Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/95659
Citations
Scopus Web of Science® Altmetric
?
?
Type: Journal article
Title: mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging
Author: Yadav, R.
Burgos, P.
Parker, A.
Iadevaia, V.
Proud, C.
Allen, R.
O'Connell, J.
Jeshtadi, A.
Stubbs, C.
Botchway, S.
Citation: BMC Cell Biology, 2013; 14(3):3-1-3-16
Publisher: Biomed Central Ltd
Issue Date: 2013
ISSN: 1471-2121
1471-2121
Statement of
Responsibility: 
Rahul B Yadav, Pierre Burgos, Anthony W Parker, Valentina Iadevaia, Christopher G Proud, Rodger A Allen, James P O'Connell, Ananya Jeshtadi, Christopher D Stubbs, and Stanley W Botchway
Abstract: Background: The mammalian target of rapamycin (mTOR) signalling pathway has a key role in cellular regulation and several diseases. While it is thought that Rheb GTPase regulates mTOR, acting immediately upstream, while raptor is immediately downstream of mTOR, direct interactions have yet to be verified in living cells, furthermore the localisation of Rheb has been reported to have only a cytoplasmic cellular localization. Results: In this study a cytoplasmic as well as a significant sub-cellular nuclear mTOR localization was shown , utilizing green and red fluorescent protein (GFP and DsRed) fusion and highly sensitive single photon counting fluorescence lifetime imaging microscopy (FLIM) of live cells. The interaction of the mTORC1 components Rheb, mTOR and raptor, tagged with EGFP/DsRed was determined using fluorescence energy transfer-FLIM. The excited-state lifetime of EGFP-mTOR of ~2400 ps was reduced by energy transfer to ~2200 ps in the cytoplasm and to 2000 ps in the nucleus when co-expressed with DsRed-Rheb, similar results being obtained for co-expressed EGFP-mTOR and DsRed-raptor. The localization and distribution of mTOR was modified by amino acid withdrawal and re-addition but not by rapamycin. Conclusions: The results illustrate the power of GFP-technology combined with FRET-FLIM imaging in the study of the interaction of signalling components in living cells, here providing evidence for a direct physical interaction between mTOR and Rheb and between mTOR and raptor in living cells for the first time.
Keywords: FLIM; FRET; mTOR; GFP; Raptor; Rheb
Rights: © 2013 Yadav et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
RMID: 0020128140
DOI: 10.1186/1471-2121-14-3
Appears in Collections:Molecular and Biomedical Science publications

Files in This Item:
File Description SizeFormat 
hdl_95659.pdfPublished version2.63 MBAdobe PDFView/Open


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.