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1.
Mol Cell ; 81(5): 905-921.e5, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33497605

RESUMEN

Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.


Asunto(s)
Antígenos CD/química , Proteínas del Tejido Nervioso/química , Péptidos/química , Receptores Acoplados a Proteínas G/química , Receptores de Péptidos/química , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Sitios de Unión , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , Expresión Génica , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteolisis , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal
2.
Nat Methods ; 19(8): 986-994, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35915194

RESUMEN

Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.


Asunto(s)
Colorantes Fluorescentes , Imagen Óptica , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos
3.
Nat Methods ; 18(3): 253-257, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33633409

RESUMEN

Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.


Asunto(s)
Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Fotoblanqueo , Artefactos , Fluorescencia , Colorantes Fluorescentes/química
4.
Angew Chem Int Ed Engl ; 62(30): e202300821, 2023 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-36971081

RESUMEN

The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1-2 receptors µm-2 . In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS-CoV-2 a high infectivity.


Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , SARS-CoV-2/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Proteínas Portadoras/metabolismo , Unión Proteica
5.
Angew Chem Int Ed Engl ; 60(51): 26685-26693, 2021 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-34606673

RESUMEN

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.


Asunto(s)
Carbocianinas/química , Fotones , Transferencia Resonante de Energía de Fluorescencia , Microscopía Fluorescente , Conformación Molecular
6.
Nat Chem Biol ; 12(8): 628-35, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27322067

RESUMEN

The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.


Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Colorantes Fluorescentes/análisis , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Movimiento , Biocatálisis , Transporte de Electrón , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Cinética , Modelos Moleculares , Conformación Proteica , Imagen Individual de Molécula , Levaduras
7.
Angew Chem Int Ed Engl ; 57(50): 16364-16369, 2018 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-30347512

RESUMEN

Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.


Asunto(s)
Carbocianinas/química , Química Clic/métodos , Colorantes Fluorescentes/química , Receptores de N-Metil-D-Aspartato/análisis , Fluorescencia , Células HEK293 , Humanos , Microscopía Fluorescente , Modelos Moleculares , Mutación , Imagen Óptica , Dominios Proteicos , Ingeniería de Proteínas , Receptores de N-Metil-D-Aspartato/genética , Coloración y Etiquetado
8.
Curr Opin Chem Biol ; 80: 102445, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38490137

RESUMEN

Super-resolution microscopy (SRM) has transformed our understanding of proteins' subcellular organization and revealed cellular details down to nanometers, far beyond conventional microscopy. While localization precision is independent of the number of fluorophores attached to a biomolecule, labeling density is a decisive factor for resolving complex biological structures. The average distance between adjacent fluorophores should be less than half the desired spatial resolution for optimal clarity. While this was not a major limitation in recent decades, the success of modern microscopy approaching molecular resolution down to the single-digit nanometer range will depend heavily on advancements in fluorescence labeling. This review highlights recent advances and challenges in labeling strategies for SRM, focusing on site-specific labeling technologies. These advancements are crucial for improving SRM precision and expanding our understanding of molecular interactions.


Asunto(s)
Colorantes Fluorescentes , Microscopía Fluorescente , Proteínas , Proteínas/química , Proteínas/metabolismo , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Humanos , Coloración y Etiquetado/métodos , Animales
9.
Adv Mater ; 36(7): e2310104, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38009560

RESUMEN

Super-resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single-molecule localization microscopy methods achieve spatial resolutions in the one-digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein-based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super-resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site-specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6 nm distances. Bioorthogonal click labeling with tetrazine-dyes and tetrazine-functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time-resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is used to resolve 6 nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super-resolution imaging techniques, particularly in complex biological environments.


Asunto(s)
ADN , Proteínas , Antígeno Nuclear de Célula en Proliferación/genética , Microscopía Fluorescente/métodos , ADN/química , Imagen Óptica , Colorantes Fluorescentes/química
10.
Protein Sci ; 32(4): e4614, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36870000

RESUMEN

The introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair enables site-specific incorporation of unnatural amino acids (uAAs) with functionalized side chains into proteins of interest. Genetic Code Expansion (GCE) via amber codon suppression confers functionalities to proteins but can also be used to temporally control the incorporation of genetically encoded elements into proteins. Here, we report an optimized GCE system (GCEXpress) for efficient and fast uAA incorporation. We demonstrate that GCEXpress can be used to efficiently alter the subcellular localization of proteins within living cells. We show that click labeling can resolve co-labeling problems of intercellular adhesive protein complexes. We apply this strategy to study the adhesion G protein-coupled receptor (aGPCR) ADGRE5/CD97 and its ligand CD55/DAF that play central roles in immune functions and oncological processes. Furthermore, we use GCEXpress to analyze the time course of ADGRE5-CD55 ligation and replenishment of mature receptor-ligand complexes. Supported by fluorescence recovery after photobleaching (FRAP) experiments our results show that ADGRE5 and CD55 form stable intercellular contacts that may support transmission of mechanical forces onto ADGRE5 in a ligand-dependent manner. We conclude that GCE in combination with biophysical measurements can be a useful approach to analyze the adhesive, mechanical and signaling properties of aGPCRs and their ligand interactions.


Asunto(s)
Aminoacil-ARNt Sintetasas , Código Genético , Ligandos , Aminoácidos/química , Clonación Molecular , Aminoacil-ARNt Sintetasas/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo
11.
ACS Nano ; 17(21): 21822-21828, 2023 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-37913789

RESUMEN

Engineered vesicular stomatitis virus (VSV) pseudotyping offers an essential method for exploring virus-cell interactions, particularly for viruses that require high biosafety levels. Although this approach has been employed effectively, the current methodologies for virus visualization and labeling can interfere with infectivity and lead to misinterpretation of results. In this study, we introduce an innovative approach combining genetic code expansion (GCE) and click chemistry with pseudotyped VSV to produce highly fluorescent and infectious pseudoviruses (clickVSVs). These clickVSVs enable robust and precise virus-cell interaction studies without compromising the biological function of the viral surface proteins. We evaluated this approach by generating VSVs bearing a unique chemical handle for click labeling and assessing the infectivity in relevant cell lines. Our results demonstrate that clickVSVs maintain their infectivity post-labeling and present an efficiency about two times higher in detecting surface proteins compared to classical immunolabeling. The utilization of clickVSVs further allowed us to visualize and track 3D virus binding and infection in living cells, offering enhanced observation of virus-host interactions. Thus, clickVSVs provide an efficient alternative for virus-associated research under the standard biosafety levels.


Asunto(s)
Virus de la Estomatitis Vesicular Indiana , Virosis , Humanos , Línea Celular , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas de la Membrana/metabolismo
12.
Commun Biol ; 5(1): 176, 2022 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-35228644

RESUMEN

G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of ß2-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm²/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbrück model.


Asunto(s)
Proteínas de la Membrana , Transducción de Señal , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Espectrometría de Fluorescencia/métodos
13.
Artículo en Inglés | MEDLINE | ID: mdl-35663461

RESUMEN

Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM.

14.
Nat Commun ; 12(1): 6715, 2021 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-34795271

RESUMEN

Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution - typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.


Asunto(s)
Aminoácidos/metabolismo , Epítopos/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Coloración y Etiquetado/métodos , Animales , Células COS , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Microscopía Confocal/métodos , Imagen Óptica/métodos , Ratas Sprague-Dawley , Receptores AMPA/metabolismo
15.
ACS Cent Sci ; 7(7): 1144-1155, 2021 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-34345667

RESUMEN

The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multistep sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.

16.
Front Synaptic Neurosci ; 13: 727406, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34899260

RESUMEN

Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.

17.
Light Sci Appl ; 9: 2, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31934334

RESUMEN

New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation, paving the way for refined multicolor super-resolution fluorescence imaging.

18.
ACS Nano ; 14(10): 12629-12641, 2020 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-32804475

RESUMEN

The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1-8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.


Asunto(s)
Colorantes Fluorescentes , Fotones , Anticuerpos , Carbocianinas , Microscopía Fluorescente
19.
Commun Biol ; 2: 261, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31341960

RESUMEN

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.


Asunto(s)
Colorantes Fluorescentes/química , Código Genético , Microscopía Confocal/instrumentación , Imagen Óptica/instrumentación , Animales , Células COS , Chlorocebus aethiops , Colorantes/química , Reacción de Cicloadición , Ciclooctanos/química , Células HEK293 , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Microscopía Confocal/métodos , Imagen Óptica/métodos , Rodaminas , Coloración y Etiquetado
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