RESUMO
Introduction of bioorthogonal functionalities (e.g., trans-cyclooctene-TCO) into a protein of interest by site-specific genetic encoding of non-canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine-functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours-long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO-ncAAs. One derivative, DOTCO-lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.
Assuntos
Aminoácidos/química , Ciclo-Octanos/química , Corantes Fluorescentes/química , Proteínas/química , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Fluorescência , Estrutura MolecularRESUMO
Super-resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine-based GCE system for click chemistry, combined with DNA-PAINT microscopy, enables the visualization of even low-abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue-specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.
Assuntos
DNA/genética , Células Eucarióticas/citologia , Código Genético , Química Click , Humanos , Microscopia de Fluorescência , Engenharia de ProteínasRESUMO
Site-specific labeling of biomolecules is rapidly advancing due to the discovery of novel mutually orthogonal reactions. Quantum chemistry studies have also increased our understanding of their relative rates, although these have until now been based on highly simplified reactants. Here we examine a set of strain-promoted click-type cycloaddition reactions of n-propyl azide, 3-benzyl tetrazine and 3-benzyl-6-methyl tetrazine with cyclooctenes/ynes, in which we aim to address all relevant structural details of the reactants. Our calculations have included the obligatory handles used to attach the label and biomolecule as these can critically influence the stereochemistry and electron demand of the reaction. We systematically computed orbital gaps, activation and distortion energies using density functional theory and determined experimental rates for validation. Our results challenge the current paradigm of the inverse electron demand for this class of reactions. We found that the ubiquitous handles, when next to the triple bond of cyclooctynes, can switch the Diels-Alder type ligations to normal electron demand, a class we term as SPINEDAC reactions. Electron donating substituents on tetrazine can enhance normal demand but also increase distortion penalties. The presence and isomeric configuration of handles thus determine the reaction speed and regioselectivity. Our findings can be directly utilized in engineering genuine cycloaddition click chemistries for biological labeling.
RESUMO
trans-Cyclooctene groups incorporated into proteins via non-canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chemistry. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans-cyclooct-2-ene isomers (1 a,b). We further show that the axially connected isomer has a half-life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as determined by stopped-flow experiments. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.
Assuntos
Aminoácidos/química , Ciclo-Octanos/química , Linhagem Celular , Estrutura MolecularRESUMO
The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.
Assuntos
Marcadores Fiduciais , Raios Infravermelhos , Lasers , Microscopia Eletrônica/métodos , Microscopia/métodos , Animais , Córtex Cerebral/ultraestrutura , Elétrons , Fluorescência , Túbulos Renais/ultraestrutura , Linfonodos/ultraestrutura , Macrófagos/ultraestrutura , Camundongos , Fixação de TecidosRESUMO
Synthesis of a set of new, azide bearing, biorthogonally applicable fluorogenic dyes with large Stokes shifts is presented herein. To assess the fluorogenic performance of these new dyes we have labeled a genetically modulated, cyclooctyne-bearing protein in lysate medium. Studies showed that the labels produce specific signal with minimal background fluorescence. We also provide theoretical insights into the design of such fluorogenic labels.
Assuntos
Azidas/química , Ciclo-Octanos/química , Corantes Fluorescentes/química , Proteínas Ligantes de Maltose/química , Imagem Óptica/métodos , Triazóis/química , Fluorescência , HumanosRESUMO
The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.
Assuntos
Corantes Fluorescentes/química , Microscopia Confocal , Aminoácidos/química , Reação de Cicloadição , Elétrons , Células HEK293 , Hemaglutininas/química , Hemaglutininas/genética , Hemaglutininas/metabolismo , Humanos , Orthomyxoviridae/metabolismo , Engenharia de Proteínas , Receptor de Insulina/química , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Synthetic procedures for the construction of fluorogenic azido-labels were developed. Photophysical properties were elaborated by experimental and theoretical investigations. Of the newly synthesized fluorogenic and bioorthogonally applicable dyes two were selected on the basis of their fluorogenic performance and further subjected to in vitro and in vivo studies. Both tags exhibited excellent fluorogenic properties as in aqueous medium, the azide form of the selected dyes is virtually non-fluorescent, while their "clicked" triazole congeners showed intense fluorescence. One of these labels showed a very large Stokes shift. To the best of our knowledge this is the first reported case of mega-Stokes type of fluorogenic labels. These studies have justified that these two fluorogenic tags are remarkably suitable for bioorthogonal tagging schemes. The developed synthetic approach together with the theoretical screen of possible fluorogenic tags will enable the generation of libraries of such tags in the future.
Assuntos
Azidas/síntese química , Corantes Fluorescentes/síntese química , Azidas/química , Fluorescência , Corantes Fluorescentes/química , Estrutura MolecularRESUMO
Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.
Assuntos
Aminoácidos/genética , Escherichia coli/genética , Genoma Bacteriano/genética , Proteínas/genética , Aminoacil-tRNA Sintetases , Códon de Terminação/genética , Corantes Fluorescentes , Genômica/métodos , RNA de Transferência/genéticaRESUMO
The envelope glycoproteins (Env) of HIV-1 mediate cell entry through fusion of the viral envelope with a target cell membrane. Intramembrane mobility and clustering of Env trimers at the viral budding site are essential for its function. Previous live-cell and super-resolution microscopy studies were limited by lack of a functional fluorescent Env derivative, requiring antibody labeling for detection. Introduction of a bio-orthogonal amino acid by genetic code expansion, combined with click chemistry, offers novel possibilities for site-specific, minimally invasive labeling. Using this approach, we established efficient incorporation of non-canonical amino acids within HIV-1 Env in mammalian cells. The engineered protein retained plasma membrane localization, glycosylation, virion incorporation, and fusogenic activity, and could be rapidly and specifically labeled with synthetic dyes. This strategy allowed us to revisit Env dynamics and nanoscale distribution at the plasma membrane close to its native state, applying fluorescence recovery after photo bleaching and STED nanoscopy, respectively.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Microscopia de Fluorescência/métodos , Movimento , Nanotecnologia/métodos , Membrana Celular/metabolismo , Sobrevivência Celular , Química Click , Corantes Fluorescentes/química , Células HEK293 , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , HIV-1/fisiologia , Humanos , Engenharia de Proteínas , Transporte ProteicoRESUMO
Oligodendrocyte damage is a central event in the pathogenesis of the common neuroinflammatory condition, multiple sclerosis (MS). Where and how oligodendrocyte damage is initiated in MS is not completely understood. Here, we use a combination of light and electron microscopy techniques to provide a dynamic and highly resolved view of oligodendrocyte damage in neuroinflammatory lesions. We show that both in MS and in its animal model structural damage is initiated at the myelin sheaths and only later spreads to the oligodendrocyte cell body. Early myelin damage itself is characterized by the formation of local myelin out-foldings-'myelinosomes'-, which are surrounded by phagocyte processes and promoted in their formation by anti-myelin antibodies and complement. The presence of myelinosomes in actively demyelinating MS lesions suggests that oligodendrocyte damage follows a similar pattern in the human disease, where targeting demyelination by therapeutic interventions remains a major open challenge.
Assuntos
Esclerose Múltipla/patologia , Bainha de Mielina/patologia , Oligodendroglia/patologia , Animais , Anticorpos/metabolismo , Proteínas do Sistema Complemento/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Humanos , Imageamento Tridimensional , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Bainha de Mielina/ultraestrutura , Oligodendroglia/ultraestrutura , Proteínas Opsonizantes/metabolismo , Organelas/metabolismo , Organelas/ultraestruturaRESUMO
Genetic code expansion is emerging as an important tool for manipulation and labeling of proteins in vitro and in vivo. In combination with click-chemistry it allows site-specific labeling of target proteins with small organic fluorophores. This is achieved by cotranslational incorporation of noncanonical amino acids (ncAAs) in target proteins via orthogonal tRNA/amino-acyl tRNA synthetase pairs. In a subsequent step, ncAAs are labeled with small dyes via click-chemistry. Small labeling tags and free choice of which fluorophore to use and where to put it into the protein are of particular importance for single molecule science and superresolution microscopy. Such genetically encoded click chemistry tools facilitate high resolution studies of protein function in live cells, viral imaging, as well as the improved design of antibody-drug conjugates.
Assuntos
Aminoácidos/química , Química Click/métodos , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Engenharia de Proteínas/métodos , Proteínas/análise , Aminoácidos/genética , Animais , Código Genético , Humanos , Imunoconjugados/análise , Imunoconjugados/genética , Modelos Moleculares , Proteínas/genética , Proteínas Virais/análise , Proteínas Virais/genética , Vírus/genética , Vírus/isolamento & purificaçãoRESUMO
We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.
Assuntos
Aminoácidos/química , Química Click/métodos , Corantes Fluorescentes/química , Proteínas/química , Alcinos/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Animais , Azidas/química , Carbocianinas/química , Química Click/instrumentação , Códon de Terminação , Reação de Cicloadição , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Mamíferos , Mutagênese Sítio-Dirigida , Proteínas/genética , Receptor de Insulina/química , Receptor de Insulina/genéticaRESUMO
The advent of super-resolution microscopy (nanoscopy) has set high standards for fluorescence tagging. Fluorescent proteins (FPs) are convenient tags in conventional imaging, but their use in nanoscopy has been questioned due to their relatively large size and propensity to form multimers. Here, we compared the nanoscale organization of proteins with or without FP tags by introducing the unnatural amino acid propargyl-L-lysine (PRK) in 26 proteins known to form multimolecular arrangements and into their FP-tagged variants. We revealed the proteins by coupling synthetic fluorophores to PRK via click chemistry and visualized them using ground-state depletion microscopy followed by individual molecule return, as well as stimulated emission depletion microscopy. The arrangements formed by the FP-tagged and nontagged proteins were similar. Mild, but statistically significant differences were observed for only six proteins (23% of all proteins tested). This suggests that FP-based nanoscopy is generally reliable. Unnatural amino acids should be a reliable alternative for the few proteins that are sensitive to FP tagging.
Assuntos
Aminoácidos/química , Química Click/métodos , Corantes Fluorescentes/química , Proteínas Luminescentes/química , Microscopia de Fluorescência/métodos , TransfecçãoRESUMO
Correlating images between light and electron microscopy is difficult especially in tissue specimens with a substantial z-dimension. To facilitate correlated light and electron microscopy (CLEM) in thick tissue specimens, we describe a basic method of using a femto-pulsed near-infrared laser to burn precise three-dimensional fiducial markers that circumscribe cells or regions of interest for easy identification between imaging methods. This rapid and reliable approach permits traditional fixation and avoids the use of electron-dense labeling methods that can obscure ultrastructural details. The versatility of the technique permits CLEM in a variety of tissue specimens to allow interpretation of highly resolved ultrastructural data in the more macroscopic and potentially dynamic context of light microscopy.
Assuntos
Imageamento Tridimensional , Animais , Córtex Cerebral/ultraestrutura , Camundongos Transgênicos , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , MicrotomiaRESUMO
Impaired axonal transport can contribute to axon degeneration and has been described in many neurodegenerative diseases. Multiple sclerosis (MS) is a common neuroinflammatory disease, which is characterized by progressive axon degeneration-whether, when, and how axonal transport is affected in this condition is unknown. Here we used in vivo two-photon imaging to directly assay transport of organelles and the stability of microtubule tracks in individual spinal axons in mouse models of MS. We found widespread transport deficits, which preceded structural alterations of axons, cargos, or microtubules and could be reversed by acute anti-inflammatory interventions or redox scavenging. Our study shows that acute neuroinflammation induces a pervasive state of reversible axonal dysfunction, which coincides with acute disease symptoms. Moreover, perpetuated transport dysfunction, as we found in a model of progressive MS, led to reduced distal organelle supply and could thus contribute to axonal dystrophy in advanced stages of the disease.
Assuntos
Transporte Axonal/fisiologia , Axônios/fisiologia , Esclerose Múltipla/patologia , Esclerose Múltipla/fisiopatologia , Animais , Transporte Axonal/efeitos dos fármacos , Modelos Animais de Doenças , Sequestradores de Radicais Livres/farmacologia , Neuroimagem Funcional , Camundongos , Microtúbulos/fisiologia , Degeneração Neural/fisiopatologia , Doadores de Óxido Nítrico/farmacologia , Organelas/fisiologia , Espermina/análogos & derivados , Espermina/farmacologia , Medula Espinal/fisiologiaRESUMO
Here we provide a protocol for rapidly labeling different cell types, distinct subcellular compartments and key injury mediators in the spinal cord of living mice. This method is based on the application of synthetic vital dyes to the surgically exposed spinal cord. Suitable vital dyes applied in appropriate concentrations lead to reliable in vivo labeling, which can be combined with genetic tags and in many cases preserved for postfixation analysis. In combination with in vivo imaging, this approach allows the direct observation of central nervous system physiology and pathophysiology at the cellular, subcellular and functional level. Surgical exposure and preparation of the spinal cord can be achieved in less than 1 h, and then dyes need to be applied for 30-60 min before the labeled spinal cord can be imaged for several hours.
Assuntos
Medula Espinal/citologia , Coloração e Rotulagem/métodos , Animais , Corantes Fluorescentes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Medula Espinal/ultraestrutura , Imagem com Lapso de Tempo/métodos , Fixação de TecidosRESUMO
In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.
Assuntos
Axônios/patologia , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/patologia , Animais , Axônios/efeitos dos fármacos , Axônios/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Sequestradores de Radicais Livres/farmacologia , Humanos , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/imunologia , Microglia/patologia , Microscopia Eletrônica de Transmissão , Microscopia de Vídeo , Bainha de Mielina/patologia , Degeneração Neural/patologia , Estresse Oxidativo , Espécies Reativas de Nitrogênio/antagonistas & inibidores , Espécies Reativas de Nitrogênio/toxicidade , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/toxicidadeRESUMO
We provide a protocol that describes imaging of single fluorescently labeled axons in the spinal cord of living mice. This method takes advantage of transgenic mouse lines in which the thy1-promoter drives the expression of variants of the green fluorescent protein in a small percentage (less than 1%) of sensory neurons. As a consequence, single axons can be resolved in the surgically exposed dorsal column using wide-field epifluorescence microscopy. This approach allows direct observation of axonal degeneration and regeneration in mouse models of spinal cord pathology for several hours or repetitively over the course of several days.