UC San Diego

Mechanistic target of rapamycin complex 1 (mTORC1) integrates growth-factor, nutrient, energy, and stress signals to regulate cell growth and homeostasis. Because conventional endpoint assays cannot fully resolve the timing, subcellular organization, or cell-to-cell variability of mTORC1 signaling, I developed genetically encoded reporters for dynamic measurement of mTORC1 activity in living cells. First, I optimized a Förster resonance energy transfer (FRET) target of rapamycin complex activity reporter (TORCAR) by screening fluorescent-protein pairs, EV-linker lengths, short flexible-linker truncations, and target of rapamycin signaling motif configurations. The final FRET TORCAR2 reporter combines truncated mTurquoise2 and YPet with a WW phosphoamino acid-binding domain, a 116-amino-acid EV linker, an engineered mTORC1 substrate, selected flexible-linker truncations, and one C-terminal target of rapamycin signaling motif. Phospho acceptor mutation, inhibitor pretreatment, and acute Torin1 addition supported phosphorylation-dependent, mTOR-dependent, and reversible reporting. I next adapted existing laboratory bioluminescence resonance energy transfer (BRET) components to create a plate-reader TORCAR platform. I established within-well normalization, screened BRET EV-linker lengths, evaluated WW- and FHA1-based reporters in transiently transfected HEK293A cells, and generated a pooled stable WW-based reporter population in NIH3T3 cells. The BRET reporters produced stimulation-associated kinetic responses, defining an initial multiwell platform while identifying response magnitude and reproducibility as priorities for further optimization. Together, FRET TORCAR2 and BRET TORCAR provide complementary strategies for single-cell imaging and scalable kinetic measurement of mTORC1 activity.