Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells

Louis Hodgson1, Feimo Shen2, Klaus Hahn2

1 Gruss‐Lipper Biophotonics Center, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, 2 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.11
DOI:  10.1002/0471143030.cb1411s46
Online Posting Date:  March, 2010
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Abstract

The biosensors developed in the authors' laboratory have been based on different designs, each imparting specific strengths and weaknesses. Here we describe detailed protocols for the application of three biosensors exemplifying different designs—first, a design in which an environmentally sensitive dye is used to report the activation of endogenous Cdc42, followed by two biosensors based on FRET, one using intramolecular and the other intermolecular FRET. The design differences lead to the need for different approaches in imaging and image analysis. Curr. Protoc. Cell Biol. 46:14.11.1‐14.11.26. © 2010 by John Wiley & Sons, Inc.

Keywords: biosensors; Rho family GTPases; FRET; live cell imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production and Use of meroCBD, Dye‐Based Biosensor for Cdc42
  • Support Protocol 1: Labeling CBD‐EGFP with Reactive Fluorophore
  • Basic Protocol 2: Imaging meroCBD in Living Cells
  • Basic Protocol 3: Expressing the RhoA Single‐Chain Biosensor
  • Basic Protocol 4: Imaging the RhoA Biosensor
  • Basic Protocol 5: Expression of Rac1 Flair, Dual‐Chain Fret Biosensor for Rac1
  • Basic Protocol 6: Imaging Rac1‐Flair
  • Basic Protocol 7: Image Processing Procedures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Production and Use of meroCBD, Dye‐Based Biosensor for Cdc42

  Materials
  • Transformation‐competent BL21(DE3) E. coli (e.g., Stratagene)
  • pET23‐CBD‐EGFP (Addgene)
  • LB medium and plates ( appendix 2A) containing 100 µg/ml carbenicillin
  • 1 M IPTG (in water), store at −20°C
  • Lysis buffer (see recipe)
  • Talon resin (Co2+ affinity, Clontech)
  • Lysis buffer (see recipe but omit 2‐ME and PMSF) containing 5 mM and 150 mM imidazole
  • 50 mM Tris⋅Cl, pH 7.5 to 8.0 ( appendix 2A)
  • 50 mM sodium phosphate buffer, pH 7.5 ( appendix 2A)
  • Storage buffer (see recipe)
  • 2000‐ml Erlenmeyer flask
  • Incubator with shaker
  • 250‐ml centrifuge bottles
  • Beckman centrifuge with JA‐10 and JA‐20 rotors (or equivalent)
  • 50‐ml conical polypropylene centrifuge tubes (Falcon)
  • Centrifuge with swinging‐bucket rotor
  • End‐over‐end rotator
  • Ultrafree Centrifugal Filtration Device (MWCO, 5000; Fisher Scientific, cat. no. UFV5BCC25)
  • Slide‐A‐Lyzer cassettes (MWCO 3500; Pierce)
  • Additional reagents and equipment for transformation of bacteria and other basic molecular biological techniques (e.g., Sambrook et al., ; Ausubel et al., ), SDS‐PAGE (unit 6.1), and dialysis ( appendix 3H)
NOTE: Use the buffers suggested in this unit. Apparently small changes have proven to greatly reduce yield.

Support Protocol 1: Labeling CBD‐EGFP with Reactive Fluorophore

  Materials
  • Dye: ISO‐IAA (Toutchkine et al., , , b)
  • Dimethylsulfoxide (DMSO)
  • CBD‐EGFP ( protocol 1)
  • 2‐mercaptoethanol (2‐ME)
  • 50 mM sodium phosphate buffer, pH 7.5 ( appendix 2A)
  • 12% SDS‐PAGE gel (unit 6.1)
  • 50 mM Tris⋅Cl, pH 8.0 ( appendix 2A)
  • Spectrophotometer
  • 2‐ml microcentrifuge tubes
  • End‐over‐end rotator
  • 0.5 cm × 6 to 8 cm column packed with Sephadex G15 gel‐filtration resin (GE Healthcare)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and spectrophotometric determination of protein concentration ( appendix 3B)

Basic Protocol 2: Imaging meroCBD in Living Cells

  Materials
  • Tet‐off stable MEF/3T3 cell system (Clontech)
  • MEF/3T3 cells transduced with the appropriate construct
  • 10 mg/ml doxycycline stock solution
  • 10 mg/ml puromycin stock solution
  • 10‐cm tissue culture dishes
  • Coverslips coated with fibronectin: immerse glass coverslips 30 to 60 min in 10 µg/ml fibronectin (e.g., Sigma), then rinse three times with PBS ( appendix 2A) and leave immersed in PBS until use
  • Additional reagents and equipment for cell culture techniques including trypsinization (unit 1.1), flow cytometric cell sorting (Robinson et al., ), and imaging the RhoA biosensor ( protocol 5)

Basic Protocol 3: Expressing the RhoA Single‐Chain Biosensor

  Materials
  • Mouse embryo fibroblast (MEF/3T3) cells (Clontech; tet‐OFF MEF/3T3)
  • Rac1 biosensor vector system (see above)
  • Fugene6 transfection reagent (Roche)
  • 10 mg/ml doxycycline stock solution
  • Coverslips coated with fibronectin: immerse glass coverslips 30 to 60 min in 10 µg/ml fibronectin (e.g., Sigma), then rinse three times with PBS ( appendix 2A) and leave immersed in PBS until use
  • Additional reagents and equipment for cell culture techniques (unit 1.1), flow cytometric cell sorting (Robinson et al., ), and imaging the Rac1‐FLAIR biosensor ( protocol 7)
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Figures

Videos

Literature Cited

Literature Cited
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