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Find video protocols related to scientific articles indexed in Pubmed.
Fluorogen Activating Proteins Provide Tunable Labeling Densities for Tracking Fc?RI Independent of IgE.
ACS Chem. Biol.
PUBLISHED: 10-25-2014
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Crosslinking of IgE bound Fc?RI on mast cells and basophils by multivalent antigen leads to degranulation and the release of key inflammatory mediators which stimulate the allergic response. Here, we present and characterize the use of Fluorogen Activating Proteins (FAPs) for single particle tracking of Fc?RI to investigate how receptor mobility is influenced after IgE induced changes in mast cell behavior. FAPs are genetically encoded tags that bind a fluorogen dye and increase its brightness upon binding up to 20,000-fold. We demonstrate that by titrating fluorogen concentration, labeling densities from ensemble to single particle can be achieved, independent of expression level and without the need for wash steps or photobleaching. The Fc?RI ?-subunit fused to a FAP (FAP-?) provides, for the first time, an IgE-independent probe for tracking this signaling subunit of Fc?RI at the single molecule level. We show that the Fc?RI ?-subunit dynamics are controlled by the IgE binding ?-subunit and that the cytokinergic IgE, SPE-7, induces mast cell activation without altering Fc?RI mobility or promoting internalization. We take advantage of the far-red emission of the malachite green (MG) fluorogen to track Fc?RI relative to dynamin-GFP and find that immobilized receptors readily correlate with locations of dynamin recruitment only under conditions that promote rapid endocytosis. These studies demonstrate the usefulness of the FAP system for single molecule studies and have provided new insights into the relationship between Fc?RI structure, activity and mobility.
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The membrane scaffold CD82 regulates cell adhesion by altering ?4 integrin stability and molecular density.
Mol. Biol. Cell
PUBLISHED: 03-12-2014
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Hematopoietic stem/progenitor cell (HSPC) interactions with the bone marrow microenvironment are important for maintaining HSPC self-renewal and differentiation. In recent work, we identified the tetraspanin protein, CD82, as a regulator of HPSC adhesion and homing to the bone marrow, although the mechanism by which CD82 mediated adhesion was unclear. In the present study, we determine that CD82 expression alters cell-matrix adhesion, as well as integrin surface expression. By combining the superresolution microscopy imaging technique, direct stochastic optical reconstruction microscopy, with protein clustering algorithms, we identify a critical role for CD82 in regulating the membrane organization of ?4 integrin subunits. Our data demonstrate that CD82 overexpression increases the molecular density of ?4 within membrane clusters, thereby increasing cellular adhesion. Furthermore, we find that the tight packing of ?4 into membrane clusters depend on CD82 palmitoylation and the presence of ?4 integrin ligands. In combination, these results provide unique quantifiable evidence of CD82's contribution to the spatial arrangement of integrins within the plasma membrane and suggest that regulation of integrin density by tetraspanins is a critical component of cell adhesion.
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Heterogeneous intracellular trafficking dynamics of brain-derived neurotrophic factor complexes in the neuronal soma revealed by single quantum dot tracking.
PLoS ONE
PUBLISHED: 01-01-2014
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Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs) are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF) binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes). Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are poised to provide understanding of how the molecular mechanisms underlying intracellular ligand-receptor trafficking shape cell signaling under conditions of both healthy and dysfunctional neurological disease models.
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ErbB3 is an active tyrosine kinase capable of homo- and hetero-interactions.
Mol. Cell. Biol.
PUBLISHED: 12-30-2013
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Often considered to be a "dead" kinase, erbB3 is implicated in escape from erbB-targeted cancer therapies. Here, heregulin stimulation is shown to markedly upregulate kinase activity in erbB3 immunoprecipitates. Intact, activated erbB3 phosphorylates tyrosine sites in an exogenous peptide substrate and this activity is abolished by mutagenesis of lysine 723 in the catalytic domain. Enhanced erbB3 kinase activity is linked to heterointeractions with catalytically active erbB2, since it is largely blocked in cells pretreated with lapatinib or pertuzumab. ErbB2 activation of erbB3 is not dependent on equal surface levels of these receptors, since it occurs even in erbB3-transfected CHO cells with disproportionally low amounts of erbB2. We tested a model in which transient erbB3/erbB2 heterointeractions set the stage for erbB3 homodimers to be signaling competent. ErbB3 homo- and hetero-dimerization events were captured in real time on live cells using single particle tracking of quantum dot probes bound to ligand or HA-tags on recombinant receptors.
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A Multiemitter Localization Comparison of 3D Superresolution Imaging Modalities.
Chemphyschem
PUBLISHED: 08-16-2013
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Single-molecule localization-based superresolution imaging is complicated by emission from multiple emitters overlapping at the detector. The potential for overlapping emitters is even greater for 3D imaging than for 2D imaging due to the large effective "volume" of the 3D point spread function. Overlapping emission can be accounted for in the estimation model, recovering the ability to localize the emitters, but with the caveat that the localization precision has a dependence on the amount of overlap from other emitters. Whether a particular 3D imaging modality has a significant advantage in facilitating the position estimation of overlapping emitters is investigated. The variants of two commonly used and easily implemented imaging modalities for 3D single-molecule imaging are compared: astigmatic imaging; dual focal plane imaging; and the combination of the two approaches, dual focal plane imaging with astigmatism. The Cramér-Rao lower bound is used to quantify the multiemitter estimation performance by calculating the theoretical best localization precision under a multiemitter estimation model. The performance of these 3D modalities is investigated under a wide range of conditions including various distributions of collected photons per emitter, background counts, pixel sizes, and camera readout noise values. Differences between modalities are small and it is therefore concluded that multiemitter fitting performance should not be a primary factor in selecting between these modalities.
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Localization Microscopy using Noncovalent Fluorogen Activation by Genetically Encoded Fluorogen-Activating Proteins.
Chemphyschem
PUBLISHED: 08-16-2013
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The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen-activating protein (FAP) can produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and super-resolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved by using physiological buffers and cellular media, in which additives and switching buffers are not required to obtain super-resolution images. We evaluate the super-resolution properties and images obtained from a selected FAP clone fused to actin, and show that the photon counts per object are between those typically reported for fluorescent proteins and switching-dye pairs, resulting in 10-30?nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures.
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Measuring image resolution in optical nanoscopy.
Nat. Methods
PUBLISHED: 03-21-2013
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Resolution in optical nanoscopy (or super-resolution microscopy) depends on the localization uncertainty and density of single fluorescent labels and on the samples spatial structure. Currently there is no integral, practical resolution measure that accounts for all factors. We introduce a measure based on Fourier ring correlation (FRC) that can be computed directly from an image. We demonstrate its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments. Our FRC resolution method makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, to optimize and rank different emitter localization and labeling strategies, to define a stopping criterion for data acquisition, to describe image anisotropy and heterogeneity, and even to estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, showing instead how the best image resolution can be achieved as fast as possible.
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Multi-color quantum dot tracking using a high-speed hyperspectral line-scanning microscope.
PLoS ONE
PUBLISHED: 01-01-2013
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Many cellular signaling processes are initiated by dimerization or oligomerization of membrane proteins. However, since the spatial scale of these interactions is below the diffraction limit of the light microscope, the dynamics of these interactions have been difficult to study on living cells. We have developed a novel high-speed hyperspectral microscope (HSM) to perform single particle tracking of up to 8 spectrally distinct species of quantum dots (QDs) at 27 frames per second. The distinct emission spectra of the QDs allows localization with ?10 nm precision even when the probes are clustered at spatial scales below the diffraction limit. The capabilities of the HSM are demonstrated here by application of multi-color single particle tracking to observe membrane protein behavior, including: 1) dynamic formation and dissociation of Epidermal Growth Factor Receptor dimers; 2) resolving antigen induced aggregation of the high affinity IgE receptor, Fc?R1; 3) four color QD tracking while simultaneously visualizing GFP-actin; and 4) high-density tracking for fast diffusion mapping.
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Determining Fc?RI diffusional dynamics via single quantum dot tracking.
Methods Mol. Biol.
PUBLISHED: 06-25-2011
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Single-particle tracking (SPT) using fluorescent quantum dots (QDs) provides high-resolution spatial-temporal information on receptor dynamics that cannot be obtained through traditional biochemical techniques. In particular, the high brightness and photostability of QDs make them ideal probes for SPT on living cells. We have shown that QD-labeled IgE can be used to characterize the dynamics of the high-affinity IgE Receptor. Here, we describe protocols for (1) coupling QDs to IgE, (2) tracking individual QD-bound receptors, and (3) analyzing one- and two-color tracking data.
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Simultaneous multiple-emitter fitting for single molecule super-resolution imaging.
Biomed Opt Express
PUBLISHED: 01-31-2011
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Single molecule localization based super-resolution imaging techniques require repeated localization of many single emitters. We describe a method that uses the maximum likelihood estimator to localize multiple emitters simultaneously within a single, two-dimensional fitting sub-region, yielding an order of magnitude improvement in the tolerance of the analysis routine with regards to the single-frame active emitter density. Multiple-emitter fitting enables the overall performance of single-molecule super-resolution to be improved in one or more of several metrics that result in higher single-frame density of localized active emitters. For speed, the algorithm is implemented on Graphics Processing Unit (GPU) architecture, resulting in analysis times on the order of minutes. We show the performance of multiple emitter fitting as a function of the single-frame active emitter density. We describe the details of the algorithm that allow robust fitting, the details of the GPU implementation, and the other imaging processing steps required for the analysis of data sets.
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ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding.
Nat. Struct. Mol. Biol.
PUBLISHED: 01-18-2011
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The extent to which ligand occupancy and dimerization contribute to erbB1 signaling is controversial. To examine this, we used two-color quantum-dot tracking for visualization of the homodimerization of human erbB1 and quantification of the dimer off-rate (k(off)) on living cells. Kinetic parameters were extracted using a three-state hidden Markov model to identify transition rates between free, co-confined and dimerized states. We report that dimers composed of two ligand-bound receptors are long-lived and their k(off) is independent of kinase activity. By comparison, unliganded dimers have a more than four times faster k(off). Transient co-confinement of receptors promotes repeated encounters and enhances dimer formation. Mobility decreases more than six times when ligand-bound receptors dimerize. Blockade of erbB1 kinase activity or disruption of actin networks results in faster diffusion of receptor dimers. These results implicate both signal propagation and the cortical cytoskeleton in reduced mobility of signaling-competent erbB1 dimers.
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Distribution and dynamics of rat basophilic leukemia immunoglobulin E receptors (FcepsilonRI) on planar ligand-presenting surfaces.
Biophys. J.
PUBLISHED: 03-22-2010
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There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. We have quantified the spatiotemporal dynamics of the redistribution of immunoglobulin E-loaded receptors (IgE-FcepsilonRI) on rat basophilic leukemia-2H3 mast cells in contact with fluid and gel-phase membranes displaying ligands for immunoglobulin E, using total internal reflection fluorescence microscopy. To clearly separate the kinetics of receptor redistribution from cell spreading, and to precisely define the initial contact time (+/-50 ms), micropipette cell manipulation was used to bring individual cells into contact with surfaces. On ligand-free surfaces, there are micron-scale heterogeneities in fluorescence that likely reflect regions of the cell that are more closely apposed to the substrate. When ligands are present, receptor clusters form with this same size scale. The initial rate of accumulation of receptors into the clusters is consistent with diffusion-limited trapping with D approximately 10(-1) microm2/s. These results support the hypothesis that clusters form by diffusion to cell-surface contact regions. Over longer timescales (>10 s), individual clusters moved with both diffusive and directed motion components. The dynamics of the cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes.
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Fast, single-molecule localization that achieves theoretically minimum uncertainty.
Nat. Methods
PUBLISHED: 02-22-2010
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We describe an iterative algorithm that converges to the maximum likelihood estimate of the position and intensity of a single fluorophore. Our technique efficiently computes and achieves the Cramér-Rao lower bound, an essential tool for parameter estimation. An implementation of the algorithm on graphics processing unit hardware achieved more than 10(5) combined fits and Cramér-Rao lower bound calculations per second, enabling real-time data analysis for super-resolution imaging and other applications.
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Formation of a mast cell synapse: Fc epsilon RI membrane dynamics upon binding mobile or immobilized ligands on surfaces.
J. Immunol.
PUBLISHED: 12-30-2009
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Fc epsilonRI on mast cells form a synapse when presented with mobile, bilayer-incorporated Ag. In this study, we show that receptor reorganization within the contacting mast cell membrane is markedly different upon binding of mobile and immobilized ligands. Rat basophilic leukemia mast cells primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand), or chemically cross-linked, multivalent DNP (immobilized ligand). Total internal reflection fluorescence imaging and electron microscopy methods were used to visualize receptor reorganization at the contact site. The spatial relationships of Fc epsilonRI to other cellular components at the synapse, such as actin, cholesterol, and linker for activation of T cells, were also analyzed. Stimulation of mast cells with immobilized polyvalent ligand resulted in typical levels of degranulation. Remarkably, degranulation also followed interaction of mast cells, with bilayers presenting mobile, monovalent ligand. Receptors engaged with mobile ligand coalesce into large, cholesterol-rich clusters that occupy the central portion of the contacting membrane. These data indicate that Fc epsilonRI cross-linking is not an obligatory step in triggering mast cell signaling and suggest that dense populations of mobile receptors are capable of initiating low-level degranulation upon ligand recognition.
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Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope.
Microsc. Res. Tech.
PUBLISHED: 02-12-2009
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Photomanipulation (photobleaching, photoactivation, or photoconversion) is an essential tool in fluorescence microscopy. Fluorescence recovery after photobleaching (FRAP) is commonly used for the determination of lateral diffusion constants of membrane proteins, and can be conveniently implemented in confocal laser scanning microscopy (CLSM). Such determinations provide important information on molecular dynamics in live cells. However, the CLSM platform is inherently limited for FRAP because of its inflexible raster (spot) scanning format. We have implemented FRAP and photoactivation protocols using structured illumination and detection in a programmable array microscope (PAM). The patterns are arbitrary in number and shape, dynamic and adjustable to and by the sample characteristics. We have used multispot PAM-FRAP to measure the lateral diffusion of the erbB3 (HER3) receptor tyrosine kinase labeled by fusion with mCitrine on untreated cells and after treatment with reagents that perturb the cytoskeleton or plasma membrane or activate coexpressed erbB1 (HER1, the EGF receptor EGFR). We also show the versatility of the PAM for photoactivation in arbitrary regions of interest, in cells expressing erbB3 fused with the photoconvertible fluorescent protein dronpa.
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Advances in high-resolution imaging--techniques for three-dimensional imaging of cellular structures.
J. Cell. Sci.
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A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.
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Single-particle tracking of immunoglobulin E receptors (Fc?RI) in micron-sized clusters and receptor patches.
FEBS Lett.
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When mast cells contact a monovalent antigen-bearing fluid lipid bilayer, IgE-loaded Fc?RI receptors aggregate at contact points and trigger degranulation and the release of immune activators. We used two-color total internal reflection fluorescence microscopy and single-particle tracking to show that most fluorescently labeled receptor complexes diffuse freely within these micron-size clusters, with a diffusion coefficient comparable to free receptors in resting cells. At later times, when the small clusters coalesce to form larger patches, receptors diffuse even more rapidly. In all cases, Monte Carlo diffusion simulations ensured that the tracking results were free of bias, and distinguished biological from statistical variation. These results show the diversity in receptor mobility in mast cells, demonstrating at least three distinct states of receptor diffusivity.
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The Spatiotemporal Organization of ErbB Receptors: Insights from Microscopy.
Cold Spring Harb Perspect Biol
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Signal transduction is regulated by protein-protein interactions. In the case of the ErbB family of receptor tyrosine kinases (RTKs), the precise nature of these interactions remains a topic of debate. In this review, we describe state-of-the-art imaging techniques that are providing new details into receptor dynamics, clustering, and interactions. We present the general principles of these techniques, their limitations, and the unique observations they provide about ErbB spatiotemporal organization.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.