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Find video protocols related to scientific articles indexed in Pubmed.
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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Phospholipid scramblase-1-induced lipid reorganization regulates compensatory endocytosis in neuroendocrine cells.
J. Neurosci.
PUBLISHED: 02-22-2013
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Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1?/? mice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis.
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Using hierarchical clustering and dendrograms to quantify the clustering of membrane proteins.
Bull. Math. Biol.
PUBLISHED: 06-03-2011
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Cell biologists have developed methods to label membrane proteins with gold nanoparticles and then extract spatial point patterns of the gold particles from transmission electron microscopy images using image processing software. Previously, the resulting patterns were analyzed using the Hopkins statistic, which distinguishes nonclustered from modestly and highly clustered distributions, but is not designed to quantify the number or sizes of the clusters. Clusters were defined by the partitional clustering approach which required the choice of a distance. Two points from a pattern were put in the same cluster if they were closer than this distance. In this study, we present a new methodology based on hierarchical clustering to quantify clustering. An intrinsic distance is computed, which is the distance that produces the maximum number of clusters in the biological data, eliminating the need to choose a distance. To quantify the extent of clustering, we compare the clustering distance between the experimental data being analyzed with that from simulated random data. Results are then expressed as a dimensionless number, the clustering ratio that facilitates the comparison of clustering between experiments. Replacing the chosen cluster distance by the intrinsic clustering distance emphasizes densely packed clusters that are likely more important to downstream signaling events.We test our new clustering analysis approach against electron microscopy images from an experiment in which mast cells were exposed for 1 or 2 minutes to increasing concentrations of antigen that crosslink IgE bound to its high affinity receptor, Fc?RI, then fixed and the Fc?RI ? subunit labeled with 5 nm gold particles. The clustering ratio analysis confirms the increase in clustering with increasing antigen dose predicted from visual analysis and from the Hopkins statistic. Access to a robust and sensitive tool to both observe and quantify clustering is a key step toward understanding the detailed fine scale structure of the membrane, and ultimately to determining the role of spatial organization in the regulation of transmembrane signaling.
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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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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.