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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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Optimal aggregation of Fc?RI with a structurally defined trivalent ligand overrides negative regulation driven by phosphatases.
ACS Chem. Biol.
PUBLISHED: 05-15-2014
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To investigate why responses of mast cells to antigen-induced IgE receptor (Fc?RI) aggregation depend nonlinearly on antigen dose, we characterized a new artificial ligand, DF3, through complementary modeling and experimentation. This ligand is a stable trimer of peptides derived from bacteriophage T4 fibritin, each conjugated to a hapten (DNP). We found low and high doses of DF3 at which degranulation of mast cells sensitized with DNP-specific IgE is minimal, but ligand-induced receptor aggregation is comparable to aggregation at an intermediate dose, optimal for degranulation. This finding makes DF3 an ideal reagent for studying the balance of negative and positive signaling in the Fc?RI pathway. We find that the lipid phosphatase SHIP and the protein tyrosine phosphatase SHP-1 negatively regulate mast cell degranulation over all doses considered. In contrast, SHP-2 promotes degranulation. With high DF3 doses, relatively rapid recruitment of SHIP to the plasma membrane may explain the reduced degranulation response. Our results demonstrate that optimal secretory responses of mast cells depend on the formation of receptor aggregates that promote sufficient positive signaling by Syk to override phosphatase-mediated negative regulatory signals.
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sdf1 Expression reveals a source of perivascular-derived mesenchymal stem cells in zebrafish.
Stem Cells
PUBLISHED: 01-29-2014
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There is accumulating evidence that mesenchymal stem cells (MSCs) have their origin as perivascular cells (PVCs) in vivo, but precisely identifying them has been a challenge, as they have no single definitive marker and are rare. We have developed a fluorescent transgenic vertebrate model in which PVC can be visualized in vivo based upon sdf1 expression in the zebrafish. Prospective isolation and culture of sdf1(DsRed) PVC demonstrated properties consistent with MSC including prototypical cell surface marker expression; mesodermal differentiation into adipogenic, osteogenic, and chondrogenic lineages; and the ability to support hematopoietic cells. Global proteomic studies performed by two-dimensional liquid chromatography and tandem mass spectrometry revealed a high degree of similarity to human MSC (hMSC) and discovery of novel markers (CD99, CD151, and MYOF) that were previously unknown to be expressed by hMSC. Dynamic in vivo imaging during fin regeneration showed that PVC may arise from undifferentiated mesenchyme providing evidence of a PVC-MSC relationship. This is the first model, established in zebrafish, in which MSC can be visualized in vivo and will allow us to better understand their function in a native environment.
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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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The architectural relationship of components controlling mast cell endocytosis.
J. Cell. Sci.
PUBLISHED: 08-28-2013
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Eukaryotic cells use multiple routes for receptor internalization. Here, we examine the topographical relationships of clathrin-dependent and clathrin-independent endocytic structures on the plasma membranes of leukemia-derived mast cells. The high affinity IgE receptor (Fc?RI) utilizes both pathways, whereas transferrin receptor serves as a marker for the classical clathrin-mediated endocytosis pathway. Both receptors were tracked by live-cell imaging in the presence or absence of inhibitors that established their differential dependence on specific endocytic adaptor proteins. The topology of antigen-bound Fc?RI, clathrin, dynamin, Arf6 and Eps15-positive structures were analyzed by 2D and 3D immunoelectron microscopy techniques, revealing their remarkable spatial relationships and unique geometry. We conclude that the mast cell plasma membrane has multiple specialized domains for endocytosis. Their close proximity might reflect shared components, such as lipids and adaptor proteins, that facilitate inward membrane curvature. Intersections between these specialized domains might represent sorting stations that direct cargo to specific endocytic pathways.
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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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Dynamic transition states of ErbB1 phosphorylation predicted by spatial stochastic modeling.
Biophys. J.
PUBLISHED: 04-09-2013
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ErbB1 overexpression is strongly linked to carcinogenesis, motivating better understanding of erbB1 dimerization and activation. Recent single-particle-tracking data have provided improved measures of dimer lifetimes and strong evidence that transient receptor coconfinement promotes repeated interactions between erbB1 monomers. Here, spatial stochastic simulations explore the potential impact of these parameters on erbB1 phosphorylation kinetics. This rule-based mathematical model incorporates structural evidence for conformational flux of the erbB1 extracellular domains, as well as asymmetrical orientation of erbB1 cytoplasmic kinase domains during dimerization. The asymmetric dimer model considers the theoretical consequences of restricted transactivation of erbB1 receptors within a dimer, where the N-lobe of one monomer docks with the C-lobe of the second monomer and triggers its catalytic activity. The dynamic nature of the erbB1 phosphorylation state is shown by monitoring activation states of individual monomers as they diffuse, bind, and rebind after ligand addition. The model reveals the complex interplay between interacting liganded and nonliganded species and the influence of their distribution and abundance within features of the membrane landscape.
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Bone marrow stromal and vascular smooth muscle cells have chemosensory capacity via bitter taste receptor expression.
PLoS ONE
PUBLISHED: 02-11-2013
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The ability of cells to detect changes in the microenvironment is important in cell signaling and responsiveness to environmental fluctuations. Our interest is in understanding how human bone marrow stromal-derived cells (MSC) and their relatives, vascular smooth muscle cells (VSMC), interact with their environment through novel receptors. We found, through a proteomics screen, that MSC express the bitter taste receptor, TAS2R46, a protein more typically localized to the taste bud. Expression was also confirmed in VSMCs. A prototypical bitter compound that binds to the bitter taste receptor class, denatonium, increased intracellular calcium release and decreased cAMP levels as well as increased the extracellular release of ATP in human MSC. Denatonium also bound and activated rodent VSMC with a change in morphology upon compound exposure. Finally, rodents given denatonium in vivo had a significant drop in blood pressure indicating a vasodilator response. This is the first description of chemosensory detection by MSC and VSMCs via a taste receptor. These data open a new avenue of research into discovering novel compounds that operate through taste receptors expressed by cells in the marrow and vascular microenvironments.
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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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Nanoscale membrane organization: where biochemistry meets advanced microscopy.
ACS Chem. Biol.
PUBLISHED: 11-14-2011
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Understanding the molecular mechanisms that shape an effective cellular response is a fundamental question in biology. Biochemical measurements have revealed critical information about the order of protein-protein interactions along signaling cascades but lack the resolution to determine kinetics and localization of interactions on the plasma membrane. Furthermore, the local membrane environment influences membrane receptor distributions and dynamics, which in turn influences signal transduction. To measure dynamic protein interactions and elucidate the consequences of membrane architecture interplay, direct measurements at high spatiotemporal resolution are needed. In this review, we discuss the biochemical principles regulating membrane nanodomain formation and protein function, ranging from the lipid nanoenvironment to the cortical cytoskeleton. We also discuss recent advances in fluorescence microscopy that are making it possible to quantify protein organization and biochemical events at the nanoscale in the living cell membrane.
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Spatio-temporal signaling in mast cells.
Adv. Exp. Med. Biol.
PUBLISHED: 06-30-2011
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This chapter summarizes the evidence for localized signaling domains in mast cells and basophils, with a particular focus on the high affinity IgE receptor, Fc?RI and its crosstalk with other membrane proteins. It is noteworthy that a literature spanning 30 years established the Fc?RI as a model receptor for studying activation-induced changes in receptor diffusion and lipid raft association. Now a combination of high resolution microscopy methods, including immunoelectron microscopy and sophisticated fluorescence-based techniques, provide new insight into the nanoscale spatial and temporal aspects of receptor topography on the mast cell plasma membrane. Physical crosslinking of Fc?RI with multivalent ligands leads to formation of IgE receptor clusters, termed "signaling patches," that recruit downstream signaling molecules. However, classes of receptors that engage solely withmono valent ligands can also form distinctive signaling patches. The dynamic relationships between receptor diffusion, aggregation state, clustering, signal initiation and signal strength are discussed in the context of these recent findings.
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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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Interleukin-4 alters early phagosome phenotype by modulating class I PI3K dependent lipid remodeling and protein recruitment.
PLoS ONE
PUBLISHED: 03-03-2011
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Phagocytosis is a complex process that involves membranelipid remodeling and the attraction and retention of key effector proteins. Phagosome phenotype depends on the type of receptor engaged and can be influenced by extracellular signals. Interleukin 4 (IL-4) is a cytokine that induces the alternative activation of macrophages (M?s) upon prolonged exposure, triggering a different cell phenotype that has an altered phagocytic capacity. In contrast, the direct effects of IL-4 during phagocytosis remain unknown. Here, we investigate the impact of short-term IL-4 exposure (1 hour) during phagocytosis of IgG-opsonized yeast particles by M?s. By time-lapse confocal microscopy of GFP-tagged lipid-sensing probes, we show that IL-4 increases the negative charge of the phagosomal membrane by prolonging the presence of the negatively charged second messenger PI(3,4,5)P3. Biochemical assays reveal an enhanced PI3K/Akt activity upon phagocytosis in the presence of IL-4. Blocking the specific class I PI3K after the onset of phagocytosis completely abrogates the IL-4-induced changes in lipid remodeling and concomitant membrane charge. Finally, we show that IL-4 direct signaling leads to a significantly prolonged retention profile of the signaling molecules Rac1 and Rab5 to the phagosomal membrane in a PI3K-dependent manner. This protracted early phagosome phenotype suggests an altered maturation, which is supported by the delayed phagosome acidification measured in the presence of IL-4. Our findings reveal that molecular differences in IL-4 levels, in the extracellular microenvironment, influence the coordination of lipid remodeling and protein recruitment, which determine phagosome phenotype and, eventually, fate. Endosomal and phagosomal membranes provide topological constraints to signaling molecules. Therefore, changes in the phagosome phenotype modulated by extracellular factors may represent an additional mechanism that regulates the outcome of phagocytosis and could have significant impact on the net biochemical output of a cell.
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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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Time-resolved three-dimensional molecular tracking in live cells.
Nano Lett.
PUBLISHED: 10-21-2010
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We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 ms to follow three-dimensional molecular motion. This method has substantial advantages over three-dimensional molecular tracking methods based upon charge-coupled device cameras, including increased Z-tracking range (10 ?m demonstrated here), substantially lower excitation powers (15 ?W used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon antibunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye-labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates. To demonstrate the power of these methods for exploring the spatiotemporal dynamics of live cells, we follow individual QD-labeled IgE-Fc?RI receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-Fc?RI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell.
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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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ERK nuclear translocation is dimerization-independent but controlled by the rate of phosphorylation.
J. Biol. Chem.
PUBLISHED: 11-17-2009
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Upon activation, ERKs translocate from the cytoplasm to the nucleus. This process is required for the induction of many cellular responses, yet the molecular mechanisms that regulate ERK nuclear translocation are not fully understood. We have used a mouse embryo fibroblast ERK1-knock-out cell line expressing green fluorescent protein (GFP)-tagged ERK1 to probe the spatio-temporal regulation of ERK1. Real time fluorescence microscopy and fluorescence correlation spectroscopy revealed that ERK1 nuclear accumulation increased upon serum stimulation, but the mobility of the protein in the nucleus and cytoplasm remained unchanged. Dimerization of ERK has been proposed as a requirement for nuclear translocation. However, ERK1-Delta4, the mutant shown consistently to be dimerization-deficient in vitro, accumulated in the nucleus to the same level as wild type (WT), indicating that dimerization of ERK1 is not required for nuclear entry and retention. Consistent with this finding, energy migration Förster resonance energy transfer and fluorescence correlation spectroscopy measurements in living cells did not detect dimerization of GFP-ERK1-WT upon activation. In contrast, the kinetics of nuclear accumulation and phosphorylation of GFP-ERK1-Delta4 were slower than that of GFP-ERK1-WT. These results indicate that the differential shuttling behavior of the mutant is a consequence of delayed phosphorylation of ERK by MEK rather than dimerization. Our data demonstrate for the first time that a delay in cytoplasmic activation of ERK is directly translated into a delay in nuclear translocation.
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Caught in the act: quantifying protein behaviour in living cells.
Trends Cell Biol.
PUBLISHED: 06-15-2009
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Protein localization and dynamics both have important roles in cell signal transduction. Biochemical studies have elucidated many details about the chain of events in signal cascades, but the poor temporal resolution and absence of spatial localization in these conventional techniques make it difficult to determine the "where and when" of protein interactions. Over the past decade, imaging technologies and biological tools have developed to a point where many fundamental questions about protein activity can be addressed at the molecular level in living cells, revealing spatio-temporal information that is not provided by traditional biochemical assays. In this review, we illustrate the power of emerging fluorescence microscopy techniques to capture and quantify protein dynamics.
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Small, mobile FcepsilonRI receptor aggregates are signaling competent.
Immunity
PUBLISHED: 03-19-2009
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Crosslinking of IgE-bound FcepsilonRI triggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that FcepsilonRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-FcepsilonRI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. FcepsilonRI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a trigger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.
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Hyperspectral confocal fluorescence imaging: exploring alternative multivariate curve resolution approaches.
Appl Spectrosc
PUBLISHED: 03-14-2009
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Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution (MCR), provides a powerful new tool for improved quantitative imaging of multi-fluorophore samples. Generally, fully non-negatively constrained models are used in the constrained alternating least squares MCR analyses of hyperspectral images since real emission components are expected to have non-negative pure emission spectra and concentrations. However, in this paper, we demonstrate four separate cases in which partially constrained models are preferred over the fully constrained MCR models. These partially constrained MCR models can sometimes be preferred when system artifacts are present in the data or where small perturbations of the major emission components are present due to environmental effects or small geometric changes in the fluorescing species. Here we demonstrate that in the cases of hyperspectral images obtained from multicomponent spherical beads, autofluorescence from fixed lung epithelial cells, fluorescence of quantum dots in aqueous solutions, and images of mercurochrome-stained endosperm portions of a wild-type corn seed, these alternative, partially constrained MCR analyses provide improved interpretability of the MCR solutions. Often the system artifacts or environmental effects are more readily described as first and/or second derivatives of the main emission components in these alternative MCR solutions since they indicate spectral shifts and/or spectral broadening or narrowing of the emission bands, respectively. Thus, this paper serves to demonstrate the need to test alternative partially constrained models when analyzing hyperspectral images with MCR methods.
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Insights into cell membrane microdomain organization from live cell single particle tracking of the IgE high affinity receptor Fc?RI of mast cells.
Bull. Math. Biol.
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Current models propose that the plasma membrane of animal cells is composed of heterogeneous and dynamic microdomains known variously as cytoskeletal corrals, lipid rafts and protein islands. Much of the experimental evidence for these membrane compartments is indirect. Recently, live cell single particle tracking studies using quantum dot-labeled IgE bound to its high affinity receptor Fc?RI, provided direct evidence for the confinement of receptors within micrometer-scale cytoskeletal corrals. In this study, we show that an innovative time-series analysis of single particle tracking data for the high affinity IgE receptor, Fc?RI, on mast cells provides substantial quantitative information about the submicrometer organization of the membrane. The analysis focuses on the probability distribution function of the lengths of the jumps in the positions of the quantum dots labeling individual IgE Fc?RI complexes between frames in movies of their motion. Our results demonstrate the presence, within the micrometer-scale cytoskeletal corrals, of smaller subdomains that provide an additional level of receptor confinement. There is no characteristic size for these subdomains; their size varies smoothly from a few tens of nanometers to a over a hundred nanometers. In QD-IGE labeled unstimulated cells, jumps of less than 70 nm predominate over longer jumps. Addition of multivalent antigen to crosslink the QD-IgE-Fc?RI complexes causes a rapid slowing of receptor motion followed by a long tail of mostly jumps less than 70 nm. The reduced receptor mobility likely reflects both the membrane heterogeneity revealed by the confined motion of the monomeric receptor complexes and the antigen-induced cross linking of these complexes into dimers and higher oligomers. In both cases, the probability distribution of the jump lengths is well fit, from 10 nm to over 100 nm, by a novel power law. The fit for short jumps suggests that the motion of the quantum dots can be modeled as diffusion in a fractal space of dimension less than two.
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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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Mast cell synapses and exosomes: membrane contacts for information exchange.
Front Immunol
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In addition to their central role in allergy, mast cells are involved in a wide variety of cellular interactions during homeostasis and disease. In this review, we discuss the ability of mast cells to extend their mechanisms for intercellular communication beyond the release of soluble mediators. These include formation of mast cell synapses on antigen presenting surfaces, as well as cell-cell contacts with dendritic cells and T cells. Release of membrane bound exosomes also provide for the transfer of antigen, mast cell proteins, and RNA to other leukocytes. With the recognition of the extended role mast cells have during immune modulation, further investigation of the processes in which mast cells are involved is necessary. This reopens mast cell research to exciting possibilities, demonstrating it to be an immunological frontier.
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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.

How does it work?

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

Video X seems to be unrelated to Abstract Y...

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.