Cheryl A. Telmer

Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins.

Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out. Protein-mediated activation of far-red fluorogenic dyes has the potential to address these challenges because the cell-permeant dye is small and nonfluorescent until bound to its activating protein, and this binding is rapid. In this study, three single chain variable fragment (scFv)-derived fluorogen activating proteins (FAPs), which activate far-red emitting fluorogens, were evaluated for targeting, brightness, and photostability in the cytosol, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum with a cell-permeant malachite green analog in cultured mammalian cells. Efficient labeling was achieved within 20–30 min for each protein upon the addition of nM concentrations of dye, producing a signal that colocalized significantly with a linked mCerulean3 (mCer3) fluorescent protein and organelle specific dyes but showed divergent photostability and brightness properties dependent on the FAP. These FAPs and the ester of malachite green dye (MGe) can be used as specific, rapid, and wash-free labels for intracellular sites in live cells with far-red excitation and emission properties, useful in a variety of multicolor experiments.

Fluorogen-activating proteins provide tunable labeling densities for tracking FcεRI independent of IgE.

Crosslinking of IgE bound FcεRI on mast cells and basophils by multivalent antigen leads to degranulation and the release of key inflammatory mediators that stimulate the allergic response. Here, we present and characterize the use of fluorogen-activating proteins (FAPs) for single particle tracking of FcεRI to investigate how receptor mobility is influenced after IgE-induced changes in mast cell behavior. FAPs are genetically encoded tags that bind a fluorogen dye and increase its brightness upon binding up to 20,000-fold. We demonstrate that, by titrating fluorogen concentration, labeling densities from ensemble to single particle can be achieved, independent of expression level and without the need for wash steps or photobleaching. The FcεRI γ-subunit fused to a FAP (FAP-γ) provides, for the first time, an IgE-independent probe for tracking this signaling subunit of FcεRI at the single molecule level. We show that the FcεRI γ-subunit dynamics are controlled by the IgE-binding α-subunit and that the cytokinergic IgE, SPE-7, induces mast cell activation without altering FcεRI mobility or promoting internalization. We take advantage of the far-red emission of the malachite green (MG) fluorogen to track FcεRI relative to dynamin–GFP and find that immobilized receptors readily correlate with locations of dynamin recruitment only under conditions that promote rapid endocytosis. These studies demonstrate the usefulness of the FAP system for single molecule studies and have provided new insights into the relationship among FcεRI structure, activity, and mobility.

Fluorogen Activating Protein–Affibody Probes: Modular, No-Wash Measurement of Epidermal Growth Factor Receptors

Fluorescence is essential for dynamic live cell imaging, and affinity reagents are required for quantification of endogenous proteins. Various fluorescent dyes can report on different aspects of biological trafficking, but must be independently conjugated to affinity reagents and characterized for specific biological readouts. Here we present the characterization of a new modular platform for small anti-EGFR affinity probes for studying rapid changes in receptor pools. A protein domain (FAP dL5**) that binds to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody ZEGFR:1907. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules. In vitro fluorescence assays demonstrated that the binding of these dyes to the FAP–affibody fusions produced thousand-fold fluorescence enhancements, with high binding affinity and fast association rates. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nanomolar surface Kd values were observed with the double-ZEGFR:1907 constructs. The application of light-harvesting fluorogens (dyedrons) significantly improved the detected fluorescence signal. Altering the order of addition of the ligand, probe, and dyes allowed differentiation between surface and endocytotic pools of receptors to reveal the rapid dynamics of endocytic trafficking. Therefore, FAP/affibody coupling provides a new approach to construct compact and modular affinity probes that label endogenous proteins on living cells and can be used for studying rapid changes in receptor pools involved in trafficking.

In Vitro Reversible Translation Control Using γPNA Probes.

On-demand regulation of gene expression in living cells is a central goal of chemical biology and antisense therapeutic development. While significant advances have allowed regulatory modulation through inserted genetic elements, on-demand control of the expression/translation state of a given native gene by complementary sequence interactions remains a technical challenge. Toward this objective, we demonstrate the reversible suppression of a luciferase gene in cell-free translation using Watson-Crick base pairing between the mRNA and a complementary γ-modified peptide nucleic acid (γPNA) sequence with a noncomplementary toehold. Exploiting the favorable thermodynamics of γPNA-γPNA interactions, the antisense sequence can be removed by hybridization of a second, fully complementary γPNA, through a strand displacement reaction, allowing translation to proceed. Complementary RNA is also shown to displace the bound antisense γPNA, opening up possibilities of in vivo regulation by native gene expression.

Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation

The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration.

Formal Analysis Provides Parameters for Guiding Hyperoxidation in Bacteria using Phototoxic Proteins

In this work, we developed a methodology to analyze a bacteria model that mimics the stages through which bacteria change when phage therapy is applied. Due to the widespread misuse and overuse of antibiotics, drug resistant bacteria now pose significant risks to health, agriculture and the environment. Therefore, we were interested in an alternative to conventional antibiotics, a phage therapy. Our model was designed according to an experimental procedure to engineer a temperate phage, Lambda (λ), and then kill bacteria via light-activated production of superoxide. We applied formal analysis to our model and the results show that such an approach can speed up evaluation of the system, which would be impractical or possibly not even feasible to study in a wet lab.

Aptamers Act as Activators for the Thrombin Mediated‐Hydrolysis of Peptide Substrates

Thrombin is the typical target in anticlotting therapy for many serious diseases such as heart attack and stroke. DNA aptamers are well‐known thrombin inhibitors that prevent fibrinogen hydrolysis. We have discovered that exosite‐targeting antithrombin aptamers enhance the activity of thrombin toward a small peptide substrate, Sar(N‐methylglycine)‐Pro‐Arg‐paranitroanilide, and that the activation of the enzyme by these aptamers is strongly inhibited by their complementary DNAs. Our study reveals that treatment with mixed aptamers or with a dual‐aptamer construct led to an 8.6‐ or 7.8‐fold enhancement in peptide hydrolysis relative to thrombin alone, a synergistic effect much higher than the activation observed with a monofunctional aptamer (1.5‐fold for Apt27 or 2.7‐fold for Apt15). In addition, we discovered that Apt27 is a biofunctional molecule for thrombin because of its activation effect. An enzyme kinetic study indicates that the binding of aptamers to exosites I and II significantly activates thrombin towards the peptide substrate, thus illustrating that binding of aptamers to exosites can allosterically regulate the active site of thrombin. Our study suggests the necessity of considering possible side effects when DNA aptamers are used for clinical applications involving the inhibition of thrombin‐mediated clotting.

PI3K class II α regulates δ-opioid receptor export from the trans-Golgi network

The δ-opioid receptor (δR) is retained in intracellular structures in neurons, but the mechanisms of retention and regulated export are not known. The atypical phosphoinositide-3 kinase C2A is required and sufficient for NGF-regulated δR export from the trans-Golgi network and surface transport.

Quantitative synapse analysis for cell-type specific connectomics

Anatomical methods for determining cell-type specific connectivity are essential to inspire and constrain our understanding of neural circuit function. We developed new genetically-encoded reagents for fluorescence-synapse labeling and connectivity analysis in brain tissue, using a fluorogen-activating protein (FAP)-or YFP-coupled, postsynaptically-localized neuroligin-1 targeting sequence (FAP/YFPpost). Sparse viral expression of FAP/YFPpost with the cell-filling, red fluorophore dTomato (dTom) enabled high-throughput, compartment-specific localization of synapses across diverse neuron types in mouse somatosensory cortex. High-resolution confocal image stacks of virally-transduced neurons were used for 3D reconstructions of postsynaptic cells and automated detection of synaptic puncta. We took advantage of the bright, far-red emission of FAPpost puncta for multichannel fluorescence alignment of dendrites, synapses, and presynaptic neurites to assess subtype-specific inhibitory connectivity onto L2 neocortical pyramidal (Pyr) neurons. Quantitative and compartment-specific comparisons show that PV inputs are the dominant source of inhibition at both the soma and across all dendritic branches examined and were particularly concentrated at the primary apical dendrite, a previously unrecognized compartment of L2 Pyr neurons. Our fluorescence-based synapse labeling reagents will facilitate large-scale and cell-type specific quantitation of changes in synaptic connectivity across development, learning, and disease states.

Methods to Expand Cell Signaling Models Using Automated Reading and Model Checking

Biomedical research results are being published at a high rate, and with existing search engines, the vast amount of published work is usually easily accessible. However, reproducing published results, either experimental data or observations is often not viable. In this work, we propose a framework to overcome some of the issues of reproducing previous research, and to ensure re-usability of published information. We present here a framework that utilizes the results from state-of-the-art biomedical literature mining, biological system modeling and analysis techniques, and provides means to scientists to assemble and reason about information from voluminous, fragmented and sometimes inconsistent literature. The overall process of automated reading, assembly and reasoning can speed up discoveries from the order of decades to the order of hours or days. Our framework described here allows for rapidly conducting thousands of in silico experiments that are designed as part of this process.