular cAMP measurements. All comparisons were performed using the same spectrofluorimetric datasets as input data, to provide a relevant comparison. Linear spectral unmixing resulted in measurements with the lowest coefficient of variation (0.10) as well as accurate fits using the Hill equation. FRET efficiency methods produced coefficients of variation of less than 0.20, while FRET indices produced coefficients of variation greater than 8.00. These results demonstrate that spectral FRET measurements provide improved response over standard, filter-based measurements. Using spectral approaches, single-cell measurements were conducted through hyperspectral confocal microscopy, linear unmixing, and cell segmentation with quantitative image analysis. Results from these studies confirmed that spectral imaging is effective for measuring subcellular, time-dependent FRET dynamics and that additional fluorescent signals can be readily separated from FRET signals, enabling multilabel studies of molecular interactions. © 2013 International Society for Advancement of Cytometry. 

Assessing FRET using spectral techniques.

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments. 

Estimating the magnitude of near-membrane PDE4 activity in living cells.

Cyclic AMP is a ubiquitous second messenger that orchestrates a variety of cellular functions over different timescales. The mechanisms underlying specificity within this signaling pathway are still not well understood. Several lines of evidence suggest the existence of spatial cAMP gradients within cells, and that compartmentalization underlies specificity within the cAMP signaling pathway. However, to date, no studies have visualized cAMP gradients in three spatial dimensions (3D: x, y, z).This is in part due to the limitations of FRET-based cAMP sensors, specifically the low signal-to-noise ratio intrinsic to all intracellular FRET probes. Here, we overcome this limitation, at least in part, by implementing spectral imaging approaches to estimate FRET efficiency when multiple fluorescent labels are used and when signals are measured from weakly expressed fluorescent proteins in the presence of background autofluorescence and stray light. Analysis of spectral image stacks in two spatial dimensions (2D) from single confocal slices indicates little or no cAMP gradients formed within pulmonary microvascular endothelial cells (PMVECs) under baseline conditions or following 10 min treatment with the adenylyl cyclase activator forskolin. However, analysis of spectral image stacks in 3D demonstrates marked cAMP gradients from the apical to basolateral face of PMVECs. Results demonstrate that spectral imaging approaches can be used to assess cAMP gradients-and in general gradients in fluorescence and FRET-within intact cells. Results also demonstrate that 2D imaging studies of localized fluorescence signals and, in particular, cAMP signals, whether using epifluorescence or confocal microscopy, may lead to erroneous conclusions about the existence and/or magnitude of gradients in either FRET or the underlying cAMP signals. Thus, with the exception of cellular structures that can be considered in one spatial dimension, such as neuronal processes, 3D measurements are required to assess mechanisms underlying compartmentalization and specificity within intracellular signaling pathways.

Andrea Britain

Center for Lung Biology,
Department of Pharmacology

University of South Alabama

Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

Andrea Britain

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center for Lung Biology,Department of Pharmacology

University of South Alabama

Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

Center for Lung Biology,
Department of Pharmacology