Cárdenas C, Escobar M, García A, Osorio-Reich M, Härtel S, Foskett JK, Franzini-Armstrong C.
J Struct Biol. 171 (3):372-381
The 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 approximately 15nm 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. Copyright © 2010 Elsevier Inc. All rights reserved.
Acknowledgments We thank Dr. Suresh Joseph for the type 1 InsP3R antibody. Cesar Cardenas would like to thank the ��Fundación Ciencias Para la Vida� (Chile). This work was support by a MDA grant to CFA and 614 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·BI (www.aibi.cl. Research in SCIAN-Lab (SH) is funded by FONDECYT 1090246, FONDEF (D07I1019), and ICM-P04-068-F (NEMO).