Yadav Rahul B, Burgos Pierre, Parker Anthony W, Iadevaia Valentina, Proud Christopher G, Allen Rodger A, O'Connell James P, Jeshtadi Ananya, Stubbs Christopher D, Botchway Stanley W
Central Laser Facility, STFC, Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot, Oxon OX110QX, UK.
BMC Cell Biol. 2013 Jan 12;14:3. doi: 10.1186/1471-2121-14-3.
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.
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.
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.
雷帕霉素的哺乳动物靶点（mTOR）信号通路在细胞调节和多种疾病中起关键作用。虽然人们认为小G蛋白Rheb在mTOR的上游发挥作用，而raptor在mTOR的下游，但它们之间的直接相互作用尚未在活细胞中得到证实，此外，据报道Rheb仅定位于细胞质中。
在本研究中，利用绿色和红色荧光蛋白（GFP和DsRed）融合以及活细胞的高灵敏度单光子计数荧光寿命成像显微镜（FLIM），显示了mTOR在细胞质以及细胞核中的显著亚细胞定位。使用荧光能量转移-FLIM确定了用EGFP/DsRed标记的mTORC1组分Rheb、mTOR和raptor之间的相互作用。当与DsRed-Rheb共表达时，EGFP-mTOR的激发态寿命约2400 ps通过能量转移降低至细胞质中的约2200 ps和细胞核中的2000 ps，共表达EGFP-mTOR和DsRed-raptor时也获得了类似结果。mTOR的定位和分布通过氨基酸去除和重新添加而改变，但不受雷帕霉素影响。
结果说明了GFP技术与FRET-FLIM成像相结合在研究活细胞中信号成分相互作用方面的强大作用，首次为活细胞中mTOR与Rheb以及mTOR与raptor之间的直接物理相互作用提供了证据。
