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

Xin Zhou1, Simin Li1, Jin Zhang2

1 Department of Pharmacology, University of California at San Diego, La Jolla, California, 2 The Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.11
Online Posting Date:  December, 2016
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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) and coordinates cell growth and proliferation. The underlying molecular mechanisms by which these stimuli regulate the activity of mTORC1 are still not fully understood. The spatial compartmentalization of mTORC1 signaling has been suggested as an important mechanism for mTORC1 to achieve the signal specificity and efficiency. To examine the spatial regulation of the activity of mTORC1 in live cells, we describe a protocol using a newly developed molecular tool, a genetically encoded fluorescence resonance energy transfer (FRET)‐based mTORC1 activity reporter, TORCAR. When expressed in the cell, TORCAR acts as a surrogate substrate of mTORC1, and exhibits a change in FRET in response to phosphorylation by mTORC1. Genetically targeting TORCAR to specific subcellular locations further allows for the characterization of spatial compartmentalized mTORC1 signaling. © 2016 by John Wiley & Sons, Inc.

Keywords: biosensor; fluorescence; live‐cell imaging; mTOR kinase

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Table of Contents

  • Introduction
  • Basic Protocol 1: Monitoring MTORC1 Activity Using Genetically Encoded FRET‐based Biosensor TORCAR
  • Support Protocol 1: Maintenance of NIH3T3 Cells in Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Monitoring MTORC1 Activity Using Genetically Encoded FRET‐based Biosensor TORCAR

  • NIH3T3 fibroblast cells plated on sterile 35‐mm glass‐bottom dishes (see protocol 2Support Protocol)
  • NIH3T3 culture medium (see recipe)
  • Opti‐MEM I reduced serum medium (Gibco, cat. no. 31985070)
  • Lipofectamine 2000 (Thermo Fisher Scientific, cat. no. 11668019)
  • TORCAR plasmid DNA (Addgene, cat. no. 64927)
  • Dulbecco's Modified Eagle's Medium, 1 g/liter glucose, no serum (Gibco, cat. no. 11885084)
  • HBSS* imaging buffer (see recipe)
  • Immersion oil, e.g., Immersol 518 F fluorescence‐free (Carl Zeiss, cat. no. 1262466A)
  • PDGF stock solution (see recipe)
  • 1 mM torin1 (TOCRIS, cat. no. 4247), in DMSO
  • 3.25 M leucine O–methyl ester (LeuOMe; EMD Chemical, cat. no. 8.54071.0005), in water
  • Inverted fluorescence microscope with appropriate objective, filters/mirrors, and detector, for example:
    • Zeiss Axiovert 200 M microscope with 40×/1.3‐NA oil‐immersion objective lens; dichroic mirror, excitation filters (CFP, YFP), and emission filters (CFP, YFP); Lambda 10‐2 filter changer (Sutter Instruments); XBO 75 W xenon arc lamp (Carl Zeiss); MicroMAX BFT512 CCD camera (Roper Scientific),
  • Computer and imaging software (e.g., METAFLUOR 6.2, Universal Imaging) to operate microscope
  • Spreadsheet application (e.g., Microsoft Excel)

Support Protocol 1: Maintenance of NIH3T3 Cells in Culture

  • NIH3T3 cells (American Type Culture Collection)
  • 70% (v/v) ethanol
  • Dulbecco's phosphate‐buffered saline without Ca2+ or Mg2+ (DPBS; Gibco, cat. no. 14190144)
  • NIH3T3 cell culture medium (see recipe)
  • 0.25% trypsin–EDTA (Thermo Fisher Scientific, cat. no. 25200072)
  • Tissue culture flasks (e.g., T‐25 cm2; BD Falcon)
  • Tissue culture incubator
  • Tissue culture hood
  • 35‐mm glass‐bottom imaging dishes (MatTEK, cat. no. P35G‐1.0‐14‐C)
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Literature Cited

Literature Cited
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