RESUMEN
The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1'-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to â¼ 31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin.
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Proteínas Bacterianas , Polarización de Fluorescencia/métodos , Proteínas Luminiscentes , Sondas Moleculares , Complejos Multiproteicos , Proteínas/análisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cartilla de ADN/genética , Escherichia coli , Transferencia Resonante de Energía de Fluorescencia , Ingeniería Genética/métodos , Hidrodinámica , Procesamiento de Imagen Asistido por Computador , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Sondas Moleculares/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Plásmidos/genética , Trombina/análisis , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
The shape of motile cells is determined by many dynamic processes spanning several orders of magnitude in space and time, from local polymerization of actin monomers at subsecond timescales to global, cell-scale geometry that may persist for hours. Understanding the mechanism of shape determination in cells has proved to be extremely challenging due to the numerous components involved and the complexity of their interactions. Here we harness the natural phenotypic variability in a large population of motile epithelial keratocytes from fish (Hypsophrys nicaraguensis) to reveal mechanisms of shape determination. We find that the cells inhabit a low-dimensional, highly correlated spectrum of possible functional states. We further show that a model of actin network treadmilling in an inextensible membrane bag can quantitatively recapitulate this spectrum and predict both cell shape and speed. Our model provides a simple biochemical and biophysical basis for the observed morphology and behaviour of motile cells.
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Movimiento Celular/fisiología , Forma de la Célula/fisiología , Cíclidos , Células Epiteliales/citología , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animales , Fenómenos Biofísicos , Biofisica , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Modelos Biológicos , Seudópodos/metabolismo , Factores de TiempoRESUMEN
Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.
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Microscopía Fluorescente/instrumentación , Dispositivos Ópticos , Diagnóstico por Imagen/métodos , Microscopía Fluorescente/métodosRESUMEN
BACKGROUND: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs. METHODS: Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells. RESULTS: Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs. CONCLUSION: We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells.
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Cell-generated extracellular vesicles (EVs) are being engineered as biologically-inspired vehicles for targeted delivery of therapeutic agents to treat difficult-to-manage human diseases, including lysosomal storage disorders (LSDs). Engineered EVs offer distinct advantages for targeted delivery of therapeutics compared to existing synthetic and semi-synthetic nanoscale systems, for example with regard to their biocompatibility, circulation lifetime, efficiencies in delivery of drugs and biologics to target cells, and clearance from the body. Here, we review literature related to the design and preparation of EVs as therapeutic carriers for targeted delivery and therapy of drugs and biologics with a focus on LSDs. First, we introduce the basic pathophysiology of LDSs and summarize current approaches to diagnose and treat LSDs. Second, we provide specific details about EVs, including subtypes, biogenesis, biological properties and their potential to treat LSDs. Third, we review state-of-the-art approaches to engineer EVs for treatments of LSDs. Finally, we summarize explorative basic research and applied applications of engineered EVs for LSDs, and highlight current challenges, and identify new directions in developing EV-based therapies and their potential impact on clinical medicine.
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Productos Biológicos , Exosomas , Vesículas Extracelulares , Enfermedades por Almacenamiento Lisosomal , Sistemas de Liberación de Medicamentos , Vesículas Extracelulares/fisiología , Humanos , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , LisosomasRESUMEN
A major challenge in cell biology is to elucidate molecular mechanisms that underlie the spatio-temporal control of cellular processes. These studies require microscope imaging techniques and associated optical probes that provide high-contrast and high-resolution images of specific proteins and their complexes. Auto-fluorescence however, can severely compromise image contrast and represents a fundamental limitation for imaging proteins within living cells. We have previously shown that optical switch probes and optical lock-in detection (OLID) image microscopy improve image contrast in high background environments. Here, we present the design, synthesis, and characterization of amino-reactive and cell permeable optical switches that integrate the highly fluorescent fluorophore, tetramethylrhodamine (TMR) and spironaphthoxazine (NISO), a highly efficient optical switch. The NISO moiety in TMR-NISO undergoes rapid and reversible, excited-state driven transitions between a colorless spiro (SP)-state and a colored merocyanine (MC)-state in response to irradiation with 365 and >530nm light. In the MC-state, the TMR (donor) emission is almost completely extinguished by Förster resonance energy transfer (FRET) to the MC probe (acceptor), whereas in the colorless SP-state, the quantum yield for TMR fluorescence is maximal. Irradiation of TMR-NISO with a defined sequence of 365 and 546nm manipulates the levels of SP and MC with concomitant modulation of FRET efficiency and the TMR fluorescence signal. High fidelity optical switching of TMR fluorescence is shown for TMR-NISO probes in vitro and for membrane permeable TMR-NISO within living cells.
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Fenómenos Fisiológicos Celulares , Colorantes Fluorescentes , Microscopía Fluorescente/métodos , Microscopía/métodos , Imagen Molecular/métodos , Oxazinas , Compuestos de Espiro , Benzopiranos/química , Benzopiranos/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Indoles/química , Indoles/metabolismo , Luz , Oxazinas/química , Rodaminas/química , Rodaminas/metabolismo , Compuestos de Espiro/químicaRESUMEN
One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed "optical switches." This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.
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Imagenología Tridimensional/métodos , Microscopía de Contraste de Fase/métodos , Actinas , Animales , Supervivencia Celular , Células Cultivadas , Colorantes Fluorescentes/química , Ratones , Microscopía Fluorescente , Músculos/citología , Células 3T3 NIH , Neuronas/citología , Ratas , Xenopus , Pez CebraRESUMEN
Spironaphthoxazine (NISO) is an efficient optical switch probe that has applications in high contrast detection of Foerster resonance energy transfer (FRET) using optical lock-in detection (OLID). NISO exists in two distinct states spiro (SP) and merocyanine (MC) that can be independently controlled by using alternate irradiation with near ultraviolet and visible light. Unfortunately, the SP-state of NISO has an absorption centered at 350 nm, which may lead to phototoxic effects when manipulating the probe within a living cell. To overcome this problem we introduce new, red-shifted amino substituted NISO probes compared to NISO that undergo an efficient SP to MC transition in response to irradiation by using 405-nm light, which is less damaging to living cells. This study details the synthesis of amino-substituted NISO and their N-hydroxysuccinimide ester and maleimide derivatives and their use in generating covalent attached protein conjugates. This study also presents a characterization of the spectroscopic and optical switching properties of these red-shifted NISO probe in solution.
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Current interest in at-home diagnostic devices derives from their potential to disrupt expensive and time-consuming hospital-based diagnostic practices. Conventional immunoassays are often touted for use in at-home diagnostic devices, although in practice they are slow, labor-intensive and require expensive equipment. Here, we introduce bead-based sensors as alternative biomarker detection platforms for at-home diagnostic devices. The immunocomplex entrapment assay (ICEA), and the related enzyme-linked ICEA (ELICEA) offer enhancements over conventional immunoassays in terms of their speed, and minimal requirements for user intervention and instrumentation. In particular, we designed bead-based sensors to entrap large molecular weight complexes between target molecules and signal-generating immunoconjugates while allowing any unbound conjugates to escape from the bead. Confocal fluorescence microscopy was used to demonstrate the sensitivity, robustness, and reproducibility of the ICEA and ELICEA platforms. For example, we showed the intensity of signals generated by entrapped immunoconjugate complexes correlate linearly with the concentration of target molecule in the sample. We employed ICEA, and ELICEA platforms to detect human forms of immunoglobulins, albumin, and κ light chain (KLC). For example, we used ICEA to detect KLC at 5 µg·mL-1 in urine, which would allow for earlier diagnosis of Bence-Jones disease compared to conventional assays. In addition, we showed bead-entrapped phosphatases (AP) in immunocomplexes generate insoluble, blue-colored dyes from AP-substrates that accumulate in beads and allow for visual and cellphone camera-based detection of IgG to 10 ng·mL-1 within 20 min. Finally, we described ICEA and ELICEA platforms to analyze multiple target proteins within individual beads.
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Biomarcadores/química , Técnicas y Procedimientos Diagnósticos/instrumentación , Enfermedad/genética , Inmunoensayo/instrumentación , HumanosRESUMEN
Exosomes are cell-derived extracellular vesicles that have great potential in the field of nano-medicine. However, a fundamental challenge in the engineering of exosomes is the design of biocompatible molecular scaffolds on their surface to enable cell targeting and therapeutic functions. CD63 is a hallmark protein of natural exosomes that is highly enriched on the external surface of the membrane. We have previously described engineering of CD63 for use as a molecular scaffold in order to introduce cell-targeting features to the exosome surface. Despite this initial success, the restrictive M-shaped topology of full-length CD63 may hinder specific applications that require N- or C-terminal display of cell-targeting moieties on the outer surface of the exosome. In this study, we describe new and topologically distinct CD63 scaffolds that enable robust and flexible surface engineering of exosomes. In particular, we conducted sequential deletions of the transmembrane helix of CD63 to generate a series of CD63 truncates, each genetically-fused to a fluorescent protein. Molecular and cellular characterization studies showed truncates of CD63 harboring the transmembrane helix 3 (TM3) correctly targeted and anchored to the exosome membrane and exhibited distinct n-, N-, Ω-, or I-shaped membrane topologies in the exosomal membrane. We further established that these truncates retained robust membrane-anchoring and exosome-targeting activities when stably expressed in the HEK293 cells. Moreover, HEK293 cells produced engineered exosomes in similar quantities to cells expressing full-length CD63. Based on the results of our systematic sequential deletion studies, we propose a model to understand molecular mechanisms that underlie membrane-anchoring and exosome targeting features of CD63. In summary, we have established new and topologically distinct scaffolds based on engineering of CD63 that enables flexible engineering of the exosome surface for applications in disease-targeted drug delivery and therapy.
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Exosomas , Vesículas Extracelulares , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Células HEK293 , Humanos , Transporte de Proteínas , Proteínas/metabolismo , Tetraspanina 30/genética , Tetraspanina 30/metabolismoRESUMEN
Ultrasensitive and sustainable near-infrared (NIR)-emitting piezoluminescence is observed from noncentrosymmetric and ferroelectric-phase Sr3 Sn2 O7 doped with rare earth Nd3+ ions. Sr3 Sn2 O7 :Nd3+ (SSN) with polar A21 am structure is demonstrated to emit piezoluminescence of wavelength of 800-1500 nm at microstrain levels, which is enhanced by the ferroelectrically polarized charges in the multipiezo material. These discoveries provide new research opportunities to study luminescence properties of multipiezo and piezo-photonic materials, and to explore their potential as novel ultrasensitive probes for deep-imaging of stress distributions in diverse materials and structures including artificial bone and other implanted structures (in vivo, in situ, etc).
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Actin polymerization and dynamics are involved in a wide range of cellular processes such as cell division and migration of tumor cells. At sites of cell lysis, such as those occurring during a stroke or inflammatory lung diseases, actin is released into the serum where it polymerizes, leading to problems with clot dissolution and sputum viscosity. Therefore, drugs that target these actin-mediated processes may provide one mechanism to treat these conditions. Marine-organism-derived macrolides, such as reidispongiolide A, can bind to, sever, and inhibit polymerization of actin. Our studies show that the function of these complex macrolides resides in their tail region, whereas the head group stabilizes the actin-drug complex. Synthetic compounds derived from this tail region could therefore be used as a mimetic of the natural product, providing a range of designer compounds to treat actin-associated diseases or as probes to study actin polymerization.
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Actinas/metabolismo , Materiales Biomiméticos/síntesis química , Materiales Biomiméticos/farmacología , Diseño de Fármacos , Macrólidos/química , Macrólidos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/antagonistas & inhibidores , Animales , Materiales Biomiméticos/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Cetonas/química , Cetonas/metabolismo , Cetonas/farmacología , Cinética , Macrólidos/farmacología , Unión Proteica/efectos de los fármacos , Ratas , Vejiga Urinaria/citologíaRESUMEN
Targeted delivery of lysosomal enzymes to the endocytic compartment of human cells represents a transformative technology for treating a large family of lysosomal storage diseases (LSDs). Gaucher disease is one of the most common types of LSDs caused by mutations to the lysosomal ß-glucocerebrosidase (GBA). Here, we describe a genetic strategy to produce engineered exosomes loaded with GBA in two different spatial configurations for targeted delivery to the endocytic compartment of recipient cells. By fusing human GBA to an exosome-anchoring protein: vesicular stomatitis virus glycoprotein (VSVG), we demonstrate that the chimeric proteins were successfully integrated into exosomes which were secreted as extracellular vesicles (EVs) by producer cells. Isolation and molecular characterization of EVs confirmed that the fusion proteins were loaded onto exosomes without altering their surface markers, particle size or distribution. Further, enzyme-loaded exosomes/EVs added to cultured medium were taken up by recipient cells. Further, the endocytosed exosomes/EVs targeted to endocytic compartments exhibited a significant increase in GBA activity. Together, we have developed a novel method for targeting and delivery of lysosomal enzymes to their natural location: the endocytic compartment of recipient cells. Since exosomes/EVs have an intrinsic ability to cross the blood-brain-barrier, our technology may provide a new approach to treat severe types of LSDs, including Gaucher disease with neurological complications.
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Sistemas de Liberación de Medicamentos/métodos , Vesículas Extracelulares/fisiología , Lisosomas/metabolismo , Transporte Biológico , Línea Celular , Endocitosis , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/enzimología , Nanopartículas , Tamaño de la PartículaRESUMEN
Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα.
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Productos Biológicos/metabolismo , Exosomas/metabolismo , Inflamación/metabolismo , Células HEK293 , Humanos , Nanopartículas , Dominios Proteicos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cryoelectron microscopy studies have identified distinct locations of tropomyosin (Tm) within the Ca(2+)-free, Ca(2+)-saturated, and myosin-S1-saturated states of the thin filament. On the other hand, steady-state Förster resonance energy transfer (FRET) studies using functional, reconstituted thin filaments under physiological conditions of temperature and solvent have failed to detect any movement of Tm upon Ca(2+) binding. In this investigation, an optimized system for FRET and anisotropy analyses of cardiac tropomyosin (cTm) dynamics was developed that employed a single tethered donor probe within a Tm dimer. Multisite FRET and fluorescence anisotropy analyses showed that S1 binding to Ca(2+) thin filaments triggered a uniform displacement of cTm toward F-actin but that Ca(2+) binding alone did not change FRET efficiency, most likely due to thermally driven fluctuations of cTm on the thin filament that decreased the effective separation of the donor probe between the blocked and closed states. Although Ca(2+) binding to the thin filament did not significantly change FRET efficiency, such a change was demonstrated when the thin filament was partially saturated with S1. FRET was also used to show that stoichiometric binding of S1 to Ca(2+)-activated thin filaments decreased the amplitude of Tm fluctuations and revealed a strong correlation between the cooperative binding of S1 to the closed state and the movement of cTm.
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Actinas/química , Calcio/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Motoras Moleculares/química , Miocardio/metabolismo , Tropomiosina/química , Anisotropía , Sitios de Unión , Movimiento (Física) , Unión Proteica , Conformación ProteicaRESUMEN
Thiol-reactive optical switch probes were used to examine conformational changes of prestin-based membrane motor. Because this motor is based on mechanoelectric coupling similar to piezoelectricity, the motile activity can be monitored by charge movements across the plasma membrane, which appears as nonlinear capacitance. When the plasma membrane is conjugated with the probes, optically induced spiro-merocyanine transition positively shifted nonlinear capacitance of outer hair cells and prestin-transfected cells by approximately 10 mV. These shifts were reversible and were eliminated by pretreatment with iodoacetamide. However, they were little affected by pretreatment with biotin maleimide, which cannot reach the cytoplasmic surface. Our results showed that merocyanine states, with a larger dipole moment, interact with the motor's extended conformation stronger than with the compact conformation by 1.6 x 10(-21) J/molecule. The interaction sites are near the cytoplasmic side of the motor protein.
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Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/metabolismo , Óptica y Fotónica , Compuestos de Sulfhidrilo/metabolismo , Animales , Biotina/análogos & derivados , Membrana Celular/metabolismo , Cisteína/farmacología , Capacidad Eléctrica , Cobayas , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/metabolismo , Humanos , Yodoacetamida/farmacología , Maleimidas/química , Maleimidas/farmacología , Conformación Proteica , Compuestos de Sulfhidrilo/química , TransfecciónRESUMEN
The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/metabolismo , Ingeniería de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Óptica y FotónicaRESUMEN
Botulinum neurotoxin (BoNT) injection into the thyroarytenoid (TA) muscle is a commonly performed medical intervention for adductor spasmodic dysphonia. The mechanism of action of BoNT at the neuromuscular junction is well understood, however, aside from reports focused on myosin heavy chain isoform abundance, there is a paucity of data addressing the effects of therapeutic BoNT injection on the TA muscle proteome. In this study, 12 adult Sprague Dawley rats underwent unilateral TA muscle BoNT serotype A injection followed by tissue harvest at 72 h, 7 days, 14 days, and 56 days postinjection. Three additional rats were reserved as controls. Proteomic analysis was performed using 2-D SDS-PAGE followed by MALDI-MS. Vocal fold movement was significantly reduced by 72 h, with complete return of function by 56 days. Twenty-five protein spots demonstrated significant protein abundance changes following BoNT injection, and were associated with alterations in energy metabolism, muscle contractile function, cellular stress response, transcription, translation, and cell proliferation. A number of protein abundance changes persisted beyond the return of gross physiologic TA function. These findings represent the first report of BoNT-induced changes in any skeletal muscle proteome, and reinforce the utility of applying proteomic tools to the study of system-wide biological processes in normal and perturbed TA muscle function.
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Toxinas Botulínicas/química , Toxinas Botulínicas/farmacología , Músculos Laríngeos/efectos de los fármacos , Músculos Laríngeos/metabolismo , Proteómica/métodos , Animales , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Masculino , Espectrometría de Masas/métodos , Modelos Anatómicos , Cadenas Pesadas de Miosina/química , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de TiempoRESUMEN
Timolol, a potent inhibitor of ß-adrenergic receptors (ßARs), is a first-line drug for decreasing the intraocular pressure (IOP) of patients with glaucoma. Timolol is administered using 0.5% eye-drop solutions at >3 × 107 times the inhibitory concentration (k i) for ßARs. This high dose is wasteful and triggers off-target effects that increase medication noncompliance. Here, we introduce contact lenses that release timolol to the eye throughout the day during passive exposures to natural daylight at a more therapeutically relevant concentration (>3000 k i). Timolol is coupled to the polymer of the contact lens via a photocleavable caged cross-linker and is released exclusively to the surrounding fluid after the 400-430 nm mediated cleavage of the cross-linking group. Studies conducted in a preclinical mouse model of glaucoma show photoreleased timolol is effective as authentic timolol in reducing IOP. Our studies highlight several advantages of daylight-mediated release of timolol from lenses compared to eye-drops. First, fitted contact lenses exposed to natural daylight release sufficient timolol to sustain the inhibition of ßARs over a 10 h period. Second, the contact lenses inhibit ßARs in the eye using only 5.7% of the timolol within a single eye-drop. Third, the lenses allow the patient to passively control the amount of timolol released from the lens-for example, early morning exposure to outdoor sunlight would release enough timolol to maximally reduce the IOP, whereas subsequent periodic exposures to indoor daylight would release sufficient timolol to overcome the effects of its spontaneous dissociation from ßARs. Fourth, our lenses are disposable, designed for single day use, and manufactured at a low cost.
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Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells.