Time-Gated Luminescence Detection of Enzymatically Produced Hydrogen Sulfide: Design, Synthesis, and Application of a Lanthanide-Based Probe.

Hydrogen sulfide (H2S) is now recognized as an important gaseous transmitter that is involved in a variety of biological processes. Here, we report the design and synthesis of a luminescent lanthanide biosensor for H2S, LP2-Cu(II)-Ln(III), a heterobinuclear metal complex that uses Cu(II) decomplexation to control millisecond-scale-lifetime-Tb(III)- or Eu(III)-emission intensity. LP2-Cu(II)-Ln(III) responded rapidly, selectively, and with high sensitivity to aqueous H2S. The probe's potential for biological applications was verified by measuring the H2S generated by the slow-releasing chemical-sulfide-donor GYY4147, by cystathionine γ-lyase (CSE), and by Na2S-stimulated HeLa cells.

Time Gated Luminescence Imaging of Immunolabeled Human Tissues.

Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded tissues is a powerful tool for investigating proteomic profiles and diagnosing disease. However, conventional immunofluorescence with organic dyes is limited in the number of colors that can be simultaneously visualized, is made less sensitive by tissue autofluorescence background, and is usually incompatible with commonly used hematoxylin and eosin staining. Herein, we demonstrate the comparative advantages of using time-gated luminescence microscopy in combination with an emissive Tb(III) complex, Lumi4-Tb, for tissue imaging in terms of sensitivity, multiplexing potential, and compatibility with common immunohistochemistry protocols. We show that time-gated detection of millisecond-scale Tb(III) emission increases signal-to-noise ratio relative to conventional steady-state detection of organic dye fluorescence and permits visualization of low-abundance tissue markers such as Bcl-6 or MSH-6. In addition, temporal separation of long- and short-lifetime (∼nanosecond) signals adds a second dimension for multiplexing and also permits detection of intermolecular Tb(III)-to-dye Förster resonance energy transfer. Furthermore, we demonstrate that the Lumi4-Tb complex is compatible with tyramide signal amplification and, unlike conventional organic dyes, can be reliably used on tissue stained with hematoxylin and eosin. Our results indicate that time-gated luminescence microscopy using Tb(III) labels can provide a sensitive and robust method to perform multiplexed immunofluorescence on archived or clinical tissue specimens.

Time-Gated Detection of Cystathionine γ-Lyase Activity and Inhibition with a Selective, Luminogenic Hydrogen Sulfide Sensor.

Herein, we report the design, synthesis, and characterization of a lanthanideIII complex-based probe for the time-gated luminescence detection of hydrogen sulfide (H2 S) in aqueous media. The probe's unique sensing mechanism relies on the selective reduction of azide to amine by sulfide, followed by intramolecular cyclization to form a quinolinone. The quinolinone is a sensitizer that absorbs near-UV light and transfers excitation energy to coordinated TbIII or EuIII ions to trigger a strong "turn-on" luminescence response with ms-scale lifetimes characteristic of lanthanide complexes. Using this probe, we developed a robust, high throughput screening (HTS) assay for detecting H2 S generated by cystathionine γ-lyase (CSE), one of the main producers of H2 S in mammalian cells. In a 240-compound screen to identify potential CSE inhibitors, the EuIII analogue of the sensor showed a low false-positive rate and high Z'-factor (>0.7).

Efficient route to pre-organized and linear polyaminopolycarboxylates: Cy-TTHA, Cy-DTPA and mono/di- reactive, tert-butyl protected TTHA/Cy-TTHA.

Pre-organized polyaminopolycarboxylate chelators Cy-TTHA and Cy-DTPA were synthesized via modular five-step syntheses from commercially available starting materials in ~ 62% and 47% overall yields, respectively. Furthermore, strategies are reported for the efficient preparation of mono- and di-reactive, tert-butyl-protected TTHA/Cy-TTHA to selectively functionalize central chelators' carboxylic acids.

Ratiometric QD-FRET Sensing of Aqueous H2S in Vitro.

We report a platform for the ratiometric fluorescent sensing of endogenously generated gaseous transmitter H2S in its aqueous form (bisulfide or hydrogen sulfide anion) based on the alteration of Förster resonance energy transfer from an emissive semiconductor quantum dot (QD) donor to a dithiol-linked organic dye acceptor. The disulfide bridge between the two chromophores is cleaved upon exposure to bisulfide, resulting in termination of FRET as the dye diffuses away from the QD. This results in enhanced QD emission and dye quenching. The resulting ratiometric response can be correlated quantitatively to the concentration of bisulfide and was found to have a detection limit as low as 1.36 ± 0.03 µM. The potential for use in biological applications was demonstrated by measuring the response of the QD-based FRET sensor microinjected into live HeLa cells upon extracellular exposure to bisulfide. The methodology used here is built upon a highly multifunctional platform that offers numerous advantages, such as low detection limit, enhanced photochemical stability, and sensing ability within a biological milieu.

Brightly Luminescent and Kinetically Inert Lanthanide Bioprobes Based on Linear and Preorganized Chelators.

The synthesis, photophysical properties, and kinetic stability of a series of water-soluble, highly emissive Tb(III) and Eu(III) complexes featuring triethylenetetraamine hexaacetic acid (TTHA) and cyclohexyl triethylenetetraamine hexaacetic acid (cyTTHA) chelator scaffolds and carbostyril sensitizers are reported. The unique and modular design of the chelators gives rise to striking quantum yields of emission in aqueous solutions (up to 54%) as well as the characteristic lanthanides' photophysical properties (long excited-state lifetimes, large effective Stokes shifts, and narrow emission peaks). Furthermore, the preorganized chelators (L3, L4, and L6) bind metal within minutes at ambient temperature yet exhibit substantial resistance to transchelation in the presence of a challenge solution (EDTA, 1 mM). Moreover, the Eu(III) complex of L4 remains stably luminescent in HeLa cells over hours, demonstrating the suitability of these compounds for live-cell imaging applications. Representative chelators suitable for derivatization and protein bioconjugation were also prepared that were functionalized with clickable azide and alkyne moieties, biotin, and trimethoprim (TMP). With exceptional long-wavelength brightness, enhanced kinetic inertness, and an adaptable synthetic route, the reported lanthanide complexes are promising probes and labels for time-gated bioanalysis, biosensing, and optical microscopy.

Evaluating the performance of time-gated live-cell microscopy with lanthanide probes.

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5-25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument's light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.

Cytoplasmic delivery and selective, multicomponent labeling with oligoarginine-linked protein tags.

Strategies that leverage bio-orthogonal interactions between small molecule ligands and genetically encoded amino acid sequences can be used to attach high-performance fluorophores to proteins in living cells. However, a major limitation of chemical protein labeling is that cells' plasma membranes are impermeable to many useful probes and biolabels. Here, we show that conjugation to nonaarginine, a cell penetrating peptide (CPP), enables passive cytoplasmic delivery of otherwise membrane-impermeant, small molecule protein labels. Heterodimers consisting of a luminescent Tb(3+) complex, Lumi4, linked to benzyl guanine, benzyl cytosine, and trimethoprim were conjugated to the peptide CysArg9 with a reducible disulfide linker. When added to culture medium, the peptide conjugates rapidly (<30 min) enter the cytoplasm and diffuse freely throughout cells. The benzyl guanine, benzyl cytosine, and trimethoprim derivatives bind selectively to fusion proteins tagged with SNAP-Tag, CLIP-Tag, and Escherichia coli dihydrofolate reductase (eDHFR), respectively. Furthermore, eDHFR and SNAP-Tag fusions can be labeled with Lumi4 analogues in the same cell, and this labeling can be detected using two-color, time-gated Förster resonance energy transfer (FRET) microscopy. Finally, we present quantitative data showing that cytoplasmic uptake of nonaarginine-conjugated probes occurs in multiple cell types (MDCK, HeLa, NIH 3T3), most cells in a culture (>75%) are loaded with probe, and the cellular probe concentration can be controlled by varying incubation conditions. CPP-mediated delivery of Lumi4-linked protein labels will greatly increase the utility of lanthanide-based FRET microscopy. Moreover, our results strongly suggest that this approach can be adapted to deliver a wide variety of protein-targeted fluorophores or other functional probes that were previously unavailable for intracellular imaging studies.

Lanthanide-based imaging of protein-protein interactions in live cells.

In order to deduce the molecular mechanisms of biological function, it is necessary to monitor changes in the subcellular location, activation, and interaction of proteins within living cells in real time. Förster resonance energy-transfer (FRET)-based biosensors that incorporate genetically encoded, fluorescent proteins permit high spatial resolution imaging of protein-protein interactions or protein conformational dynamics. However, a nonspecific fluorescence background often obscures small FRET signal changes, and intensity-based biosensor measurements require careful interpretation and several control experiments. These problems can be overcome by using lanthanide [Tb(III) or Eu(III)] complexes as donors and green fluorescent protein (GFP) or other conventional fluorophores as acceptors. Essential features of this approach are the long-lifetime (approximately milliseconds) luminescence of Tb(III) complexes and time-gated luminescence microscopy. This allows pulsed excitation, followed by a brief delay, which eliminates nonspecific fluorescence before the detection of Tb(III)-to-GFP emission. The challenges of intracellular delivery, selective protein labeling, and time-gated imaging of lanthanide luminescence are presented, and recent efforts to investigate the cellular uptake of lanthanide probes are reviewed. Data are presented showing that conjugation to arginine-rich, cell-penetrating peptides (CPPs) can be used as a general strategy for the cellular delivery of membrane-impermeable lanthanide complexes. A heterodimer of a luminescent Tb(III) complex, Lumi4, linked to trimethoprim and conjugated to nonaarginine via a reducible disulfide linker rapidly (∼10 min) translocates into the cytoplasm of Maden Darby canine kidney cells from the culture medium. With this reagent, the intracellular interaction between GFP fused to FK506 binding protein 12 (GFP-FKBP12) and the rapamycin binding domain of mTOR fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) were imaged at high signal-to-noise ratio with fast (1-3 s) image acquisition using a time-gated luminescence microscope. The data reviewed and presented here show that lanthanide biosensors enable fast, sensitive, and technically simple imaging of protein-protein interactions in live cells.

Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells.

Förster resonance energy transfer (FRET) with fluorescent proteins permits high spatial resolution imaging of protein-protein interactions in living cells. However, substantial non-FRET fluorescence background can obscure small FRET signals, making many potential interactions unobservable by conventional FRET techniques. Here we demonstrate time-resolved microscopy of luminescence resonance energy transfer (LRET) for live-cell imaging of protein-protein interactions. A luminescent terbium complex, TMP-Lumi4, was introduced into cultured cells using two methods: (i) osmotic lysis of pinocytic vesicles; and (ii) reversible membrane permeabilization with streptolysin O. Upon intracellular delivery, the complex was observed to bind specifically and stably to transgenically expressed Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins. LRET between the eDHFR-bound terbium complex and green fluorescent protein (GFP) was detected as long-lifetime, sensitized GFP emission. Background signals from cellular autofluorescence and directly excited GFP fluorescence were effectively eliminated by imposing a time delay (10 micros) between excitation and detection. Background elimination made it possible to detect interactions between the first PDZ domain of ZO-1 (fused to eDHFR) and the C-terminal YV motif of claudin-1 (fused to GFP) in single microscope images at subsecond time scales. We observed a highly significant (P<10(-6)), six-fold difference between the mean, donor-normalized LRET signal from cells expressing interacting fusion proteins and from control cells expressing noninteracting mutants. The results show that time-resolved LRET microscopy with a selectively targeted, luminescent terbium protein label affords improved speed and sensitivity over conventional FRET methods for a variety of live-cell imaging and screening applications.

Cytoskeletal coherence requires myosin-IIA contractility.

Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in 'C' or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading.

An adaptable luminescence resonance energy transfer assay for measuring and screening protein-protein interactions and their inhibition.

Protein-protein interactions (PPIs) are central to biological processes and represent an important class of therapeutic targets. Here we show that the interaction between FK506-binding protein 12 fused to green fluorescent protein (GFP-FKBP) and the rapamycin-binding domain of mTor fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) can be sensitively detected (signal-to-background ratio (S/B)>100) and accurately quantified within an impure cell lysate matrix using a luminescence resonance energy transfer (LRET) assay. Ascomycin-mediated inhibition of GFP-FKBP-rapamycin-FRB-eDHFR complex formation was also detected at high S/B ratio (>80) and Z'-factor (0.89). The method leverages the selective, stable binding of trimethoprim (TMP)-terbium complex conjugates to eDHFR, and time-resolved, background-free detection of the long-lifetime (∼ms) terbium-to-GFP LRET signal that indicates target binding. TMP-eDHFR labeling can be adapted to develop high-throughput screening assays and complementary, quantitative counter-screens for a wide variety of PPI targets with a broad range of affinities that may not be amenable to purification.

Cell-penetrating peptides as delivery vehicles for a protein-targeted terbium complex.

Release after transmission: Arginine-rich, cell-penetrating peptides (CPPs) mediate cytoplasmic delivery of trimethoprim (TMP)-terbium complex conjugates and selective, intracellular labeling of E. coli dihydrofolate reductase (eDHFR) fusion proteins. A disulfide bond linking CPP and cargo is reduced following uptake. CPP conjugation can be used to deliver otherwise cell-impermeable, ligand-fluorophore conjugates.

Förster resonance energy transfer biosensors for fluorescence and time-gated luminescence analysis of rac1 activity.

Genetically encoded, Förster resonance energy transfer (FRET) biosensors enable live-cell optical imaging of signaling molecules. Small conformational changes often limit the dynamic range of biosensors that combine fluorescent proteins (FPs) and sensing domains into a single polypeptide. To address this, we developed FRET and lanthanide-based FRET (LRET) biosensors of Rac1 activation with two key features that enhance sensitivity and dynamic range. For one, alpha helical linker domains separate FRET partners and ensure a large conformational change and FRET increase when activated Rac1 at the biosensor C-terminus interacts with an amino-terminal Rac binding domain. Incorporation of a luminescent Tb(III) complex with long (~ ms) excited state lifetime as a LRET donor enabled time-gated luminescence measurements of Rac1 activity in cell lysates. The LRET dynamic range increased with ER/K linker length up to 1100% and enabled robust detection of Rac1 inhibition in 96-well plates. The ER/K linkers had a less pronounced, but still significant, effect on conventional FRET biosensors (with FP donors and acceptors), and we were able to dynamically image Rac1 activation at cell edges using fluorescence microscopy. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays.

Labeling proteins with long-lifetime emitting lanthanide (III) chelate reporters enables sensitive, time-resolved luminescence bioaffinity assays. Heterodimers of trimethoprim (TMP) covalently linked to various cs124-sensitized, polyaminocarboxylate chelates stably retain lanthanide ions and exhibit quantum yields of europium emission up to 20% in water. A time-resolved, luminescence resonance energy transfer (LRET) assay showed that TMP-polyaminocarboxylates bind to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins with nanomolar affinity in purified solutions and in bacterial lysates. The ability to selectively impart terbium or europium luminescence to fusion proteins in complex physiological mixtures bypasses the need for specific antibodies and simplifies sample preparation.

Lanthanide-based resonance energy transfer biosensors for live-cell applications.

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enable sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living mammalian cells. LRET biosensors are polypeptides that consist of an alpha-helical linker sequence sandwiched between a lanthanide complex-binding domain and a fluorescent protein (FP) with two interacting domains residing at each terminus. Interaction between the terminal affinity domains brings the lanthanide complex and FP in close proximity such that lanthanide-to-FP, LRET-sensitized emission is increased. A recent proof-of-concept study examined model biosensors that incorporated the affinity partners FKBP12 and the rapamycin-binding domain of m-Tor (FRB) as well as p53 (1-92) and HDM2 (1-128). The sensors contained an Escherichia coli dihydrofolate reductase (eDHFR) domain that binds with high selectivity and affinity to Tb(III) complexes coupled to the ligand trimethoprim (TMP). When cell lines that stably expressed the sensors were treated with TMP-Tb(III), TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. Much larger signal changes (>2500%) and Z'-factors of 0.5 or more were observed when cells were grown in 96-well or 384-well plates and analyzed using a TGL plate reader. In this chapter, we elaborate on the design and performance of LRET biosensors and provide detailed protocols to guide their use for live-cell microscopic imaging studies and high-throughput library screening.

Time-resolved microscopy for imaging lanthanide luminescence in living cells.

Time-resolved luminescence (TRL) microscopy can image signals from lanthanide coordination complexes or other probes with long emission lifetimes, thereby eliminating short-lifetime (<100 ns) autofluorescence background from biological specimens. However, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes, making it difficult to rapidly acquire high quality images at probe concentrations that are relevant to live cell experiments. This article describes the development and characterization of a TRL microscope that employs a light-emitting diode (LED, λ(em) = 365 nm) for pulsed epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection. Europium chelate-impregnated microspheres were used to evaluate instrument performance in terms of short-lifetime fluorescence background rejection, photon collection efficiency, image contrast, and signal-to-noise ratio (SNR). About 200 nm microspheres were imaged within the time resolution limit of the ICCD (66.7 ms) with complete autofluorescence suppression. About 40 nm microspheres containing ~400 chelate molecules were detected within ~1-s acquisition times. A luminescent terbium complex, Lumi4-Tb®, was introduced into the cytoplasm of cultured cells at an estimated concentration of 300 nM by the method of osmotic lysis of pinocytic vesicles. Time-resolved images of the living, terbium complex-loaded cells were acquired within acquisition times as short as 333 ms, and the effects of increased exposure time and frame summing on image contrast and SNR were evaluated. The performance analyses show that TRL microscopy is sufficiently sensitive and precise to allow high-resolution, quantitative imaging of lanthanide luminescence in living cells under physiologically relevant experimental conditions.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1-92) and HDM2 (1-128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z'-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Luminescent terbium protein labels for time-resolved microscopy and screening.

Brilliance of terbium: Heterodimeric conjugates of trimethoprim covalently linked to sensitized terbium chelates bind to Escherichia coli dihydrofolate reductase fusion proteins with nanomolar affinity (see picture). Terbium luminescence enables sensitive and time-resolved detection of labeled proteins in vitro and on the surface of living mammalian cells.

Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis.

Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.