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EVpedia: A Community Web Portal for Extracellular Vesicles Research.
Dae-Kyum Kim, Jaewook Lee, Sae Rom Kim, Dong-Sic Choi, Yae Jin Yoon, Ji Hyun Kim, Gyeongyun Go, Dinh Nhung, Kahye Hong, Su Chul Jang, Si-Hyun Kim, Kyong-Su Park, Oh Youn Kim, Hyun Taek Park, Ji Hye Seo, Elena Aikawa, Monika Baj-Krzyworzeka, Bas W M van Balkom, Mattias Belting, Lionel Blanc, Vincent Bond, Antonella Bongiovanni, Francesc E Borràs, Luc Buée, Edit I Buzás, Lesley Cheng, Aled Clayton, Emanuele Cocucci, Charles S Dela Cruz, Dominic M Desiderio, Dolores Di Vizio, Karin Ekström, Juan M Falcon-Perez, Chris Gardiner, Bernd Giebel, David W Greening, Julia Christina Gross, Dwijendra Gupta, An Hendrix, Andrew F Hill, Michelle M Hill, Esther Nolte-'t Hoen, Do Won Hwang, Jameel Inal, Medicharla V Jagannadham, Muthuvel Jayachandran, Young-Koo Jee, Malene Jørgensen, Kwang Pyo Kim, Yoon-Keun Kim, Thomas Kislinger, Cecilia Lässer, Dong Soo Lee, Hakmo Lee, Johannes van Leeuwen, Thomas Lener, Ming-Lin Liu, Jan Lötvall, Antonio Marcilla, Suresh Mathivanan, Andreas Möller, Jess Morhayim, François Mullier, Irina Nazarenko, Rienk Nieuwland, Diana N Nunes, Ken Pang, Jaesung Park, Tushar Patel, Gabriella Pocsfalvi, Hernando Del Portillo, Ulrich Putz, Marcel I Ramirez, Marcio L Rodrigues, Tae-Young Roh, Felix Royo, Susmita Sahoo, Raymond Schiffelers, Shivani Sharma, Pia Siljander, Richard J Simpson, Carolina Soekmadji, Philip Stahl, Allan Stensballe, Ewa Stępień, Hidetoshi Tahara, Arne Trummer, Hadi Valadi, Laura J Vella, Sun Nyunt Wai, Kenneth Witwer, María Yáñez-Mó, Hyewon Youn, Reinhard Zeidler, Yong Song Gho.
Bioinformatics
PUBLISHED: 11-13-2014
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Extracellular vesicles are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for extracellular vesicle-related publications and vesicular components are currently challenging.
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Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit.
Mol. Biol. Cell
PUBLISHED: 09-17-2014
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Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission.
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ARFGAP1 promotes AP-2-dependent endocytosis.
Nat. Cell Biol.
PUBLISHED: 02-03-2011
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COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.
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Distinct dynamics of endocytic clathrin-coated pits and coated plaques.
PLoS Biol.
PUBLISHED: 03-04-2009
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Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then pinch off to become traffic carriers. These carriers are the principal vehicles of receptor-mediated endocytosis and are the major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of endocytic clathrin coat formation, which we designate as "coated pits" and "coated plaques." Coated pits are small, rapidly forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane--classical coated pits and coated plaques--and that these two assemblies interact quite differently with other intracellular structures.
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Shedding microvesicles: artefacts no more.
Trends Cell Biol.
PUBLISHED: 01-12-2009
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The small vesicles shed from the surface of many cells upon stimulation, considered for a long time to be artefacts, are now recognized as specific structures that are distinct from the exosomes released upon exocytosis of multivesicular bodies. Recent reports indicate that shedding vesicles participate in important biological processes, such as the surface-membrane traffic and the horizontal transfer of protein and RNAs among neighboring cells, which are necessary for the rapid phenotype adjustments in a variety of conditions. In addition, shedding vesicles have important physiological and pathological roles: in coagulation, by mediating the coordinate contribution of platelets, macrophages and neutrophils; in inflammatory diseases, via the release of cytokines; and in tumor progression, facilitating the spreading and release of cancer cells to generate metastases.
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Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.
PLoS Biol.
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Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.
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Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.
Proc. Natl. Acad. Sci. U.S.A.
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G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the ?(1)-adrenergic receptor (?(1)AR), the ?(2)-adrenergic receptor (?(2)AR), and the ?-aminobutyric acid (GABA(B)) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for ?(1)ARs (monomers/dimers) and highest for GABA(B) receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas ?(1)-/?(2)ARs were apparently freely diffusing on the cell surface, GABA(B) receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA(B) receptor complexes. These data provide a spatiotemporal characterization of ?(1)-/?(2)ARs and GABA(B) receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.
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Dynamics of intracellular clathrin/AP1- and clathrin/AP3-containing carriers.
Cell Rep
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Clathrin/AP1- and clathrin/AP3-coated vesicular carriers originate from endosomes and the trans-Golgi network. Here, we report the real-time visualization of these structures in living cells reliably tracked by rapid, three-dimensional imaging with the use of a spinning-disk confocal microscope. We imaged relatively sparse, diffraction-limited, fluorescent objects containing chimeric fluorescent protein (clathrin light chain, ? adaptor subunits, or dynamin2) with a spatial precision of up to ~30 nm and a temporal resolution of ~1 s. The dynamic characteristics of the intracellular clathrin/AP1 and clathrin/AP3 carriers are similar to those of endocytic clathrin/AP2 pits and vesicles; the clathrin/AP1 coats are, on average, slightly shorter-lived than their AP2 and AP3 counterparts. We confirmed that although dynamin2 is recruited as a burst to clathrin/AP2 pits immediately before their budding from the plasma membrane, we found no evidence supporting a similar association of dynamin2 with clathrin/AP1 or clathrin/AP3 carriers at any stage during their lifetime. We found no effects of chemical inhibitors of dynamin function or the K44A dominant-negative mutant of dynamin on AP1 and AP3 dynamics. This observation suggests that an alternative budding mechanism, yet to be discovered, is responsible for the scission step of clathrin/AP1 and clathrin/AP3 carriers.
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The first five seconds in the life of a clathrin-coated pit.
Cell
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Coated pits assemble by growth of a clathrin lattice, which is linked by adaptors to the underlying membrane. How does this process start? We used live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution to detect arrival of the clathrin triskelions and AP2 adaptors that initiate coat assembly. Unbiased object identification and trajectory tracking, together with a statistical model, yield the arrival times and numbers of individual proteins, as well as experimentally confirmed estimates of the extent of substitution of endogenous by expressed, fluorescently tagged proteins. Pits initiate by coordinated arrival of clathrin and AP2, which is usually detected as two sequential steps, each of one triskelion with two adaptors. PI-4,5-P2 is essential for initiation. The accessory proteins FCHo1/2 are not; instead, they are required for sustained growth. This objective picture of coated pit initiation also shows that methods outlined here will be broadly useful for studies of dynamic assemblies in living cells.
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Limited transferrin receptor clustering allows rapid diffusion of canine parvovirus into clathrin endocytic structures.
J. Virol.
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Viral pathogens usurp cell surface receptors to access clathrin endocytic structures, yet the mechanisms of virus incorporation into these structures remain incompletely understood. Here we used fluorescence microscopy to directly visualize the association of single canine parvovirus (CPV) capsids with cellular transferrin receptors (TfR) on the surfaces of live feline cells and to monitor how these CPV-TfR complexes access endocytic structures. We found that most capsids associated with fewer than five TfRs and that ?25% of TfR-bound capsids laterally diffused into assembling clathrin-coated pits less than 30 s after attachment. Capsids that did not encounter a coated pit dissociated from the cell surface with a half-life of ?30 s. Together, our results show how CPV exploits the natural mechanism of TfR endocytosis to engage the clathrin endocytic pathway and reveal that the low affinity of capsids for feline TfRs limits the residence time of capsids on the cell surface and thus the efficiency of virus internalization.
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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.