RESUMEN
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.
Asunto(s)
ADN/química , Epigénesis Genética , Heterocromatina/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Ciclo Celular/genética , Línea Celular , ADN/genética , ADN/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Heterocromatina/ultraestructura , Histonas/genética , Humanos , Microscopía/métodos , Nucleosomas/ultraestructuraRESUMEN
Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore's response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as "frequency multiplexed DNA-PAINT," or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.
Asunto(s)
Color , ADN/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Fluorescencia , Colorantes Fluorescentes , HumanosRESUMEN
Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization-condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients.
Asunto(s)
Efrinas/metabolismo , Multimerización de Proteína , Receptores de la Familia Eph/metabolismo , Transducción de Señal , Células HEK293 , Humanos , Polimerizacion , Receptores de la Familia Eph/químicaRESUMEN
Vesicle transport is regulated at multiple levels, including regulation by scaffolding proteins and the cytoskeleton. This tight regulation is essential, since slowing or stoppage of transport can cause accumulation of obstacles and has been linked to diseases. Understanding the mechanisms by which transport is regulated as well as how motor proteins overcome obstacles can give important clues as to how these mechanisms break down in disease states. Here, we describe that the cytoskeleton architecture impacts transport in a vesicle-size-dependent manner, leading to pausing of vesicles larger than the separation of the microtubules. We further develop methods capable of following 3D transport processes in living cells. Using these methods, we show that vesicles move using two different modes along the microtubule. Off-axis motion, which leads to repositioning of the vesicle in 3D along the microtubule, correlates with the presence of steric obstacles and may help in circumventing them.
Asunto(s)
Vesículas Citoplasmáticas/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/ultraestructura , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Microesferas , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Nocodazol/farmacología , Imagen Óptica , Paclitaxel/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tubulina (Proteína)/genética , Proteína Fluorescente RojaRESUMEN
The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Virus de la Influenza A/inmunología , Fusión de Membrana/inmunología , Virión/inmunología , Anticuerpos Neutralizantes/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H1N1 del Virus de la Influenza A/ultraestructura , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/ultraestructura , Virus de la Influenza A/fisiología , Virus de la Influenza A/ultraestructura , Gripe Humana/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Cinética , Fusión de Membrana/efectos de los fármacos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Método de Montecarlo , Unión Proteica , Virión/efectos de los fármacos , Virión/ultraestructura , Internalización del Virus/efectos de los fármacosRESUMEN
Protein-mediated fusion between phospholipid bilayers is a fundamental and necessary mechanism for many cellular processes. The short-lived nature of the intermediate states visited during fusion makes it challenging to capture precise kinetic information using classical, ensemble-averaging biophysical techniques. Recently, a number of single-particle fluorescence microscopy-based assays that allow researchers to obtain highly quantitative data about the fusion process by observing individual fusion events in real time have been developed. These assays depend upon changes in the acquired fluorescence signal to provide a direct readout for transitions between the various fusion intermediates. The resulting data yield meaningful and detailed kinetic information about the transitory states en route to productive membrane fusion. In this review, we highlight recent in vitro and in vivo studies of membrane fusion at the single-particle level in the contexts of viral membrane fusion and SNARE-mediated synaptic vesicle fusion. These studies afford insight into mechanisms of coordination between fusion-mediating proteins as well as coordination of the overall fusion process with other cellular processes. The development of single-particle approaches to investigate membrane fusion and their successful application to a number of model systems have resulted in a new experimental paradigm and open up considerable opportunities to extend these methods to other biological processes that involve membrane fusion.
Asunto(s)
Fusión de Membrana/fisiología , Animales , Fenómenos Biofísicos , Humanos , Cinética , Membrana Dobles de Lípidos/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Fosfolípidos/metabolismo , Proteínas SNARE/metabolismo , Vesículas Sinápticas/metabolismo , Internalización del VirusRESUMEN
Fundamental life science and pharmaceutical research are continually striving to provide physiologically relevant context for their biological studies. Zebrafish present an opportunity for high-content screening (HCS) to bring a true in vivo model system to screening studies. Zebrafish embryos and young larvae are an economical, human-relevant model organism that are amenable to both genetic engineering and modification, and direct inspection via microscopy. The use of these organisms entails unique challenges that new technologies are overcoming, including artificial intelligence (AI). In this perspective article, we describe the state-of-the-art in terms of automated sample handling, imaging, and data analysis with zebrafish during early developmental stages. We highlight advances in orienting the embryos, including the use of robots, microfluidics, and creative multi-well plate solutions. Analyzing the micrographs in a fast, reliable fashion that maintains the anatomical context of the fluorescently labeled cells is a crucial step. Existing software solutions range from AI-driven commercial solutions to bespoke analysis algorithms. Deep learning appears to be a critical tool that researchers are only beginning to apply, but already facilitates many automated steps in the experimental workflow. Currently, such work has permitted the cellular quantification of multiple cell types in vivo, including stem cell responses to stress and drugs, neuronal myelination and macrophage behavior during inflammation and infection. We evaluate pro and cons of proprietary versus open-source methodologies for combining technologies into fully automated workflows of zebrafish studies. Zebrafish are poised to charge into HCS with ever-greater presence, bringing a new level of physiological context.
Asunto(s)
Inteligencia Artificial , Pez Cebra , Algoritmos , Animales , Programas Informáticos , Flujo de Trabajo , Pez Cebra/fisiologíaRESUMEN
Zebrafish provide a unique opportunity for drug screening in living animals, with the fast-developing, transparent embryos allowing for relatively high-throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed an easy-to-use, customisable automated screening procedure suitable for high-throughput phenotype-based screens of live zebrafish. We utilised the WiScan® Hermes High Content Imaging System to rapidly acquire brightfield and fluorescent images of embryos, and the WiSoft® Athena Zebrafish Application for analysis, which harnesses an Artificial Intelligence-driven algorithm to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions and exclusively select the desired side-oriented fish. Our initial validation combined structural analysis with fluorescence images to enumerate GFP-tagged haematopoietic stem and progenitor cells in the tails of embryos, which correlated with manual counts. We further validated this system to assess the effects of genetic mutations and X-ray irradiation in high content using a wide range of assays. Further, we performed simultaneous analysis of multiple cell types using dual fluorophores in high throughput. In summary, we demonstrate a broadly applicable and rapidly customisable platform for high-content screening in zebrafish. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Embrión no Mamífero/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Modelos Animales , Pez Cebra/embriología , Algoritmos , Animales , FenotipoRESUMEN
Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour.
Asunto(s)
Efrina-B1/ultraestructura , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Microscopía Fluorescente/métodos , Proteínas Recombinantes de Fusión/ultraestructura , Programas Informáticos , Efrina-B1/genética , Efrina-B1/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Células HEK293 , Humanos , Microscopía Confocal/métodos , Agregado de Proteínas , Multimerización de Proteína , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptor EphB2/ultraestructura , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Señal-RuidoRESUMEN
Human monoclonal antibodies have been identified which neutralize broad spectra of influenza A or B viruses. Here, we dissect the mechanisms by which such antibodies interfere with infectivity. We distinguish four mechanisms that link the conserved hemagglutinin (HA) epitopes of broadly neutralizing antibodies to critical processes in the viral life cycle. HA-stem binding antibodies can act intracellularly by blocking fusion between the viral and endosomal membranes and extracellularly by preventing the proteolytic activation of HA. HA-head binding antibodies prevent viral attachment and release. These insights into newly identified ways by which the human immune system can interfere with influenza virus infection may aid the development of novel universal vaccines and antivirals.
Asunto(s)
Hemaglutininas/metabolismo , Subtipo H1N1 del Virus de la Influenza A/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Perros , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Humanos , Immunoblotting , Virus de la Influenza A/inmunología , Células de Riñón Canino Madin Darby , Microscopía Electrónica de Rastreo , Microscopía Electrónica de TransmisiónRESUMEN
'Forehead to forehead I meet thee, this third time, Moby Dick!' [Ahab (Melville, 1851)] Herman Melville's fictional portrayal of the sinking of the Pequod was inspired by instances in which large sperm whales sank whaling ships by ramming the ships with their heads. Observations of aggression in species of the four major clades of cetacean and the artiodactyl outgroup suggest that head-butting during male-male aggression is a basal behavior for cetaceans. We hypothesize that the ability of sperm whales to destroy stout wooden ships, 3-5 times their body mass, is a product of specialization for male-male aggression. Specifically, we suggest that the greatly enlarged and derived melon of sperm whales, the spermaceti organ, evolved as a battering ram to injure an opponent. To address this hypothesis, we examined the correlation between relative melon size and the level of sexual dimorphism in body size among cetaceans. We also modeled impacts between two equal-sized sperm whales to determine whether it is physically possible for the spermaceti organ to function as an effective battering ram. We found (i) that the evolution of relative melon size in cetaceans is positively correlated with the evolution of sexual dimorphism in body size and (ii) that the spermaceti organ of a charging sperm whale has enough momentum to seriously injure an opponent. These observations are consistent with the hypothesis that the spermaceti organ has evolved to be a weapon used in male-male aggression.