Ca2+ and cAMP open differentially dilating synaptic fusion pores.

Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins that affect synaptic growth and plasticity. Rather than using full collapse exocytosis that commonly mediates peptide hormone release by endocrine cells, DCVs at the Drosophila neuromuscular junction release their contents via fusion pores formed by kiss-and-run exocytosis. Here, we used fluorogen-activating protein (FAP) imaging to reveal the permeability range of synaptic DCV fusion pores and then show that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca2+-independent full fusions require PKA-R2, a PKA phosphorylation site on Complexin and the acute presynaptic function of Rugose, the homolog of mammalian neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca2+-independent cAMP signaling opens dilating fusion pores to release large cargoes that cannot pass through the narrower fusion pores that mediate spontaneous and activity-dependent neuropeptide release. These results imply that the fusion pore is a variable filter that differentially sets the composition of proteins released at the synapse by independent exocytosis triggers responsible for routine peptidergic transmission (Ca2+) and synaptic development (cAMP).

A Chemoptogenetic Tool for Spatiotemporal Induction of Oxidative DNA Lesions In Vivo.

Oxidative nuclear DNA damage increases in all tissues with age in multiple animal models, as well as in humans. However, the increase in DNA oxidation varies from tissue to tissue, suggesting that certain cells/tissues may be more vulnerable to DNA damage than others. The lack of a tool that can control dosage and spatiotemporal induction of oxidative DNA damage, which accumulates with age, has severely limited our ability to understand how DNA damage drives aging and age-related diseases. To overcome this, here we developed a chemoptogenetic tool that produces 8-oxoguanine (8-oxoG) at DNA in a whole organism, Caenorhabditis elegans. This tool uses di-iodinated malachite green (MG-2I) photosensitizer dye that generates singlet oxygen, 1O2, upon fluorogen activating peptide (FAP) binding and excitation with far-red light. Using our chemoptogenetic tool, we are able to control generation of singlet oxygen ubiquitously or in a tissue-specific manner, including in neurons and muscle cells. To induce oxidative DNA damage, we targeted our chemoptogenetic tool to histone, his-72, that is expressed in all cell types. Our results show that a single exposure to dye and light is able to induce DNA damage, promote embryonic lethality, lead to developmental delay, and significantly reduce lifespan. Our chemoptogenetic tool will now allow us to assess the cell autonomous versus non-cell autonomous role of DNA damage in aging, at an organismal level.

Esterase-Activated, pH-Responsive, and Genetically Targetable Nano-Prodrug for Cancer Cell Photo-Ablation.

Activatable prodrugs have drawn considerable attention for cancer cell ablation owing to their high specificity in drug delivery systems. However, phototheranostic prodrugs with dual organelle-targeting and synergistic effects are still rare due to low intelligence of their structures. Besides, the cell membrane, exocytosis, and diffusional hindrance by the extracellular matrix reduce drug uptake. Moreover, the up-regulation of heat shock protein and short singlet-oxygen lifetime in cancer cells hamper photo-ablation efficacy, especially in the mono-therapeutic model. To overcome those obstacles, we prepare an esterase-activated DM nano-prodrug, which is conjugated by diiodine-substituted fluorogenic malachite green derivative (MG-2I) and phototherapeutic agent DPP-OH via hydrolyzable ester linkage, having pH-responsiveness and genetically targetable activity for dual organelles-targeting to optimize photo-ablation efficacy. The DM nanoparticles (NPs) present improved pH-responsive photothermal/photodynamic property by the protonation of diethylaminophenyl units in acidic environment. More importantly, the MG-2I and DPP-OH moieties can be released from DM nano-prodrug through overexpressed esterase; then specifically target lysosomes and mitochondria in CT-26 Mito-FAP cells. Hence, near-infrared DM NPs can trigger parallel damage in dual-organelles with strong fluorescence and effective phototoxicity, thus inducing serious mitochondrial dysfunction and apoptotic death, showing excellent photo-ablation effect based on esterase-activated, pH-responsive, and genetically targetable activities.

Bottom-Up Design: A Modular Golden Gate Assembly Platform of Yeast Plasmids for Simultaneous Secretion and Surface Display of Distinct FAP Fusion Proteins.

A need in synthetic biology is the ability to precisely and efficiently make flexible fully designed vectors that addresses challenging cloning strategies of single plasmids that rely on combinatorial co-expression of a multitude of target and bait fusion reporters useful in projects like library screens. For these strategies, the regulatory elements and functional components need to correspond perfectly to project specific sequence elements that facilitate easy exchange of these elements. This requires systematic implementation and building on recent improvements in Golden Gate (GG) that ensures high cloning efficiency for such complex vectors. Currently, this is not addressed in the variety of molecular GG cloning techniques in synthetic biology. Here, we present the bottom-up design and plasmid synthesis to prepare 10 kb functional yeast secrete and display plasmids that uses an optimized version of GG in combination with fluorogen-activating protein reporter technology. This allowed us to demonstrate nanobody/target protein interactions in a single cell, as detected by cell surface retention of secreted target proteins by cognate nanobodies. This validates the GG constructional approach and suggests a new approach for discovering protein interactions. Our GG assembly platform paves the way for vector-based library screening and can be used for other recombinant GG platforms.

The strawberry-derived permeation enhancer pelargonidin enables oral protein delivery.

Although patients generally prefer oral drug delivery to injections, low permeability of the gastrointestinal tract makes this method impossible for most biomacromolecules. One potential solution is codelivery of macromolecules, including therapeutic proteins or nucleic acids, with intestinal permeation enhancers; however, enhancer use has been limited clinically by modest efficacy and toxicity concerns surrounding long-term administration. Here, we hypothesized that plant-based foods, which are well tolerated by the gastrointestinal tract, may contain compounds that enable oral macromolecular absorption without causing adverse effects. Upon testing more than 100 fruits, vegetables, and herbs, we identified strawberry and its red pigment, pelargonidin, as potent, well-tolerated enhancers of intestinal permeability. In mice, an oral capsule formulation comprising pelargonidin and a 1 U/kg dose of insulin reduced blood glucose levels for over 4 h, with bioactivity exceeding 100% relative to subcutaneous injection. Effects were reversible within 2 h and associated with actin and tight junction rearrangement. Furthermore, daily dosing of mice with pelargonidin for 1 mo resulted in no detectable side effects, including weight loss, tissue damage, or inflammatory responses. These data suggest that pelargonidin is an exceptionally effective enhancer of oral protein uptake that may be safe for routine pharmaceutical use.

Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis.

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

Protein Proximity Observed Using Fluorogen Activating Protein and Dye Activated by Proximal Anchoring (FAP-DAPA) System.

The development and function of tissues, blood, and the immune system is dependent upon proximity for cellular recognition and communication. However, the detection of cell-to-cell contacts is limited due to a lack of reversible, quantitative probes that can function at these dynamic sites of irregular geometry. Described here is a novel chemo-genetic tool developed for fluorescent detection of protein-protein proximity and cell apposition that utilizes the Fluorogen Activating Protein (FAP) in combination with a Dye Activated by Proximal Anchoring (DAPA). The FAP-DAPA system has two protein components, the HaloTag and FAP, expressed on separate protein targets or in separate cells. The proteins function to bind and activate a compound that has the hexyl chloride (HexCl) ligand connected to malachite green (MG), the FAP fluorogen, via a poly(ethylene glycol) spacer spanning up to 28 nm. The dehalogenase protein, HaloTag, covalently binds the HexCl ligand, locally concentrating the attached MG. If the FAP is within range of the anchored fluorogen, it will bind and activate MG specifically when the bath concentration is too low to saturate the FAP receptor. A new FAP variant was isolated with a 1000-fold reduced KD of ∼10-100 nM so that the fluorogen activation reports proximity without artificially enhancing it. The system was characterized using purified FRB and FKBP fusion proteins and showed a doubling of fluorescence upon rapamycin induced complex formation. In cocultured HEK293 cells (HaloTag and FAP-expressing) fluorescence increased at contact sites across a broad range of labeling conditions, more reliably providing contact-specific fluorescence activation with the lower-affinity FAP variant. When combined with suitable targeting and expression constructs, this labeling system may offer significant improvements in on-demand detection of intercellular contacts, potentially applicable in neurological and immunological synapse measurements and other transient, dynamic biological appositions that can be perturbed using other labeling methods that stabilize these interactions.

Activity-evoked and spontaneous opening of synaptic fusion pores.

Synaptic release of neuropeptides packaged in dense-core vesicles (DCVs) regulates synapses, circuits, and behaviors including feeding, sleeping, and pain perception. Here, synaptic DCV fusion pore openings are imaged without interference from cotransmitting small synaptic vesicles (SSVs) with the use of a fluorogen-activating protein (FAP). Activity-evoked kiss and run exocytosis opens synaptic DCV fusion pores away from active zones that readily conduct molecules larger than most native neuropeptides (i.e., molecular weight [MW] up to, at least, 4.5 kDa). Remarkably, these synaptic fusion pores also open spontaneously in the absence of stimulation and extracellular Ca2+ SNARE perturbations demonstrate different mechanisms for activity-evoked and spontaneous fusion pore openings with the latter sharing features of spontaneous small molecule transmitter release by active zone-associated SSVs. Fusion pore opening at resting synapses provides a mechanism for activity-independent peptidergic transmission.

Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis.

Previously we used gene-editing to label endogenous EGF receptor (EGFR) with GFP and demonstrate that picomolar concentrations of EGFR ligand drive signaling and endocytosis of EGFR in tumors in vivo (Pinilla-Macua et al., 2017). We now use gene-editing to insert a fluorogen activating protein (FAP) in the EGFR extracellular domain. Binding of the tandem dye pair MG-Bis-SA to FAP-EGFR provides a ratiometric pH-sensitive model with dual fluorescence excitation and a single far-red emission. The excitation ratio of fluorescence intensities was demonstrated to faithfully report the fraction of FAP-EGFR located in acidic endosomal/lysosomal compartments. Coupling native FAP-EGFR expression with the high method sensitivity has allowed development of a high-throughput assay to measure the rates of clathrin-mediated FAP-EGFR endocytosis stimulated with physiological EGF concentrations. The assay was utilized to screen a phosphatase siRNA library. These studies highlight the utility of endogenous pH-sensitive FAP-receptor chimeras in high-throughput analysis of endocytosis.

High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue.

The most promising F508del-CFTR corrector, VX-809, has been unsuccessful as an effective, stand-alone treatment for CF patients, but the rescue effect in combination with other drugs may confer an acceptable level of therapeutic benefit. Targeting cellular factors that modify trafficking may act to enhance the cell surface density of F508-CFTR with VX-809 correction. Our goal is to identify druggable kinases that enhance F508del-CFTR rescue and stabilization at the cell surface beyond that achievable with the VX-809 corrector alone. To achieve this goal, we implemented a new high-throughput screening paradigm that quickly and quantitatively measures surface density and total protein in the same cells. This allowed for rapid screening for increased surface targeting and proteostatic regulation. The assay utilizes fluorogen-activating-protein (FAP) technology with cell excluded and cell permeant fluorogenic dyes in a quick, wash-free fluorescent plate reader format on live cells to first measure F508del-CFTR expressed on the surface and then the total amount of F508del-CFTR protein present. To screen for kinase targets, we used Dharmacon's ON-TARGET plus SMARTpool siRNA Kinase library (715 target kinases) with and without 10 µM VX-809 treatment in triplicate at 37 °C. We identified several targets that had a significant interaction with VX-809 treatment in enhancing surface density with siRNA knockdown. Select small-molecule inhibitors of the kinase targets demonstrated augmented surface expression with VX-809 treatment.

Structural analyses of NEAT1 lncRNAs suggest long-range RNA interactions that may contribute to paraspeckle architecture.

Paraspeckles are nuclear bodies that regulate multiple aspects of gene expression. The long non-coding RNA (lncRNA) NEAT1 is essential for paraspeckle formation. NEAT1 has a highly ordered spatial organization within the paraspeckle, such that its 5' and 3' ends localize on the periphery of paraspeckle, while central sequences of NEAT1 are found within the paraspeckle core. As such, the structure of NEAT1 RNA may be important as a scaffold for the paraspeckle. In this study, we used SHAPE probing and computational analyses to investigate the secondary structure of human and mouse NEAT1. We propose a secondary structural model of the shorter (3,735 nt) isoform hNEAT1_S, in which the RNA folds into four separate domains. The secondary structures of mouse and human NEAT1 are largely different, with the exception of several short regions that have high structural similarity. Long-range base-pairing interactions between the 5' and 3' ends of the long isoform NEAT1 (NEAT1_L) were predicted computationally and verified using an in vitro RNA-RNA interaction assay. These results suggest that the conserved role of NEAT1 as a paraspeckle scaffold does not require extensively conserved RNA secondary structure and that long-range interactions among NEAT1 transcripts may have an important architectural function in paraspeckle formation.

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged ß2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

A versatile optical tool for studying synaptic GABAA receptor trafficking.

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABAAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABAAR γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2pHFAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2pHFAP GABAARs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2pHFAP GABAARs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2pHFAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2pHFAP-MG dye approach reveals enhanced GABAAR turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor measurements. To our knowledge, this is the first application of the FAP-MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABAAR trafficking.

Tethered Fluorogen Assay to Visualize Membrane Apposition in Living Cells.

We describe proof-of-concept for a novel approach for visualizing regions of close apposition between the surfaces of living cells. A membrane-anchored protein with high affinity for a chemical ligand is expressed on the surface of one set of cells, and the cells are co-cultured with a second set of cells that express a membrane-anchored fluorogen-activating protein (FAP). The co-cultured cells are incubated with a bivalent reagent composed of fluorogen linked to the high-affinity ligand, with the concentration of the bivalent reagent chosen to be less than the binding constant for the FAP-fluorogen pair but greater than the binding constant for the ligand-high-affinity protein pair. In these conditions, strong FAP signal is observed only in regions of close proximity between membranes of the two classes of cell, where high local concentration of fluorogen favors binding to the FAP.

A PTEN-Regulated Checkpoint Controls Surface Delivery of δ Opioid Receptors.

The δ opioid receptor (δR) is a promising alternate target for pain management because δR agonists show decreased abuse potential compared with current opioid analgesics that target the µ opioid receptor. A critical limitation in developing δR as an analgesic target, however, is that δR agonists show relatively low efficacy in vivo, requiring the use of high doses that often cause adverse effects, such as convulsions. Here we tested whether intracellular retention of δR in sensory neurons contributes to this low δR agonist efficacy in vivo by limiting surface δR expression. Using direct visualization of δR trafficking and localization, we define a phosphatase and tensin homolog (PTEN)-regulated checkpoint that retains δR in the Golgi and decreases surface delivery in rat and mice sensory neurons. PTEN inhibition releases δR from this checkpoint and stimulates delivery of exogenous and endogenous δR to the neuronal surface both in vitro and in vivo PTEN inhibition in vivo increases the percentage of TG neurons expressing δR on the surface and allows efficient δR-mediated antihyperalgesia in mice. Together, we define a critical role for PTEN in regulating the surface delivery and bioavailability of the δR, explain the low efficacy of δR agonists in vivo, and provide evidence that active δR relocation is a viable strategy to increase δR antinociception.SIGNIFICANCE STATEMENT Opioid analgesics, such as morphine, which target the µ opioid receptor (µR), have been the mainstay of pain management, but their use is highly limited by adverse effects and their variable efficacy in chronic pain. Identifying alternate analgesic targets is therefore of great significance. Although the δ opioid receptor (δR) is an attractive option, a critical limiting factor in developing δR as a target has been the low efficacy of δR agonists. Why δR agonists show low efficacy is still under debate. This study provides mechanistic and functional data that intracellular localization of δR in neurons is a key factor that contributes to low agonist efficacy, and presents a proof of mechanism that relocating δR improves efficacy.

Affibody-targeted fluorogen activating protein for in vivo tumor imaging.

Molecular imaging using near-infrared (NIR) fluorescence is useful for intraoperative imaging and real-time margin identification. Directly conjugated IR dyes possess useful properties for in vivo imaging, but conjugation often substantially alters the circulation dynamics of targeting moieties. We developed and characterized a new tumor-targeting probe, affiFAP, which consists of a protein that specifically binds EGFR (affibody) and a fluorogen activating protein (FAP). This compact molecular recognition reagent can reversibly bind and activate fluorescence of otherwise nonfluorescent dyes and allows tumor visualization with low nonspecific tissue staining. We demonstrate molecular pre-targeting of affiFAPs and subsequent systemic or topical application of fluorogenic dye to achieve high contrast, fast clearance, and good tissue penetration that may be used in clinical settings to molecularly define tumor margins.

Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.

Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics.

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.

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules.

Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nanomolar KD values for their respective proteins, >95% energy transfer efficiency and high fluorescence quantum yields.

Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins.

Agonist-promoted G-protein coupled receptor (GPCR) endocytosis and recycling plays an important role in many signaling events in the cell. However, the approaches that allow fast and quantitative analysis of such processes still remain limited. Here we report an improved labeling approach based on the genetic fusion of a fluorogen activating protein (FAP) to a GPCR and binding of a sulfonated analog of the malachite green (MG) fluorogen to rapidly and selectively label cell surface receptors. Fluorescence microscopy and flow cytometry demonstrate that this dye does not cross the plasma membrane, binds with high affinity to a dL5** FAP-GPCR fusion construct, activating tagged surface receptors within seconds of addition. The ability to rapidly and selectively label cell surface receptors with a fluorogenic genetically encoded tag allows quantitative imaging and analysis of highly dynamic processes like receptor endocytosis and recycling.