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1.
Nat Commun ; 12(1): 2005, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790271

RESUMEN

Förster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral bandwidth required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent protein kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for extracellular signal-regulated kinase (ERK) activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Algoritmos , Animales , Técnicas Biosensibles/métodos , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Imagen de Lapso de Tiempo/métodos
2.
Nat Commun ; 12(1): 1478, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674570

RESUMEN

The recently introduced minimal photon fluxes (MINFLUX) concept pushed the resolution of fluorescence microscopy to molecular dimensions. Initial demonstrations relied on custom made, specialized microscopes, raising the question of the method's general availability. Here, we show that MINFLUX implemented with a standard microscope stand can attain 1-3 nm resolution in three dimensions, rendering fluorescence microscopy with molecule-scale resolution widely applicable. Advances, such as synchronized electro-optical and galvanometric beam steering and a stabilization that locks the sample position to sub-nanometer precision with respect to the stand, ensure nanometer-precise and accurate real-time localization of individually activated fluorophores. In our MINFLUX imaging of cell- and neurobiological samples, ~800 detected photons suffice to attain a localization precision of 2.2 nm, whereas ~2500 photons yield precisions <1 nm (standard deviation). We further demonstrate 3D imaging with localization precision of ~2.4 nm in the focal plane and ~1.9 nm along the optic axis. Localizing with a precision of <20 nm within ~100 µs, we establish this spatio-temporal resolution in single fluorophore tracking and apply it to the diffusion of single labeled lipids in lipid-bilayer model membranes.


Asunto(s)
Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Difusión , Diseño de Equipo , Fluorescencia , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador , Fotones
3.
Nat Commun ; 12(1): 1559, 2021 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-33692354

RESUMEN

Structured illumination microscopy (SIM) is one of the most powerful and versatile optical super-resolution techniques. Compared with other super-resolution methods, SIM has shown its unique advantages in wide-field imaging with high temporal resolution and low photon damage. However, traditional SIM only has about 2 times spatial resolution improvement compared to the diffraction limit. In this work, we propose and experimentally demonstrate an easily-implemented, low-cost method to extend the resolution of SIM, named speckle metamaterial-assisted illumination nanoscopy (speckle-MAIN). A metamaterial structure is introduced to generate speckle-like sub-diffraction-limit illumination patterns in the near field with improved spatial frequency. Such patterns, similar to traditional SIM, are then used to excite objects on top of the surface. We demonstrate that speckle-MAIN can bring the resolution down to 40 nm and beyond. Speckle-MAIN represents a new route for super-resolution, which may lead to important applications in bio-imaging and surface characterization.


Asunto(s)
Microscopía/métodos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos
4.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-33672911

RESUMEN

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (ΔsprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ΔsprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Flavobacterium/fisiología , Locomoción/fisiología , Proteínas Bacterianas/genética , Flavobacterium/genética , Flavobacterium/ultraestructura , Locomoción/genética , Microscopía Electrónica de Transmisión/métodos , Microscopía Fluorescente/métodos , Mutación , Imagen de Lapso de Tiempo/métodos
5.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-33672992

RESUMEN

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.


Asunto(s)
Cromosomas de las Plantas/genética , Hordeum/genética , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Cromosomas de las Plantas/química , Cromosomas de las Plantas/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Colorantes Fluorescentes/química , Hordeum/citología , Indoles/química , Metafase/genética , Reproducibilidad de los Resultados
6.
Nat Commun ; 12(1): 1748, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741958

RESUMEN

Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.


Asunto(s)
Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Actinas/metabolismo , Animales , Células CHO , Membrana Celular , Cricetulus , Difusión , Receptores ErbB/metabolismo , Fluorescencia , Humanos , Espectrometría de Fluorescencia/métodos
7.
Nat Commun ; 12(1): 1809, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753744

RESUMEN

Dynamic membraneless compartments formed by protein condensates have multifunctional roles in cellular biology. Tools that inducibly trigger condensate formation have been useful for exploring their cellular function, however, there are few tools that provide inducible control over condensate disruption. To address this need we developed DisCo (Disassembly of Condensates), which relies on the use of chemical dimerizers to inducibly recruit a ligand to the condensate-forming protein, triggering condensate dissociation. We demonstrate use of DisCo to disrupt condensates of FUS, associated with amyotrophic lateral sclerosis, and to prevent formation of polyglutamine-containing huntingtin condensates, associated with Huntington's disease. In addition, we combined DisCo with a tool to induce condensates with light, CRY2olig, achieving bidirectional control of condensate formation and disassembly using orthogonal inputs of light and rapamycin. Our results demonstrate a method to manipulate condensate states that will have broad utility, enabling better understanding of the biological role of condensates in health and disease.


Asunto(s)
Proteínas Fluorescentes Verdes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Multimerización de Proteína , Proteínas/química , Animales , Células COS , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Microscopía Fluorescente/métodos , Proteínas/genética , Proteínas/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo
8.
Methods Mol Biol ; 2246: 237-247, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33576993

RESUMEN

A method for measuring mRNA copies in intact bacterial cells by fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) is presented. Unlike conventional single-molecule FISH, where the presence of a transcript is determined by fluorescence intensity, fliFISH relies on On-Off duty cycles of photo-switching dyes to set a predetermined threshold for distinguishing true signals from background noise. The method provides a quantitative approach for detecting and counting true mRNA copies and rejecting false signals with high accuracy.


Asunto(s)
Bacterias/genética , Hibridación Fluorescente in Situ/métodos , ARN Mensajero/genética , Fluorescencia , Colorantes Fluorescentes/metabolismo , Microscopía Fluorescente/métodos
9.
Methods Mol Biol ; 2218: 303-317, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606241

RESUMEN

Protein-protein interactions (PPIs) play a central role in all cellular processes. The discovery of green fluorescent protein (GFP) and split varieties, which are functionally reconstituted by complementation, led to the development of the bimolecular fluorescence complementation (BiFC) assay for the investigation of PPI in vivo. BiFC became a popular tool, as it is a convenient and quick technology to directly visualize PPI in a wide variety of living cells. In combination with the transparency of the early zebrafish embryo, it also permits detection of PPI in the context of an entire living organism, which performs all spatial and temporal regulations missing in in vitro systems like tissue culture. However, the application of BiFC in some research areas including the study of zebrafish is limited due to the lack of efficient and convenient BiFC expression vectors. Here, we describe the engineering of a novel set of Gateway®-adapted BiFC destination vectors to investigate PPI with all possible permutations for BiFC experiments. Moreover, we demonstrate the versatility of these destination vectors by confirming the interaction between zebrafish Bucky ball and RNA helicase Vasa in living embryos.


Asunto(s)
Bioensayo/métodos , Microscopía Fluorescente/métodos , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Embrión no Mamífero/metabolismo , Fluorescencia , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/metabolismo , Mapeo de Interacción de Proteínas/métodos , ARN Helicasas/metabolismo
10.
J Vis Exp ; (168)2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33616114

RESUMEN

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.


Asunto(s)
Actinas/metabolismo , Ensayos de Migración Celular , Movimiento Celular , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Miosinas/metabolismo , Animales , Miosinas/química , Células Sf9 , Spodoptera
11.
Nat Commun ; 12(1): 1180, 2021 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-33608524

RESUMEN

3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.


Asunto(s)
Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Conformación Molecular , Imagen Individual de Molécula/instrumentación , Imagen Individual de Molécula/métodos , Vesículas Cubiertas por Clatrina/ultraestructura , ADN/ultraestructura , Fluorescencia , Colorantes Fluorescentes/química , Microtúbulos/ultraestructura , Modelos Biológicos
12.
J Vis Exp ; (167)2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33522506

RESUMEN

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to. Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres. This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.


Asunto(s)
Drosophila melanogaster/citología , Imagenología Tridimensional , Microscopía Fluorescente/métodos , Animales , Técnicas de Cultivo de Célula , Supervivencia Celular , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Microtúbulos/metabolismo , Células Madre/citología , Fracciones Subcelulares/metabolismo , Testículo/citología , Imagen de Lapso de Tiempo , Tubulina (Proteína)/metabolismo
13.
Nat Protoc ; 16(3): 1647-1713, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33619390

RESUMEN

Chromatin conformation capture (3C) methods and fluorescent in situ hybridization (FISH) microscopy have been used to investigate the spatial organization of the genome. Although powerful, both techniques have limitations. Hi-C is challenging for low cell numbers and requires very deep sequencing to achieve its high resolution. In contrast, FISH can be done on small cell numbers and capture rare cell populations, but typically targets pairs of loci at a lower resolution. Here we detail a protocol for optical reconstruction of chromatin architecture (ORCA), a microscopy approach to trace the 3D DNA path within the nuclei of fixed tissues and cultured cells with a genomic resolution as fine as 2 kb and a throughput of ~10,000 cells per experiment. ORCA can identify structural features with comparable resolution to Hi-C while providing single-cell resolution and multimodal measurements characteristic of microscopy. We describe how to use this DNA labeling in parallel with multiplexed labeling of dozens of RNAs to relate chromatin structure and gene expression in the same cells. Oligopaint probe design, primary probe making, sample collection, cryosectioning and RNA/DNA primary probe hybridization can be completed in 1.5 weeks, while automated RNA/DNA barcode hybridization and RNA/DNA imaging typically takes 2-6 d for data collection and 2-7 d for the automated steps of image analysis.


Asunto(s)
Hibridación Fluorescente in Situ/métodos , Microscopía Fluorescente/métodos , Mapeo de Restricción Óptica/métodos , Línea Celular , Núcleo Celular/genética , Células Cultivadas , Cromatina/metabolismo , Inmunoprecipitación de Cromatina/métodos , Cromosomas/genética , ADN/química , ADN/genética , Sondas de ADN , Colorantes Fluorescentes/química , Técnicas Genéticas , Genoma/genética , Genómica/métodos , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , ARN/química , ARN/genética
14.
Nat Protoc ; 16(3): 1600-1628, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33627844

RESUMEN

Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). We describe the technical details for handling and imaging the samples for sptPALM, with a particular emphasis on the specificity of imaging plant cells, such as their thick cell walls or high degree of autofluorescence. We then provide a practical guide from data collection to image analyses. In particular, we introduce our sptPALM_viewer software and describe how to install and use it for analyzing sptPALM experiments. Finally, we report an R statistical analysis pipeline to analyze and compare sptPALM experiments. Altogether, this protocol should enable plant researchers to perform sptPALM using a benchmarked reproducible protocol. Routinely, the procedure takes 3-4 h of imaging followed by 3-4 d of image processing and data analysis.


Asunto(s)
Proteínas de la Membrana/metabolismo , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Arabidopsis/metabolismo , Difusión , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Proteínas de la Membrana/aislamiento & purificación , Imagen Óptica/métodos , Células Vegetales/química , Plantas/química , Plantas/metabolismo , Espectrometría de Fluorescencia/métodos
15.
Nat Commun ; 12(1): 501, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33479249

RESUMEN

DNA-PAINT is a versatile optical super-resolution technique relying on the transient binding of fluorescent DNA 'imagers' to target epitopes. Its performance in biological samples is often constrained by strong background signals and non-specific binding events, both exacerbated by high imager concentrations. Here we describe Repeat DNA-PAINT, a method that enables a substantial reduction in imager concentration, thus suppressing spurious signals. Additionally, Repeat DNA-PAINT reduces photoinduced target-site loss and can accelerate sampling, all without affecting spatial resolution.


Asunto(s)
ADN/química , Microscopía Fluorescente/métodos , Nanoestructuras/química , Nanotecnología/métodos , Animales , Ácidos Nucleicos/química , Oligonucleótidos/química , Reproducibilidad de los Resultados
16.
Nat Commun ; 12(1): 517, 2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33483489

RESUMEN

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.


Asunto(s)
Fluorescencia , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Imagen Individual de Molécula/métodos , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Microtúbulos/metabolismo , Fotometría/métodos
17.
Nat Commun ; 12(1): 563, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33495456

RESUMEN

Myosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. Here, we identify a binding protein for Drosophila myosin-7a termed M7BP, and describe how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex's processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Miosina VIIa/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Movimiento Celular/genética , Movimiento Celular/fisiología , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Microscopía Fluorescente/métodos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Miosina VIIa/química , Miosina VIIa/genética , Unión Proteica , Multimerización de Proteína , Seudópodos/genética , Seudópodos/fisiología , Estereocilios/genética , Estereocilios/fisiología
18.
Nat Commun ; 12(1): 107, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33398061

RESUMEN

Rapid 3D imaging of entire organs and organisms at cellular resolution is a recurring challenge in life science. Here we report on a computational light-sheet microscopy able to achieve minute-timescale high-resolution mapping of entire macro-scale organs. Through combining a dual-side confocally-scanned Bessel light-sheet illumination which provides thinner-and-wider optical sectioning of deep tissues, with a content-aware compressed sensing (CACS) computation pipeline which further improves the contrast and resolution based on a single acquisition, our approach yields 3D images with high, isotropic spatial resolution and rapid acquisition over two-order-of-magnitude faster than conventional 3D microscopy implementations. We demonstrate the imaging of whole brain (~400 mm3), entire gastrocnemius and tibialis muscles (~200 mm3) of mouse at ultra-high throughput of 5~10 min per sample and post-improved subcellular resolution of ~ 1.5 µm (0.5-µm iso-voxel size). Various system-level cellular analyses, such as mapping cell populations at different brain sub-regions, tracing long-distance projection neurons over the entire brain, and calculating neuromuscular junction occupancy across whole muscle, are also readily accomplished by our method.


Asunto(s)
Imagenología Tridimensional , Microscopía Fluorescente/métodos , Especificidad de Órganos , Animales , Encéfalo/diagnóstico por imagen , Mapeo Encefálico , Recuento de Células , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Músculos/diagnóstico por imagen , Unión Neuromuscular/diagnóstico por imagen , Neuronas/metabolismo , Fracciones Subcelulares
19.
Nat Commun ; 12(1): 669, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33510146

RESUMEN

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.


Asunto(s)
Arabidopsis/metabolismo , Pared Celular/metabolismo , Microtúbulos/metabolismo , Xilema/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Hipocótilo/citología , Hipocótilo/genética , Hipocótilo/metabolismo , Microscopía Fluorescente/métodos , Plantas Modificadas Genéticamente , Análisis de la Célula Individual/métodos , Imagen de Lapso de Tiempo/métodos , Xilema/citología , Xilema/genética
20.
Methods Mol Biol ; 2251: 73-89, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33481232

RESUMEN

The dynamic phosphorylation of phosphatidylinositol produces seven distinct but interconvertible phosphatidylinositol phosphates (PIPs). Each PIP exhibits specific enrichment in a subset of membrane compartments as a result of dynamic phosphorylation and dephosphorylation by lipid kinases and phosphatases, and/or by vesicle-mediated transport. Several PIPs are found within the plasma membrane, such as phosphatidylinositol-4-phosphate [PI(4)P], phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate (PIP3), and these control many aspects of cell physiology, including receptor signaling and membrane traffic. As a result, measurement of the cell surface abundance of these PIPs is a valuable resource to allow understanding of the regulation and function of these cell surface lipids. Here, we describe methods based on quantification of the localization of genetically encoded fluorescent PIP probes to the cell surface by either spinning disc confocal microscopy or total internal reflection fluorescence microscopy that allow detection of changes in cell surface levels of PI(4,5)P2, PI(3,4)P2, and PIP3. These methods can also be applied to the measurement of other PIPs or lipid species at the cell surface, and thus represent a useful resource for the study of the cell biology of PIPs.


Asunto(s)
Técnicas Biosensibles/métodos , Membrana Celular/química , Fosfatidilinositoles/análisis , Animales , Técnicas de Cultivo de Célula/métodos , Membrana Celular/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Humanos , Microscopía Fluorescente/métodos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/química , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismo , Transporte de Proteínas/fisiología
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