Visualization of inositol 1,4,5-trisphosphate receptors on the nuclear envelope outer membrane by freeze-drying and rotary shadowing for electron microscopy

JOURNAL OF STRUCTURAL BIOLOGY (PRINT);
J. Struct. Biol.

dc.creatorGarcía-Acevedo, Alejandra
dc.creatorHartel-Grundler, Steffen
dc.creatorCardenas-Alarcón, César
dc.creatorEscobar, Matias
dc.creatorFoskett, Kevin
dc.creatorFranzini-Amstrong- Clara
dc.creatorOsorio-Reich, María
dc.date2017-04-27T18:53:25Z
dc.date2022-07-07T02:26:20Z
dc.date2017-04-27T18:53:25Z
dc.date2022-07-07T02:26:20Z
dc.date2010
dc.date.accessioned2023-08-23T00:21:58Z
dc.date.available2023-08-23T00:21:58Z
dc.identifier0
dc.identifierD07I1019
dc.identifierD07I1019
dc.identifierWOS:000280680100014
dc.identifier1047-8477
dc.identifierhttps://hdl.handle.net/10533/197886
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8354859
dc.descriptionThe receptors for the second messenger InsP(3) comprise a family of closely related ion channels that release Ca(2+) from intracellular stores, most prominently the endoplasmic reticulum and its extension into the nuclear envelope. The precise sub-cellular localization of InsP(3)Rs and the spatial relationships among them are important for the initiation, spatial and temporal properties and propagation of local and global Ca(2+) signals, but the spatial organization of InsP(3)Rs in Ca(2+) stores is poorly characterized. Using nuclei isolated from insect Sf9 cells and freeze-dry rotary shadowing, we have addressed this by directly visualizing the cytoplasmic domain of InsP(3)R located on the cytoplasmic side of the nuclear envelope. Identification of similar to 15 nm structures as the cytoplasmic domain of InsP(3)R was indirectly supported by a marked increase in their frequency after transient transfections with cDNAs for rat types 1 and 3 InsP(3)R, and directly confirmed by gold labeling either with heparin or a specific anti-InsP(3)R antibody. Over-expression of InsP(3)R did not result in the formation of arrays or clusters with channels touching each other. Gold-labeling suggests that the channel amino terminus resides near the center of the cytoplasmic tetrameric quaternary structure. The combination of nuclear isolation with freeze-drying and rotary shadow techniques allows direct visualization of InsP(3)Rs in native nuclear envelopes and can be used to determine their spatial distribution and density. (C) 2010 Elsevier Inc. All rights reserved.
dc.descriptionWe thank Dr. Suresh Joseph for the type 1 InsP<INF>3</INF>R antibody. Cesar Cardenas would like to thank the "Fundacion Ciencias Para la Vida" (Chile). This work was support by a MDA grant to CFA and NIH GM065830 to JKF. SCIAN-Lab is a member of the German-Chilean Center of Excellence Initiative for Medical Informatics (DAAD) and the Advanced Imaging & Bioinformatics Initiative AI center dot BI (www.aibi.cl. Research in SCIAN-Lab (SH) is funded by FONDECYT 1090246, FONDEF (D0711019), and ICM-P04-068-F (NEMO).
dc.description8
dc.descriptionFONDEF
dc.descriptionfoskett@mail.med.upenn.edu
dc.descriptionMDA; NIH [GM065830]; FONDECYT [1090246]; FONDEF [D0711019]; NEMO [ICM-P04-068-F]
dc.description3
dc.descriptionFONDEF
dc.description171
dc.languageENG
dc.publisherACADEMIC PRESS INC ELSEVIER SCIENCE
dc.relationinstname: Conicyt
dc.relationreponame: Repositorio Digital RI2.0
dc.relationinstname: Conicyt
dc.relationreponame: Repositorio Digital RI2.0
dc.relationinfo:eu-repo/grantAgreement/Fondef/D07I1019
dc.relationinfo:eu-repo/semantics/dataset/hdl.handle.net/10533/93477
dc.relationhttps://doi.org/10.1016/j.jsb.2010.05.003
dc.rightsinfo:eu-repo/semantics/openAccess
dc.titleVisualization of inositol 1,4,5-trisphosphate receptors on the nuclear envelope outer membrane by freeze-drying and rotary shadowing for electron microscopy
dc.titleJOURNAL OF STRUCTURAL BIOLOGY (PRINT)
dc.titleJ. Struct. Biol.
dc.typeArticulo
dc.typeinfo:eu-repo/semantics/article
dc.typeinfo:eu-repo/semantics/publishedVersion
dc.coverageSAN DIEGO


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