Emmanuel Oppong

Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells

Raster image correlation spectroscopy is a powerful tool to study fast molecular dynamics such as protein diffusion or receptor-ligand interactions inside living cells and tissues. By analysing spatio-temporal correlations of fluorescence intensity fluctuations from raster-scanned microscopy images, molecular motions can be revealed in a spatially resolved manner. Because of the diffraction-limited optical resolution, however, conventional raster image correlation spectroscopy can only distinguish larger regions of interest and requires low fluorophore concentrations in the nanomolar range. Here, to overcome these limitations, we combine raster image correlation spectroscopy with stimulated emission depletion microscopy. With imaging experiments on model membranes and live cells, we show that stimulated emission depletion-raster image correlation spectroscopy offers an enhanced multiplexing capability because of the enhanced spatial resolution as well as access to 10-100 times higher fluorophore concentrations.

Allergen Arrays for Antibody Screening and Immune Cell Activation Profiling Generated by Parallel Lipid Dip‐Pen Nanolithography

Multiple-allergen testing for high throughput and high sensitivity requires the development of miniaturized immunoassays that allow for a large test area and require only a small volume of the test analyte, which is often available only in limited amounts. Developing such miniaturized biochips containing arrays of test allergens needs application of a technique able to deposit molecules at high resolution and speed while preserving its functionality. Lipid dip-pen nanolithography (L-DPN) is an ideal technique to create such biologically active surfaces, and it has already been successfully applied for the direct, nanoscale deposition of functional proteins, as well as for the fabrication of biochemical templates for selective adsorption. The work presented here shows the application of L-DPN for the generation of arrays of the ligand 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] onto glass surfaces as a model system for detection of allergen-specific Immunoglobin E (IgE) antibodies and for mast cell activation profiling.

A potent and selective small molecule inhibitor for the lymphoid-specific tyrosine phosphatase (LYP), a target associated with autoimmune diseases

Lymphoid-specific tyrosine phosphatase (LYP), a member of the protein tyrosine phosphatase (PTP) family of signaling enzymes, is associated with a broad spectrum of autoimmune diseases. Herein we describe our structure-based lead optimization efforts within a 6-hydroxy-benzofuran-5-carboxylic acid series culminating in the identification of compound 8b, a potent and selective inhibitor of LYP with a K(i) value of 110 nM and more than 9-fold selectivity over a large panel of PTPs. The structure of LYP in complex with 8b was obtained by X-ray crystallography, providing detailed information about the molecular recognition of small-molecule ligands binding LYP. Importantly, compound 8b possesses highly efficacious cellular activity in both T- and mast cells and is capable of blocking anaphylaxis in mice. Discovery of 8b establishes a starting point for the development of clinically useful LYP inhibitors for treating a wide range of autoimmune disorders.

Localization and Dynamics of Glucocorticoid Receptor at the Plasma Membrane of Activated Mast Cells

In addition to their actions in the cell nucleus, glucocorticoids exhibit rapid non-nuclear responses that are mechanistically not well understood. To explain these effects, the localization of a glucocorticoid receptor (GR) expressed in mast cells as a GFP fusion was analyzed after activation of the cells on allergenic lipid arrays. These arrays were produced on glass slides by dip-pen nanolithography (DPN) and total internal reflection (TIRF) microscopy was used to visualize the GR. A rapid glucocorticoid-independent and -dependent recruitment of the GR-GFP to the plasma cell membrane was observed following contact of the cells with the allergenic array. In addition, the mobility of the GR at the membrane was monitored by fluorescence recovery after photobleaching (FRAP) and shown to follow binding kinetics demonstrating interactions of the receptor with membrane-bound factors. Furthermore the recruitment of the GR to the cell membrane was shown to result in a glucocorticoid-mediated increase in Erk phosphorylation. This is evidenced by findings that destruction of the membrane composition of the mast cells by cholesterol depletion impairs the membrane localization of the GR and subsequent glucocorticoid-mediated enhancement of Erk phosphorylation. These results demonstrate a membrane localization and function of the GR in mast cell signaling.

Molecular mechanisms of glucocorticoid action in mast cells

Glucocorticoids are compounds that have successfully been used over the years in the treatment of inflammatory disorders. They are known to exhibit their effects through the glucocorticoid receptor (GR) that acts to downregulate the action of proinflammatory transcription factors such as AP-1 and NF-κB. The GR also exerts anti-inflammatory effects through activation of distinct genes. In addition to their anti-inflammatory actions, glucocorticoids are also potent antiallergic compounds that are widely used in conditions such as asthma and anaphylaxis. Nevertheless the mechanism of action of this hormone in these disorders is not known. In this article, we have reviewed reports on the effects of glucocorticoids in mast cells, one of the important immune cells in allergy. Building on the knowledge of the molecular action of glucocorticoids and the GR in the treatment of inflammation in other cell types, we have made suggestions as to the likely mechanisms of action of glucocorticoids in mast cells. We have further identified some important questions and research directions that need to be addressed in future studies to improve the treatment of allergic disorders.

Effects of Glucocorticoids in the Immune System

Glucocorticoids (GCs) are steroid hormones with widespread effects. They control intermediate metabolism by stimulating gluconeogenesis in the liver, mobilize amino acids from extra hepatic tissues, inhibit glucose uptake in muscle and adipose tissue, and stimulate fat breakdown in adipose tissue. They also mediate stress response. They exert potent immune-suppressive and anti-inflammatory effects particularly when administered pharmacologically. Understanding these diverse effects of glucocorticoids requires a detailed knowledge of their mode of action. Research over the years has uncovered several details on the molecular action of this hormone, especially in immune cells. In this chapter, we have summarized the latest findings on the action of glucocorticoids in immune cells with a view of identifying important control points that may be relevant in glucocorticoid therapy.

An Amyloidogenic Sequence at the N-Terminus of the Androgen Receptor Impacts Polyglutamine Aggregation

The human androgen receptor (AR) is a ligand inducible transcription factor that harbors an amino terminal domain (AR-NTD) with a ligand-independent activation function. AR-NTD is intrinsically disordered and displays aggregation properties conferred by the presence of a poly-glutamine (polyQ) sequence. The length of the polyQ sequence as well as its adjacent sequence motifs modulate this aggregation property. AR-NTD also contains a conserved KELCKAVSVSM sequence motif that displays an intrinsic property to form amyloid fibrils under mild oxidative conditions. As peptide sequences with intrinsic oligomerization properties are reported to have an impact on the aggregation of polyQ tracts, we determined the effect of the KELCKAVSVSM on the polyQ stretch in the context of the AR-NTD using atomic force microscopy (AFM). Here, we present evidence for a crosstalk between the amyloidogenic properties of the KELCKAVSVSM motif and the polyQ stretch at the AR-NTD.

STED-RICS - A Versatile Method for Studying Biomolecular Dynamics in Live Cells

By analyzing spatio-temporal correlations of fluorescence intensity fluctuations from raster-scanned microscopy images, raster image correlation spectroscopy (RICS) provides spatially resolved information on fast molecular dynamics such as protein diffusion or receptor-ligand interactions inside living cells and tissues [1]. Conventional RICS can only distinguish larger regions of interest, however, and requires low fluorophore concentrations in the nanomolar range, due to its diffraction-limited optical resolution. We have recently combined RICS with stimulated emission depletion (STED) microscopy to remove these limitations [2]. STED-RICS yields an enhanced multiplexing capability due to the increase in spatial resolution and can accommodate up to ∼100-fold higher fluorophore concentrations.[1] Rossow, M.J.; Sasaki, J. M.; Digman, M. A.; Gratton, E., Nat. Protoc. 2010, 5, 1761-1774.[2] Hedde, P. N.; Dorlich, R. M.; Blomley, R.; Gradl, D.; Oppong, E.; Cato, A. C.; Nienhaus, G. U., Nat Commun. 2013, 4, 2093.

Micropatterned surfaces as tools for the study of the rapid non-genomic actions of steroid receptors

Steroid hormones control several developmental and physiological processes by binding to intracellular receptors that, in turn, interact with DNA to alter gene expression. These processes typically take at least 30 to 60 min for an increase in mRNA expression to be observed. In contrast, other regulatory actions of steroid hormones such as increases in activity of mitogen activated protein kinases are manifested within seconds to a few minutes and are far too rapid to be due to changes at the genomic level. Because these effects are not impaired by inhibitors of mRNA transcription, they are referred to non-genomic or rapid actions to distinguish them from the classical genomic effects at the transcriptional level. The non-genomic effects are thought to occur at the plasma membrane but have proven difficult to analyse in detail because of technical problems arising from capturing the receptors at the membrane due to their dynamic behaviour, subcellular sizes and complexity of action. Here we describe a novel technique for studying the non-genomic action of steroid hormones making use of dip-pen nanolithography (DPN) for patterning supported lipid bilayers containing haptenated lipids onto glass surfaces. Mast cells have been chosen for these studies because of the crucial role they play in allergic reactions and because the non-genomic action of steroid hormones have been reported as one of the means whereby allergy is regulated in these cells. Since mast cells express IgE receptors on their surfaces, they are treated with an anti-IgE antibody and allowed to settle on the patterned surfaces. The IgE receptor is then cross-linked through interaction with the haptenated lipids and this leads to the recruitment of different signalling molecules including steroid receptors to the patterned lipids. The DPN approach allows a nano-scale characterisation of the activating events afforded by the lipid bilayer. The patterns enable quantitative evaluation of co-localised cellular components and the steroid receptors to be assessed. This assay also allows visualisation and analysis of the interacting proteins to be made on a single cell level as well as receptor-proximal events triggered by allergens and regulation by steroid receptors to be measured. This method could be adapted for studying the rapid action of steroid hormones in other cell types.