RESUMO
The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Transcrição Gênica/fisiologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Laminas/genética , Laminas/metabolismo , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos , CoesinasRESUMO
Transcription and genome architecture are interdependent, but it is still unclear how nucleosomes in the chromatin fiber interact with nascent RNA, and which is the relative nuclear distribution of these RNAs and elongating RNA polymerase II (RNAP II). Using super-resolution (SR) microscopy, we visualized the nascent transcriptome, in both nucleoplasm and nucleolus, with nanoscale resolution. We found that nascent RNAs organize in structures we termed RNA nanodomains, whose characteristics are independent of the number of transcripts produced over time. Dual-color SR imaging of nascent RNAs, together with elongating RNAP II and H2B, shows the physical relation between nucleosome clutches, RNAP II, and RNA nanodomains. The distance between nucleosome clutches and RNA nanodomains is larger than the distance measured between elongating RNAP II and RNA nanodomains. Elongating RNAP II stands between nascent RNAs and the small, transcriptionally active, nucleosome clutches. Moreover, RNA factories are small and largely formed by few RNAP II. Finally, we describe a novel approach to quantify the transcriptional activity at an individual gene locus. By measuring local nascent RNA accumulation upon transcriptional activation at single alleles, we confirm the measurements made at the global nuclear level.
Assuntos
Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Nucleossomos/ultraestrutura , TranscriptomaRESUMO
Chromatin organization is crucial for regulating gene expression. Previously, we showed that nucleosomes form groups, termed clutches. Clutch size correlated with the pluripotency grade of mouse embryonic stem cells and human induced pluripotent stem cells. Recently, it was also shown that regions of the chromatin containing activating epigenetic marks were composed of small and dispersed chromatin nanodomains with lower DNA density compared to the larger silenced domains. Overall, these results suggest that clutch size may regulate DNA packing density and gene activity. To directly test this model, we carried out 3D, two-color super-resolution microscopy of histones and DNA with and without increased histone tail acetylation. Our results showed that lower percentage of DNA was associated with nucleosome clutches in hyperacetylated cells. We further showed that the radius and compaction level of clutch-associated DNA decreased in hyperacetylated cells, especially in regions containing several neighboring clutches. Importantly, this change was independent of clutch size but dependent on the acetylation state of the clutch. Our results directly link the epigenetic state of nucleosome clutches to their DNA packing density. Our results further provide in vivo support to previous in vitro models that showed a disruption of nucleosome-DNA interactions upon hyperacetylation.
Assuntos
DNA/química , Epigênese Genética , Heterocromatina/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Ciclo Celular/genética , Linhagem Celular , DNA/genética , DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Heterocromatina/ultraestrutura , Histonas/genética , Humanos , Microscopia/métodos , Nucleossomos/ultraestruturaRESUMO
The diffusivity of macromolecules in the cytoplasm of eukaryotic cells varies over orders of magnitude and dictates the kinetics of cellular processes. However, a general description that associates the Brownian or anomalous nature of intracellular diffusion to the architectural and biochemical properties of the cytoplasm has not been achieved. Here we measure the mobility of individual fluorescent nanoparticles in living mammalian cells to obtain a comprehensive analysis of cytoplasmic diffusion. We identify a correlation between tracer size, its biochemical nature and its mobility. Inert particles with size equal or below 50 nm behave as Brownian particles diffusing in a medium of low viscosity with negligible effects of molecular crowding. Increasing the strength of non-specific interactions of the nanoparticles within the cytoplasm gradually reduces their mobility and leads to subdiffusive behaviour. These experimental observations and the transition from Brownian to subdiffusive motion can be captured in a minimal phenomenological model.
Assuntos
Citosol/metabolismo , Nanopartículas/química , Difusão , Células HeLa , Humanos , Tamanho da Partícula , Pontos Quânticos/química , Pontos Quânticos/metabolismoRESUMO
In the version of this Article originally published, Supplementary Videos 3-5 were incorrectly labelled; 3 should have been 5, 4 should have been 3 and 5 should have been 4. This has now been corrected.
RESUMO
The mechanical manipulation of magnetic nanoparticles is a powerful approach to probing and actuating biological processes in living systems. Implementing this technique in high-throughput assays can be achieved using biocompatible micromagnet arrays. However, the magnetic properties of these arrays are usually indirectly inferred from simulations or Stokes drag measurements, leaving unresolved questions about the actual profile of the magnetic fields at the micrometer scale and the exact magnetic forces that are applied. Here, we exploit the magnetic field sensitivity of nitrogen-vacancy color centers in diamond to map the 3D stray magnetic field produced by a single soft ferromagnetic microstructure. By combining this wide-field optical magnetometry technique with magneto-optic Kerr effect microscopy, we fully analyze the properties of the micromagnets, including their magnetization saturation and their size-dependent magnetic susceptibility. We further show that the high magnetic field gradients produced by the micromagnets, greater than 104 T·m-1 under an applied magnetic field of about 100 mT, enables the manipulation of magnetic nanoparticles smaller than 10 nm inside living cells. This work paves the way for quantitative and parallelized experiments in magnetogenetics and magnetomechanics in cell biology.
Assuntos
Materiais Biocompatíveis/química , Diamante/química , Magnetometria/métodos , Imãs/química , Fenômenos Biomecânicos , Desenho de Equipamento , Células HeLa , Humanos , Lasers , Campos Magnéticos , Magnetometria/instrumentação , Microscopia/instrumentação , Microscopia/métodos , Nanopartículas/química , Nitrogênio/química , Dispositivos Ópticos , Tamanho da PartículaRESUMO
Tools for controlling the spatial organization of proteins are a major prerequisite for deciphering mechanisms governing the dynamic architecture of living cells. Here, we have developed a generic approach for inducing and maintaining protein gradients inside living cells by means of biofunctionalized magnetic nanoparticles (MNPs). For this purpose, we tailored the size and surface properties of MNPs in order to ensure unhindered mobility in the cytosol. These MNPs with a core diameter below 50 nm could be rapidly relocalized in living cells by exploiting biased diffusion at weak magnetic forces in the femto-Newton range. In combination with MNP surface functionalization for specific in situ capturing of target proteins as well as efficient delivery into the cytosplasm, we here present a comprehensive technology for controlling intracellular protein gradients with a temporal resolution of a few tens of seconds.
Assuntos
Citosol/efeitos dos fármacos , Nanopartículas de Magnetita/química , Proteínas/química , Linhagem Celular , Citosol/química , Humanos , Nanopartículas de Magnetita/administração & dosagem , Tamanho da Partícula , Proteínas/administração & dosagem , Propriedades de SuperfícieRESUMO
A label-free biosensor device based on registration of photonic crystal surface waves is described. Angular interrogation of the optical surface wave resonance is used to detect changes in the thickness of an adsorbed layer, while an additional simultaneous detection of the critical angle of total internal reflection provides independent data of the liquid refractive index. The abilities of the device are demonstrated by measuring of biotin molecule binding to a streptavidin monolayer, and by measuring association and dissociation kinetics of immunoglobulin G proteins. Additionally, deposition of PSS / PAH polyelectrolytes is recorded in situ resulting calculation of PSS and PAH monolayer thicknesses separately.
Assuntos
Técnicas Biossensoriais/instrumentação , Óptica e Fotônica/instrumentação , Animais , Biotina/metabolismo , Biotinilação , Cristalização , Eletrólitos/química , Proteínas Imobilizadas/metabolismo , Imunoglobulina G/metabolismo , Ligantes , Camundongos , Coelhos , Processamento de Sinais Assistido por Computador , Estreptavidina/metabolismo , Propriedades de SuperfícieRESUMO
Here, we describe three complementary microscopy-based approaches to quantify morphological changes of chromatin organization in cultured adherent cells: the analysis of the coefficient of variation of DNA, the measurement of DNA-free nuclear areas, and the quantification of chromatin-associated proteins at the nuclear edge. These approaches rely on confocal imaging and stochastic optical reconstruction microscopy and allow a fast and robust quantification of chromatin compaction. These approaches circumvent inter-variability between imaging conditions and apply to every type of adherent cells. For complete details on the use and execution of this protocol, please refer to Neguembor et al. (2021).
Assuntos
Cromatina/química , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Técnicas de Cultura de Células , Núcleo Celular/química , Células Cultivadas , Células HeLa , HumanosRESUMO
[This corrects the article DOI: 10.1039/C7SC01462G.].
RESUMO
Remote control of cellular functions is a key challenge in biomedical research. Only a few tools are currently capable of manipulating cellular events at distance, at spatial and temporal scales matching their naturally active range. A promising approach, often referred to as 'magnetogenetics', is based on the use of magnetic fields, in conjunction with targeted biofunctional magnetic nanoparticles. By triggering molecular stimuli via mechanical, thermal or biochemical perturbations, magnetic actuation constitutes a highly versatile tool with numerous applications in fundamental research as well as exciting prospects in nano- and regenerative medicine. Here, we highlight recent studies, comment on the advancement of magnetic manipulation, and discuss remaining challenges.
RESUMO
Magnetogenetics is emerging as a novel approach for remote-controlled manipulation of cellular functions in tissues and organisms with high spatial and temporal resolution. A critical, still challenging issue for these techniques is to conjugate target proteins with magnetic probes that can satisfy multiple colloidal and biofunctional constraints. Here, semisynthetic magnetic nanoparticles are tailored based on human ferritin coupled to monomeric enhanced green fluorescent protein (mEGFP) for magnetic manipulation of proteins inside living cells. This study demonstrates efficient delivery, intracellular stealth properties, and rapid subcellular targeting of those magnetic nanoparticles via GFP-nanobody interactions. By means of magnetic field gradients, rapid spatial reorganization in the cytosol of proteins captured to the nanoparticle surface is achieved. Moreover, exploiting efficient nanoparticle targeting to intracellular membranes, remote-controlled arrest of mitochondrial dynamics using magnetic fields is demonstrated. The studies establish subcellular control of proteins and organelles with unprecedented spatial and temporal resolution, thus opening new prospects for magnetogenetic applications in fundamental cell biology and nanomedicine.