RESUMEN
Essential biological functions, such as mitosis, require tight coordination of hundreds of proteins in space and time. Localization, the timing of interactions and changes in cellular structure are all crucial to ensure the correct assembly, function and regulation of protein complexes1-4. Imaging of live cells can reveal protein distributions and dynamics but experimental and theoretical challenges have prevented the collection of quantitative data, which are necessary for the formulation of a model of mitosis that comprehensively integrates information and enables the analysis of the dynamic interactions between the molecular parts of the mitotic machinery within changing cellular boundaries. Here we generate a canonical model of the morphological changes during the mitotic progression of human cells on the basis of four-dimensional image data. We use this model to integrate dynamic three-dimensional concentration data of many fluorescently knocked-in mitotic proteins, imaged by fluorescence correlation spectroscopy-calibrated microscopy5. The approach taken here to generate a dynamic protein atlas of human cell division is generic; it can be applied to systematically map and mine dynamic protein localization networks that drive cell division in different cell types, and can be conceptually transferred to other cellular functions.
Asunto(s)
Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/metabolismo , Mitosis , Edición Génica , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Imagenología Tridimensional , Microscopía Fluorescente , Imagen Molecular , Factores de TiempoRESUMEN
In Drosophila, two Piwi proteins, Aubergine (Aub) and Argonaute-3 (Ago3), localize to perinuclear "nuage" granules and use guide piRNAs to target and destroy transposable element transcripts. We find that Aub and Ago3 are recruited to nuage by two different mechanisms. Aub requires a piRNA guide for nuage recruitment, indicating that its localization depends on recognition of RNA targets. Ago3 is recruited to nuage independently of a piRNA cargo and relies on interaction with Krimper, a stable component of nuage that is able to aggregate in the absence of other nuage proteins. We show that Krimper interacts directly with Aub and Ago3 to coordinate the assembly of the ping-pong piRNA processing (4P) complex. Symmetrical dimethylated arginines are required for Aub to interact with Krimper, but they are dispensable for Ago3 to bind Krimper. Our study reveals a multi-step process responsible for the assembly and function of nuage complexes in piRNA-guided transposon repression.
Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Drosophila/química , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Cinética , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , ARN Interferente Pequeño/metabolismoRESUMEN
Clathrin-mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein-protein interactions between up to 60 different proteins. However, little is known about the spatio-temporal regulation of these interactions. Using fluorescence (cross)-correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self-interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein-protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo.
Asunto(s)
Endocitosis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citosol/fisiología , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/química , Espectrometría de FluorescenciaRESUMEN
Dynamic actin filaments are a crucial component of clathrin-mediated endocytosis when endocytic proteins cannot supply enough energy for vesicle budding. Actin cytoskeleton is thought to provide force for membrane invagination or vesicle scission, but how this force is transmitted to the plasma membrane is not understood. Here we describe the molecular mechanism of plasma membrane-actin cytoskeleton coupling mediated by cooperative action of epsin Ent1 and the HIP1R homolog Sla2 in yeast Saccharomyces cerevisiae. Sla2 anchors Ent1 to a stable endocytic coat by an unforeseen interaction between Sla2's ANTH and Ent1's ENTH lipid-binding domains. The ANTH and ENTH domains bind each other in a ligand-dependent manner to provide critical anchoring of both proteins to the membrane. The C-terminal parts of Ent1 and Sla2 bind redundantly to actin filaments via a previously unknown phospho-regulated actin-binding domain in Ent1 and the THATCH domain in Sla2. By the synergistic binding to the membrane and redundant interaction with actin, Ent1 and Sla2 form an essential molecular linker that transmits the force generated by the actin cytoskeleton to the plasma membrane, leading to membrane invagination and vesicle budding.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Clatrina/metabolismo , Citoesqueleto/metabolismo , Endocitosis , Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Glutatión Transferasa/metabolismo , Lípidos/química , Modelos Biológicos , Fenotipo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
To quantify more precisely and more reliably diffusion and reaction properties of biomolecules in living cells, a novel closed description in 3D of both the bleach and the post-bleach segment of fluorescence recovery after photobleaching (FRAP) data acquired at a point, i.e., a diffraction-limited observation area, termed point FRAP, is presented. It covers a complete coupled reaction-diffusion scheme for mobile molecules undergoing transient or long-term immobilization because of binding. We assess and confirm the feasibility with numerical solutions of the differential equations. By applying this model to free EYFP expressed in HeLa cells using a customized confocal laser scanning microscope that integrates point FRAP and fluorescence correlation spectroscopy (FCS), the applicability is validated by comparison with results from FCS. We show that by taking diffusion during bleaching into consideration and/or by employing a global analysis of series of bleach times, the results can be improved significantly. As the point FRAP approach allows to obtain data with diffraction-limited positioning accuracy, diffusion and binding properties of the exon-exon junction complex (EJC) components REF2-II and Magoh are obtained at different localizations in the nucleus of MCF7 cells and refine our view on the position-dependent association of the EJC factors with a maturating mRNP complex. Our findings corroborate the concept of combining point FRAP and FCS for a better understanding of the underlying diffusion and binding processes.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Espectrometría de Fluorescencia/métodos , Proteínas Bacterianas/química , Línea Celular Tumoral , Células HeLa , Histonas/química , Humanos , Proteínas Luminiscentes/química , Células MCF-7 , Unión Proteica , Proteínas Recombinantes de Fusión/químicaRESUMEN
The genome of eukaryotes is organized into a dynamic nucleoprotein complex referred to as chromatin, which can adopt different functional states. Both the DNA and the protein component of chromatin are subject to various post-translational modifications that define the cell's gene expression program. Their readout and establishment occurs in a spatio-temporally coordinated manner that is controlled by numerous chromatin-interacting proteins. Binding to chromatin in living cells can be measured by a spatially resolved analysis of protein mobility using fluorescence microscopy based approaches. Recent advancements in the acquisition of protein mobility data using fluorescence bleaching and correlation methods provide data sets on diffusion coefficients, binding kinetics, and cellular concentrations on different time and length scales. The combination of different techniques is needed to dissect the complex interplay of diffusive translocations, binding events, and mobility constraints of the chromatin environment. While bleaching techniques have their strength in the characterization of particles that are immobile on the second/minute time scale, a correlation analysis is advantageous to characterize transient binding events with millisecond residence time. The application and synergy effects of the different approaches to obtain protein mobility and interaction maps in the nucleus are illustrated for the analysis of heterochromatin protein 1.
Asunto(s)
Cromatina/metabolismo , Mapeo de Interacción de Proteínas/métodos , Procesamiento Proteico-Postraduccional , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/análisis , ADN , Difusión , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Expresión Génica , Microscopía Fluorescente , Modelos Biológicos , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
The exon-exon junction complex (EJC) forms via association of proteins during splicing of mRNA in a defined manner. Its organization provides a link between biogenesis, nuclear export, and translation of the transcripts. The EJC proteins accumulate in nuclear speckles alongside most other splicing-related factors. We followed the establishment of the EJC on mRNA by investigating the mobility and interactions of a representative set of EJC factors in vivo using a complementary analysis with different fluorescence fluctuation microscopy techniques. Our observations are compatible with cotranscriptional binding of the EJC protein UAP56 confirming that it is involved in the initial phase of EJC formation. RNPS1, REF/Aly, Y14/Magoh, and NXF1 showed a reduction in their nuclear mobility when complexed with RNA. They interacted with nuclear speckles, in which both transiently and long-term immobilized factors were identified. The location- and RNA-dependent differences in the mobility between factors of the so-called outer shell and inner core of the EJC suggest a hypothetical model, in which mRNA is retained in speckles when EJC outer-shell factors are missing.
Asunto(s)
Empalme del ARN , ARN Mensajero/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Exones , Humanos , Modelos Moleculares , ARN Mensajero/químicaRESUMEN
The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.
Asunto(s)
Microscopía Confocal/métodos , Óptica y Fotónica , Espectrometría de Fluorescencia/métodos , Algoritmos , Difusión , Proteínas Fluorescentes Verdes/química , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Rayos Láser , Modelos Estadísticos , Probabilidad , Factores de TiempoRESUMEN
We introduce a fast spectral imaging system using an electron-multiplying charge-coupled device (EM-CCD) as a detector. Our system is combined with a custom-built two-photon excitation laser scanning microscope and has 80 detection channels, which allow for high spectral resolution and fast frame acquisition without any loss of spectral information. To demonstrate the efficiency of our approach, we applied this technology to monitor fluorescent proteins and quantum dot-labeled G protein-coupled receptors in living cells as well as autofluorescence in tissue samples.
Asunto(s)
Aumento de la Imagen/instrumentación , Microscopía Confocal/instrumentación , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Espectrometría de Fluorescencia/instrumentación , Diseño de Equipo , Análisis de Falla de EquipoRESUMEN
We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.
RESUMEN
The ATPase ISWI is the molecular motor of several nucleosome remodeling complexes including ACF. We analyzed the ACF-nucleosome interactions and determined the characteristics of ACF-dependent nucleosome remodeling. In contrast to ISWI, ACF interacts symmetrically with DNA entry sites of the nucleosome. Two-color fluorescence cross-correlation spectroscopy measurements show that ACF can bind four DNA duplexes simultaneously in a complex that contains two Acf1 and ISWI molecules. Using bead-bound nucleosomal substrates, nucleosome movement by mechanisms involving DNA twisting was excluded. Furthermore, an ACF-dependent local detachment of DNA from the nucleosome was demonstrated in a novel assay based on the preferred intercalation of ethidium bromide to free DNA. The findings suggest a loop recapture mechanism in which ACF introduces a DNA loop at the nucleosomal entry site that propagates over the histone octamer surface and leads to nucleosome repositioning.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , ADN/metabolismo , Proteínas de Drosophila/metabolismo , Modelos Genéticos , Nucleosomas/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Huella de ADN , Drosophila , Ensayo de Cambio de Movilidad Electroforética , Etidio , Histonas/metabolismo , Nucleosomas/fisiología , Reacción en Cadena de la Polimerasa , Espectrometría de FluorescenciaRESUMEN
Cancer clone evolution takes place within tissue ecosystem habitats. But, how exactly tumors arise from a few malignant cells within an intact epithelium is a central, yet unanswered question. This is mainly due to the inaccessibility of this process to longitudinal imaging together with a lack of systems that model the progression of a fraction of transformed cells within a tissue. Here, we developed a new methodology based on primary mouse mammary epithelial acini, where oncogenes can be switched on in single cells within an otherwise normal epithelial cell layer. We combine this stochastic breast tumor induction model with inverted light-sheet imaging to study single-cell behavior for up to four days and analyze cell fates utilizing a newly developed image-data analysis workflow. The power of this integrated approach is illustrated by us finding that small local clusters of transformed cells form tumors while isolated transformed cells do not.
There are now drugs to treat many types of cancer, but questions still remain around how these diseases start in the first place. Researchers think that tumor growth begins when a single cell suffers damage to certain sites in its DNA that eventually cause it to divide uncontrollably. That damaged cell, and its descendants, go on to form a lump, or tumor. The trouble with proving this theory is that it is hard to watch it happening in real time. Doctors usually only meet people with cancer when their tumors start to cause health problems. By this point, the tumors contain millions of cells. A way to watch the very beginnings of a cancer could reveal risk factors within a tissue that foster the growth of a tumor. But first, researchers need to test their theory about how the disease begins in the first place. One way to do this is to surround a single cancer cell with healthy cells and watch what happens next. To do this, Alladin, Chaible et al. took healthy cells from the breast tissue of mice and grew them in the laboratory into mini-organs called organoids. These organoids share a lot of features with actual mouse breast tissue; they can even make milk if given the right hormones. Once the organoids were ready, Alladin, Chaible et al then started modifying a small number of single cells inside them by switching on genes called oncogenes, which are known to drive cancer formation in humans. Using fluorescent proteins and a sheet of laser light it was possible to watch what happened to the cells over time. This revealed that, even though all the oncogene-driven single cells received the same signals, not all of them started to divide uncontrollably. In fact, a single modified cell had a low chance of forming a tumor on its own. The more oncogene-driven cells there were near to each other, the more likely they were to form tumors. Alladin, Chaible et al. think that this is because the healthy tissue interacts with the modified, oncogene-driven cells to suppress tumor formation. It is only when a larger number of modified cells group together and start to communicate with each other that they can override the inhibitory messages of the healthy tissue. How healthy tissue stops single modified cells from forming tumors is not yet clear. But, with this new mini-organ system, researchers now have the tools to investigate. In the future, this could lead to new strategies to stop cancer before it has a chance to get started.
Asunto(s)
Células Acinares/citología , Neoplasias de la Mama/genética , Neoplasias de la Mama/fisiopatología , Rastreo Celular/métodos , Células Epiteliales/citología , Glándulas Mamarias Humanas/citología , Células Madre Neoplásicas/citología , Animales , Femenino , Humanos , Ratones , Microscopía/métodos , Modelos AnimalesRESUMEN
How tissues migrate robustly through changing guidance landscapes is poorly understood. Here, quantitative imaging is combined with inducible perturbation experiments to investigate the mechanisms that ensure robust tissue migration in vivo. We show that tissues exposed to acute "chemokine floods" halt transiently before they perfectly adapt, i.e., return to the baseline migration behavior in the continued presence of elevated chemokine levels. A chemokine-triggered phosphorylation of the atypical chemokine receptor Cxcr7b reroutes it from constitutive ubiquitination-regulated degradation to plasma membrane recycling, thus coupling scavenging capacity to extracellular chemokine levels. Finally, tissues expressing phosphorylation-deficient Cxcr7b migrate normally in the presence of physiological chemokine levels but show delayed recovery when challenged with elevated chemokine concentrations. This work establishes that adaptation to chemokine fluctuations can be "outsourced" from canonical GPCR signaling to an autonomously acting scavenger receptor that both senses and dynamically buffers chemokine levels to increase the robustness of tissue migration.
Asunto(s)
Movimiento Celular , Quimiocinas/metabolismo , Embrión no Mamífero/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Comunicación Celular , Quimiocinas/genética , Embrión no Mamífero/citología , Fosforilación , Receptores CXCR/genética , Receptores CXCR4/genética , Transducción de Señal , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genéticaRESUMEN
Heterochromatin protein 1 (HP1) is a central factor in establishing and maintaining the repressive heterochromatin state. To elucidate its mobility and interactions, we conducted a comprehensive analysis on different time and length scales by fluorescence fluctuation microscopy in mouse cell lines. The local mobility of HP1alpha and HP1beta was investigated in densely packed pericentric heterochromatin foci and compared with other bona fide euchromatin regions of the nucleus by fluorescence bleaching and correlation methods. A quantitative description of HP1alpha/beta in terms of its concentration, diffusion coefficient, kinetic binding, and dissociation rate constants was derived. Three distinct classes of chromatin-binding sites with average residence times t(res) Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo
, Animales
, Línea Celular
, Supervivencia Celular
, Homólogo de la Proteína Chromobox 5
, Difusión
, Epigénesis Genética
, Recuperación de Fluorescencia tras Fotoblanqueo
, Heterocromatina/metabolismo
, N-Metiltransferasa de Histona-Lisina/metabolismo
, Cinética
, Ratones
, Microscopía Fluorescente
, Movimiento
, Transporte de Proteínas
, Espectrometría de Fluorescencia
RESUMEN
The cell needs to stably maintain its genome and protect it from uncontrolled modifications that would compromise its function. At the same time, the genome has to be a plastic structure that can dynamically (re)organize to allow the cell to adopt different functional states. These dynamics occur on the nanometer to micrometer length scale, i.e. ranging from the level of single proteins up to that of whole chromosomes, and on a microsecond to hour time scale. Here, we review different contributions to the dynamic features of the genome, describe how they are determined experimentally, and discuss the results of these measurements in terms of how the requirements for stability and plasticity are accommodated with specific activities in the nucleus.
Asunto(s)
Genoma , Inestabilidad Genómica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Transferencia Resonante de Energía de Fluorescencia , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Matemática , Modelos Teóricos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
The spatial and temporal fluctuation microscope (STFM) presented here extends the concept of a fluorescence confocal laser scanning microscope to illumination and detection along a line. The parallel multichannel acquisition of the fluorescence signal was accomplished by using a single line of an electron-multiplying charge-coupled device camera at 14 mus time resolution for detection of the fluorescence signal. The STFM system provided fast confocal imaging (30 images per second) and allowed for the spatially resolved detection of particle concentration fluctuations in fluorescence correlation spectroscopy experiments. For the application of the STFM, an approximated theoretical description of the beam geometry, the point-spread function, and the fluorescence auto- and cross-correlation functions were derived. The STFM was applied to studies of the dynamics of promyelocytic leukemia nuclear bodies, green fluorescent protein, and chromatin-remodeling complexes in living cells. The results demonstrate the unique capabilities of the STFM for characterizing the position-dependent translocations and interactions of proteins in the cell.
Asunto(s)
Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Microscopía Fluorescente/métodos , Proteínas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Algoritmos , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Difusión , Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal/métodos , Microscopía Fluorescente/instrumentación , Modelos Teóricos , Movimiento (Física) , Nanosferas , Poliestirenos , Puntos Cuánticos , Espectrometría de Fluorescencia/métodos , Factores de Tiempo , TransfecciónRESUMEN
The ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables quantitative understanding of protein dynamics at the physiological level. Genome-editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, thereby opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool for investigating spatial and temporal protein dynamics; however, it lacks the required quantitative power to make the kind of absolute and comparable measurements required for systems analysis. In contrast, fluorescence correlation spectroscopy (FCS) provides quantitative proteomic and biophysical parameters such as protein concentration, hydrodynamic radius, and oligomerization but lacks the capability for high-throughput application in 4D spatial and temporal imaging. Here we present an automated experimental and computational workflow that integrates both methods and delivers quantitative 4D imaging data in high throughput. These data are processed to yield a calibration curve relating the fluorescence intensities (FIs) of image voxels to the absolute protein abundance. The calibration curve allows the conversion of the arbitrary FIs to protein amounts for all voxels of 4D imaging stacks. Using our workflow, users can acquire and analyze hundreds of FCS-calibrated image series to map their proteins of interest in four dimensions. Compared with other protocols, the current protocol does not require additional calibration standards and provides an automated acquisition pipeline for FCS and imaging data. The protocol can be completed in 1 d.
Asunto(s)
Células/química , Imagenología Tridimensional/métodos , Imagen Óptica/métodos , Proteínas/análisis , Proteoma/análisis , Proteómica/métodos , Coloración y Etiquetado/métodos , Automatización de Laboratorios/métodos , Edición Génica/métodos , Análisis Espacio-TemporalRESUMEN
BACKGROUND: Genome organization into subchromosomal topologically associating domains (TADs) is linked to cell-type-specific gene expression programs. However, dynamic properties of such domains remain elusive, and it is unclear how domain plasticity modulates genomic accessibility for soluble factors. RESULTS: Here, we combine and compare a high-resolution topology analysis of interacting chromatin loci with fluorescence correlation spectroscopy measurements of domain dynamics in single living cells. We identify topologically and dynamically independent chromatin domains of ~1 Mb in size that are best described by a loop-cluster polymer model. Hydrodynamic relaxation times and gyration radii of domains are larger for open (161 ± 15 ms, 297 ± 9 nm) than for dense chromatin (88 ± 7 ms, 243 ± 6 nm) and increase globally upon chromatin hyperacetylation or ATP depletion. CONCLUSIONS: Based on the domain structure and dynamics measurements, we propose a loop-cluster model for chromatin domains. It suggests that the regulation of chromatin accessibility for soluble factors displays a significantly stronger dependence on factor concentration than search processes within a static network.
RESUMEN
Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3-Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3-Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3-Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis.
Asunto(s)
Mesodermo/embriología , Proteína Homeótica Nanog/análisis , Factor 3 de Transcripción de Unión a Octámeros/análisis , Proteínas de Pez Cebra/análisis , Pez Cebra/embriología , Animales , Microscopía Intravital , Mesodermo/química , Microscopía Fluorescente , Unión Proteica , Análisis Espacio-Temporal , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture (T2C), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. RESULTS: The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. CONCLUSIONS: This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.