While DNA-directed nanotechnology is now a well-established platform for bioinspired nanoscale assembly in vitro, the direct targeting of various nanomaterials in living biological systems remains a significant challenge. Hybrid biological systems with integrated and targeted nanomaterials may have interesting and exploitable properties, so methods for targeting various nanomaterials to precise biological locations are required. Fluorescence imaging has benefited from the use of nanoparticles with superior optical properties compared to fluorescent organic dyes or fluorescent proteins. While single-particle tracking (SPT) in living cells with genetically encoded proteins is limited to very short trajectories, the high photon output of genetically targeted and multiplexed quantum dots (QDs) would enable long-trajectory analysis of multiple proteins. However, challenges with genetic targeting of QDs limit their application in these experiments. In this report, we establish a modular method for targeting QD nanoparticles selectively to multiple genetically encoded tags by precomplexing QD-streptavidin conjugates with cognate biotinylated hapten molecules. This approach enables labeling and SPT of multiple genetically encoded proteins on living cells at high speed and can label expressed proteins in the cytosol upon microinjection into living cells. While we demonstrate labeling with three distinct QD conjugates, the approach can be extended to other specific hapten-affinity molecule interactions and alternative nanoparticles, enabling precise directed targeting of nanoparticles in living biological systems.
Crosslinking of IgE bound Fc?RI on mast cells and basophils by multivalent antigen leads to degranulation and the release of key inflammatory mediators which stimulate the allergic response. Here, we present and characterize the use of Fluorogen Activating Proteins (FAPs) for single particle tracking of Fc?RI 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 Fc?RI ?-subunit fused to a FAP (FAP-?) provides, for the first time, an IgE-independent probe for tracking this signaling subunit of Fc?RI at the single molecule level. We show that the Fc?RI ?-subunit dynamics are controlled by the IgE binding ?-subunit and that the cytokinergic IgE, SPE-7, induces mast cell activation without altering Fc?RI mobility or promoting internalization. We take advantage of the far-red emission of the malachite green (MG) fluorogen to track Fc?RI 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 between Fc?RI structure, activity and mobility.
Biomolecular detection and imaging methods provide quantitative measurements essential for biological research. In this context, molecular beacon based sensors have emerged as powerful, no-wash imaging agents, providing target-specific fluorescent activation for nucleic acids, proteins, and small molecules. Conventional molecular beacons require double-labeled DNA sequences, which are costly and time-consuming to prepare. To address this issue, we developed DNA based label-free molecular beacons consisting of two regions: a signal-generating region based on human telomeric G-quadruplex sequence that activates Thioflavin T fluorescence and a target recognition sequence designed to interact in a molecular beacon format. We demonstrated the utility of these probes for the selective detection of DNA, RNA, and protein. Multiple probes were applied against a single target to achieve improved brightness in fluorescence detection of nucleic acid targets. This label-free strategy provides a straightforward, cost-effective alternative to fluorescently labeled oligonucleotides in biomolecular detection and imaging.
Monoclonal antibodies are one of the most useful and ubiquitous affinity reagents used in the biological sciences. Immunostaining of fixed and live cells for microscopy or cytometry measurements frequently employs fluorescently labeled antibodies, in particular fluorescein-labeled antibodies. This dye emits light at a wavelength overlapping with cellular autofluorescence, making it difficult to measure antibody binding to proteins of relatively low copy number or in cells of high green autofluorescence. A number of high affinity fluorescein binding antibodies and antibody domains have been developed that quench the dye's fluorescence. Using a fluorescein-binding recombinant antibody domain genetically fused to a fluorogen activating protein (FAP), we demonstrate a molecular converter capable of binding and quenching fluorescein, while binding and activating a fluorogenic triarylmethane dye. This reagent converts fluorescein conjugates to far-red fluorescent probes, where cellular autofluorescence is low, improving signal-to-background of cell-based antibody binding measurements by ?7-fold. Microscopy experiments show colocalization of both fluorescein and MG fluorescence. This dual affinity fluorescein-quenching-FAP can also be used to convert fluorescein to the red fluorescing MG fluorogen on biological molecules other than antibodies.
Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (?90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.
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.
The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen-activating protein (FAP) can produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and super-resolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved by using physiological buffers and cellular media, in which additives and switching buffers are not required to obtain super-resolution images. We evaluate the super-resolution properties and images obtained from a selected FAP clone fused to actin, and show that the photon counts per object are between those typically reported for fluorescent proteins and switching-dye pairs, resulting in 10-30?nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures.
With the advent of superresolution imaging methods, fast dynamic imaging of biological processes in live cells remains a challenge. A subset of these methods requires the cellular targets to be labeled with spontaneously blinking probes. The delivery and specific targeting of cytosolic targets and the control of the probes blinking properties are reviewed for three types of blinking probes: quantum dots, synthetic dyes, and fluorescent proteins.
We report that a symmetric small-molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* fluorogen activating protein is a VL domain that binds malachite green (MG) dye to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity-determining regions are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high-affinity protein tags and capture reagents.
The integration of synthetic and cell-free biology has made tremendous strides towards creating artificial cellular nanosystems using concepts from solution-based chemistry, where only the concentrations of reacting species modulate gene expression rates. However, it is known that macromolecular crowding, a key feature in natural cells, can dramatically influence biochemical kinetics via volume exclusion effects, which reduce diffusion rates and enhance binding rates of macromolecules. Here, we demonstrate that macromolecular crowding can increase the robustness of gene expression by integrating synthetic cellular components of biological circuits and artificial cellular nanosystems. Furthermore, we reveal how ubiquitous cellular modules, including genetic components, a negative feedback loop and the size of the crowding molecules can fine-tune gene circuit response to molecular crowding. By bridging a key gap between artificial and living cells, our work has implications for efficient and robust control of both synthetic and natural cellular circuits.
The rapid accumulation of neuroproteomics data in recent years has prompted the emergence of novel antibody-based imaging methods that aim to understand the anatomical and functional context of the multitude of identified proteins. The pioneering field of ultrastructural multiplexed proteomic imaging now includes a number of high resolution methods, such as array tomography, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy and automated transmission electron microscopy, which allow a detailed molecular characterization of individual synapses and subsynaptic structures within brain tissues for the first time. While all of these methods still face considerable limitations, a combined complementary approach building on the respective strengths of each method is possible and will enable fascinating research into the proteomic diversity of the nervous system.
Thirteen years after the demonstration of quantum dots as biological imaging agents, and nine years after the initial commercial introduction of bioconjugated quantum dots, the brightness and photostability of the quantum dots has enabled a range of investigations using single molecule tracking. These materials are being routinely utilized by a number of groups to track the dynamics of single molecules in reconstituted biophysical systems and on living cells, and are especially powerful for investigations of single molecules over long timescales with short exposure times and high pointing accuracy. New approaches are emerging where the quantum dots are used as hard-sphere probes for intracellular compartments. Innovations in quantum dot surface modification are poised to substantially expand the utility of these materials.
The valency of quantum dot nanoparticles conjugated with biomolecules is closely related to their performance in cell tagging, tracking, and imaging experiments. Commercially available streptavidin conjugates (SAv QDs) are the most commonly used tool for preparing QD-biomolecule conjugates. The fluorescence quenching of biotin-4-fluorscein (B4F) provides a straightforward assay to quantify the number of biotin binding sites per SAv QD. The utility of this method was demonstrated by quantitatively characterizing the biotin binding capacity of commercially available amphiphilic poly(acrylic acid) Qdot ITK SAv conjugates and poly(ethylene glycol) modified Qdot PEG SAv conjugates with emission wavelengths of 525, 545, 565, 585, 605, 625, 655, 705, and 800 nm. Results showed that 5- to 30-fold more biotin binding sites are available on ITK SAv QDs compared to PEG SAv QDs of the same color with no systematic variation of biotin binding capacity with size.
We have developed a class of dendron-based fluorogenic dyes (termed dyedrons) comprised of multiple cyanine (Cy3) donors coupled to a single malachite green (MG) acceptor that fluoresce only when the MG is noncovalently but specifically bound to a cognate single chain antibody (scFv). These cell-impermeant dyedrons exploit efficient intramolecular energy transfer from Cy3 donors to stoichiometrically amplify the fluorescence of MG chromophores that are activated by binding to the scFv. These chromophore enhancements, coupled with our optimized scFv, can significantly increase fluorescence emission generated by the dyedron/scFv complex to brightness levels several-fold greater than that for single fluorescent proteins and targeted small molecule fluorophores. Efficient intramolecular quenching of free dyedrons enables sensitive homogeneous (no wash) detection under typical tissue culture conditions, with undetectable nonspecific activation.
Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (beta2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.
Quantum dots are ideal probes for fluorescent imaging of vascular and lymphatic tissues. On injection into appropriate sites, red- and near-infrared-emitting quantum dots provide excellent definition of vasculature, lymphoid organs, and lymph nodes draining both normal tissues and tumors. We detail methods for use with commercially available quantum dots and discuss common difficulties.
Observations of quantum dot (QD) labeled cells in biomedical research are mainly qualitative in nature, which limits the ability of researchers to compare results experiment-to-experiment and lab-to-lab to improve the state-of-the-art. Labeled cells are useful in a range of in vitro and in vivo assays where tracking behavior of administered cells is integral for answering research questions in areas such as tissue engineering and stem cell therapy. Before the full potential of QD based toolsets can be realized in the clinic, uptake of QDs by cells must be quantified and standardized. This unit describes a novel, simple method to assess the number of QDs per cell using flow cytometry and commercially available standards. This quick and easy method can be used by all researchers to calibrate their flow cytometry instruments and settings, and quantify QD uptake by cells for in vitro and in vivo experimentation for comparable results across QD conjugate types, cell types, research groups, lots of commercial QDs, and homemade QDs.
Quantum dots are a powerful fluorophore family with desirable attributes for fluorescence imaging. They have been used in several animal models with direct clinical relevance, including sentinel lymph node mapping, tracing vasculature and lymphatics, and targeting specific lesions for diagnosis and removal. (1-12) Despite significant interest for use in translational applications, little is known about the persistence and long-term fate of quantum dots in vivo. We have observed fluorescence of quantum dots injected into Balb/c and nude mice for up to two-years post injection using both whole-body and microscopic fluorescence techniques. Two-photon spectral microscopy was used to verify the existence of quantum dots within two-year tissues, but also revealed a range of significantly blue-shifted emission peaks with increased bandwidths. Systemically administered quantum dots persist and retain fluorescence for up to two-years in vivo, but with significantly blue-shifted emission.
We demonstrate the effectiveness of a genetically encoded Malachite Green (MG) binding fluorogen activating protein (FAP) for live cell stimulated emission depletion nanoscopy (STED). Both extracellular and intracellular FAPs were tested in living cells using fluorogens with either membrane expressed FAP or as an intracellular FAP-actin fusion. Structures with FWHM of 110-122nm were observed. Depletion data however suggests a resolution of 70nm with the given instrument.
To test the feasibility of localized intravaginal therapy directed to neighboring lymph nodes, the transport of quantum dots across the vaginal wall was investigated. Quantum dots instilled into the mouse vagina were transported across the vaginal mucosa into draining lymph nodes, but not into distant nodes. Most of the particles were transported to the lumbar nodes; far fewer were transported to the inguinal nodes. A low level of transport was evident at 4 hr after intravaginal instillation, and transport peaked at about 36 hr after instillation. Transport was greatly enhanced by prior vaginal instillation of Nonoxynol-9. Hundreds of micrograms of nanoparticles/kg tissue (ppb) were found in the lumbar lymph nodes at 36 hr post-instillation. Our results imply that targeted transport of microbicides or immunogens from the vagina to local lymph organs is feasible. They also offer an in vivo model for assessing the toxicity of compounds intended for intravaginal use.
Color bind: We have developed a probe TMR-para-MG that switches its fluorescence emission upon binding to a fluorogen-activating protein (FAP). In cells that express FAP, this dye labels target sites in one color and mitochondria in another color, thus it might be a suitable tool for monitoring changes in mitochondrial membrane potential.
Localization-based super resolution imaging is presently limited by sampling requirements for dynamic measurements of biological structures. Generating an image requires serial acquisition of individual molecular positions at sufficient density to define a biological structure, increasing the acquisition time. Efficient analysis of biological structures from sparse localization data could substantially improve the dynamic imaging capabilities of these methods. Using a feature extraction technique called the Hough Transform simple biological structures are identified from both simulated and real localization data. We demonstrate that these generative models can efficiently infer biological structures in the data from far fewer localizations than are required for complete spatial sampling. Analysis at partial data densities revealed efficient recovery of clathrin vesicle size distributions and microtubule orientation angles with as little as 10% of the localization data. This approach significantly increases the temporal resolution for dynamic imaging and provides quantitatively useful biological information.
Traffic cam: a tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of ?(2)-adrenergic receptors. It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown).
The ability to detect single molecules over the electronic noise requires high performance detector systems. Electron Multiplying Charge-Coupled Device (EMCCD) cameras have been employed successfully to image single molecules. Recently, scientific Complementary Metal Oxide Semiconductor (sCMOS) based cameras have been introduced with very low read noise at faster read out rates, smaller pixel sizes and a lower price compared to EMCCD cameras. In this study, we have compared the two technologies using two EMCCD and three sCMOS cameras to detect single Cy5 molecules. Our findings indicate that the sCMOS cameras perform similar to EMCCD cameras for detecting and localizing single Cy5 molecules.
The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit ?4 alters channel Ca(++)- and voltage-sensitivity, and ?4 knock-out animals exhibit spontaneous seizures. Here we investigate ?4s effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BK? subunit in living cells, we find that ?4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high ?4 expression, whole-cell BK channel currents display none of the characteristic properties of BK?+?4 channels observed in heterologous cells. Finally, ?4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that ?4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific ?4 subunit in CA3 neurons is control of surface trafficking.
The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g., EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extracellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes (activate the fluorogen). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000-fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (?2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well.
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