In Vivo Imaging of Signal Transduction Cascades with Probes Based on Förster Resonance Energy Transfer (FRET)

Takeshi Nakamura1, Michiyuki Matsuda1

1 Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.10
DOI:  10.1002/0471143030.cb1410s45
Online Posting Date:  December, 2009
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Genetically encoded FRET probes enable us to visualize a variety of signaling events such as protein phosphorylation and G‐protein activation in living cells. This unit focuses on FRET probes wherein both the donor and acceptor are fluorescence proteins and incorporated into a single molecule, i.e., a unimolecular probe. Advantages of these probes lie in their easy loading into cells, simple acquisition of FRET images, and clear evaluation of data. We have developed FRET probes for Ras‐superfamily GTPases, designated Ras and interacting protein chimeric unit (Raichu) probes. We hereby describe strategies to develop Raichu‐type FRET probes, procedures for their characterization, and acquisition and processing of images. Although improvements upon FRET probes are still based on trial‐and‐error, we provide practical tips for their optimization and briefly discuss the theory and applications of unimolecular FRET probes. Curr. Protoc. Cell Biol. 45:14.10.1‐14.10.12. © 2009 by John Wiley & Sons, Inc.

Keywords: FRET; unimolecular probe; CFP; YFP; Ras GTPase; Rho GTPase

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Table of Contents

  • Introduction
  • Basic Protocol 1: Development of Raichu Fret Probes
  • Basic Protocol 2: Imaging with FRET Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Development of Raichu Fret Probes

  • Needed DNA constructs:
    • Plasmid for the Raichu probe (available from Matsuda Lab,‐‐phogemon/index.htm)
    • cDNA of the GTPase of interest (can either be purchased from a public depository or cloned by PCR from cDNA libraries)
    • cDNA of GTPase‐binding domains of effector proteins (can either be purchased from a public depository or cloned by PCR from cDNA libraries; try at least a few known effectors for initial experiments)
  • Ion‐exchange resin for DNA purification (e.g., Qiagen; also see Ausubel et al., )
  • Transfection reagent for calcium phosphate coprecipitation (unit 20.3)
  • 293T cells (ATCC cat. no. CRL‐11268), cultured in 100‐mm‐diameter collagen‐coated dishes, 80% confluent
  • MEM (Invitrogen, cat. no. 10370) containing 10% fetal bovine serum (FBS)
  • Phenol‐red‐free MEM (Invitrogen, cat. no. 11054) containing 10% FBS
  • Lysis buffer (see recipe)
  • Fluorescence spectrophotometer (for example, JASCO FP‐750) and 3‐ml cuvettes
  • Additional reagents and equipment for basic molecular biology techniques including restriction digestion, PCR, plasmid preparation, cloning of DNA, and purification of DNA (Ausubel et al., ), and calcium phosphate transfection of DNA (unit 20.3)

Basic Protocol 2: Imaging with FRET Probes

  • Cells for experiment [e.g., HeLa cells (ATCC cat. no. CRL‐2) or COS7 cells (ATCC cat. no. CRK‐1651)] and appropriate medium
  • Expression plasmid for FRET probe ( protocol 1)
  • Transfection reagent
  • Phenol red‐free medium
  • Mineral oil (Sigma)
  • 35‐mm glass‐base dish (Asahi Techno Glass; with a 10‐mm‐diameter glass coverslip mounted on the bottom
  • Temperature‐controlled chamber with thermostat and/or a CO 2 controller
  • A fluorescence microscope with a CCD camera including:
    • IX81 inverted microscope
    • 75‐W xenon arc lamp
    • 60× oil immersion objective lens, PlanApo 60×/1.4 (Olympus)
    • Cool SNAP‐HQ cooled CCD camera (Roper Scientific)
    • Laser‐based auto‐focusing system, IX2‐ZDC (Olympus)
    • Automatically programmable XY stage, MD‐XY30100T‐Meta (SIGMA KOKI).
  • Excitation and emission filter wheels: Filter wheel 99A354 and MAC5000 shutter controller (Ludl Electronic Products)
  • Filters:
    • XF1071 (Omega Optical; cat. no. 440AF21) excitation filter
    • XF2034 (Omega Optical; cat. no. 455DRLP) dichroic mirror
    • XF3075 (Omega Optical; cat. no. 480AF30) emission filter for CFP
    • XF3079 (Omega Optical; cat. no. 535AF26) emission filter for FRET
    • Neutral‐density (ND) filters
  • Software for operation of the microscope and analysis of acquired images; e.g., MetaMorph software (Universal Imaging)
  • Additional reagents and equipment for construction of the FRET probe ( protocol 1)
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Key References
   Miyawaki, 2003. See above.
  Comprehensive review of GFP‐based FRET technology.
Internet Resources‐‐phogemon/index.htm
  Web site for further information about the Raichu‐type FRET probe. Setup of the FRET imaging system is described.
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