RESUMEN
A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.
Asunto(s)
Cromatina/química , Microscopía Fluorescente/métodos , Células Madre Embrionarias de Ratones/metabolismo , Imagen Individual de Molécula/métodos , Animales , Línea Celular , Cromatina/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Células Madre Embrionarias de Ratones/ultraestructura , FotoblanqueoRESUMEN
Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.
Asunto(s)
Cromatina/ultraestructura , Cromosomas/ultraestructura , Biología Molecular/métodos , Conformación Molecular , Células Madre Embrionarias de Ratones , Imagen Óptica/métodos , Animales , Células Cultivadas , Imagenología Tridimensional/métodos , Ratones , Análisis de la Célula Individual/métodosRESUMEN
The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.
Asunto(s)
Ensamble y Desensamble de Cromatina , Genoma , Imagen Molecular/métodos , Nucleosomas/química , Análisis de la Célula Individual/métodos , Animales , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/química , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Fase G1 , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genoma/genética , Haploidia , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones , Modelos Moleculares , Conformación Molecular , Imagen Molecular/normas , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Reproducibilidad de los Resultados , Análisis de la Célula Individual/normas , CohesinasRESUMEN
HIV-1 genomic RNA has a noncoding 5' region containing sequential conserved structural motifs that control many parts of the life cycle. Very limited data exist on their three-dimensional (3D) conformation and, hence, how they work structurally. To assemble a working model, we experimentally reassessed secondary structure elements of a 240-nt region and used single-molecule distances, derived from fluorescence resonance energy transfer, between defined locations in these elements as restraints to drive folding of the secondary structure into a 3D model with an estimated resolution below 10 Å. The folded 3D model satisfying the data is consensual with short nuclear-magnetic-resonance-solved regions and reveals previously unpredicted motifs, offering insight into earlier functional assays. It is a 3D representation of this entire region, with implications for RNA dimerization and protein binding during regulatory steps. The structural information of this highly conserved region of the virus has the potential to reveal promising therapeutic targets.
