Two-Photon Imaging of the Interaction of mTORC1 Components using Fluorescence Energy Transfer between Gfp-Expressing Proteins in a Spheroid Tumor Cell Model

Abstract

The interaction of key mTORC1 signalling components controlling cell growth was investigated in living cells using fluorescence energy transfer (FRET) between GFP and DsRed tagged proteins in a spheroid cell model with HEK 293 cells. mTORC1 is responsible for controlling cell growth and the interaction of its components is a target for controlling this process. The spheroid cell model offers a number of advantages over traditional monolayer culture models, since it is a more realistic model of cells in tumor form. The challenge is to be able to image deep inside the spheroid to study and eventually manipulate cell signalling to control the fate of a particular cell and its neighbouring cells. We have been exploring this using fluorescence lifetime quenching of GFP-mTOR expressed in the cells by its adjacent DsRed-labelled Rheb and DsRed-raptor proteins. It was found that standard transfection of HEK 293 cells could be achieved simultaneously with the initiation of the formation of spheroids of ∼400 μm diameter with both Rheb-mTOR/mTOR-raptor interactions observable deep into the spheroid after 24-48 h. The combination of two-photon lifetime imaging and the spheroid model offers a powerful approach to the study of signalling of cells deep within tumors and eventually the potential of their manipulation.