Tracking the Activity of mTORC1 in Living Cells Using Genetically Encoded FRET-based Biosensor TORCAR.

摘要: Mechanistic target of rapamycin complex 1 (mTORC1) is a highly conserved serine/threonine protein kinase that responds to multiple distinct signals (e.g., growth factors, amino acids, stress, and energy level) coordinates cell proliferation. The underlying molecular mechanisms by which these stimuli regulate the activity mTORC1 are still not fully understood. spatial compartmentalization signaling has been suggested as an important mechanism for achieve signal specificity efficiency. To examine regulation in live cells, we describe protocol using newly developed tool, genetically encoded fluorescence resonance transfer (FRET)-based reporter, TORCAR. When expressed cell, TORCAR acts surrogate substrate mTORC1, exhibits change FRET response phosphorylation mTORC1. Genetically targeting specific subcellular locations further allows characterization compartmentalized signaling. © 2016 John Wiley & Sons, Inc. Keywords: biosensor; fluorescence; live-cell imaging; mTOR