Multicolor FRET-FLIM Microscopy to Analyze Multiprotein Interactions in Live Cells
The need to describe and understand signaling pathways in live cell is seen as a primary route to identifying and developing targeted medicines. Signaling cascade is also seen as a complex communication and involves interactions between multiple interconn
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Introduction All cellular processes are driven by the action of proteins, from ion channels across phospholipid membranes to DNA synthesis and cell growth. For example, the mechanistic or mammalian target of rapamycin (mTOR) pathway functions in the coordination of energy, nutrients, and growth factor availability to regulate key biological processes including cellular growth, metabolism, and protein synthesis through the phosphorylation of downstream substrates, ribosomal protein, S6 Kinase 1 (S6K1), and 4E-binding
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247, https://doi.org/10.1007/978-1-0716-1126-5_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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protein 1 (4E-BP1/eIF4E) [1–4]. The phosphorylation of these effectors is to regulate cell growth, aging and adiposity [5], memory [6], immunity [7], and muscle hypertrophy [8]. The human mTOR protein exists as a multi-protein complex with Rheb, raptor, mLST8, PRAS40, and DEPTOR proteins termed mTOR Complex 1 (mTORC1). This complex is the main rapamycin target via the FKBP12 protein. Furthermore, rapamycin differentially inhibits S6Ks and 4E-BP1 toward mRNA translation [9]. It has also been observed that the concentration of rapamycin required for an effect varies significantly between cell lines with a range of nM-mM reported [10]. Majority of our understanding of the mTOR signaling pathway and protein interaction has come from classical cell disruption pull-down assays. Hence, information where within the cell these assemblies are localized that lead to subsequent downstream targets phosphorylation in real time is lost. Cell immunofluorescence staining following cell fixation also has several drawbacks and limitations such as mislocalization of proteins after fixation, poor antibody sensitivity and specificity in some cases, as well as antibodies not reaching their target of interest. Fo¨rster or fluorescence resonance energy transfer (FRET) is an excellent and powerful technique to determine relevant distances in biological processes under physiological conditions [11]. It relies on the non-radiative energy transfer from an excited fluorescent donor molecule to an acceptor molecule with non-excited fluorescence in its close vicinity through a dipole–dipole interaction and may be used to investigate physical interactions between two or more small or macro-molecules. Thus molecular scale distances (1–10 nm) can be measured using energy transfer processes. The very short distances required for this process to occur (
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