Tools for Controlling Protein Interactions Using Light

Chandra L. Tucker1, Justin D. Vrana1, Matthew J. Kennedy1

1 Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.16
DOI:  10.1002/0471143030.cb1716s64
Online Posting Date:  September, 2014
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Abstract

Genetically encoded actuators that allow control of protein‐protein interactions using light, termed ‘optical dimerizers’, are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and the coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use in controlling transcription, protein localization, and protein secretion using light. Additionally, we provide instructions and software for constructing a pulse‐controlled LED device for use in experiments requiring extended light treatments. Curr. Protoc. Cell Biol. 64:17.16.1‐17.16.20. © 2014 by John Wiley & Sons, Inc.

Keywords: optogenetics; photoreceptor; UVR8; cryptochrome; protein interactions; protein secretion

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Controlling Protein Secretion in Cultured Cells Using Light
  • Basic Protocol 2: Controlling Protein Localization in Cultured Cells Using Light
  • Basic Protocol 3: Controlling Transcription in Yeast Using Light
  • Support Protocol 1: Construction of a Programmable LED Device
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Controlling Protein Secretion in Cultured Cells Using Light

  Materials
  • Lipofectamine 2000
  • Dulbecco's modified Eagle medium (DMEM)
  • VSVG‐YFP‐2xUVR8 plasmid DNA (Addgene plasmid no. 49800) or other engineered tandem UVR8 construct
  • Suitable adherent cell line (e.g., COS‐7, HeLa) grown on 18‐mm coverslips (#1 or 1.5) in 12‐well culture dishes
  • Fetal bovine serum (FBS)
  • HEPES imaging buffer (see recipe)
  • Tissue culture incubator, 5% CO 2 and 37°C
  • Coverglass chamber for live cell imaging (e.g., Ludin chamber type 1, Life Imaging Services)
  • Confocal or epifluorescence microscope
  • UVB light source (e.g., model EB280C UV, Spectroline, 312 nm)

Basic Protocol 2: Controlling Protein Localization in Cultured Cells Using Light

  Materials
  • Lipofectamine 2000
  • Dulbecco's modified Eagle medium (DMEM)
  • DNA isolated from:
  • CIBN‐pmGFP (CIBN fused to an EGFP‐CaaX prenylation sequence allowingmembrane localization; Addgene plasmid 26867)
  • CRY2PHR‐mCherry‐target protein fusion (if target fusion protein is normallylocalized to the plasma membrane, a mutant version must be used thatdisrupts normal localization; Addgene plasmid 26866)
  • Suitable adherent cell line (e.g., COS‐7, HeLa) grown in 35‐mm live cell imaging dishes (e.g., MatTek 35‐mm glass‐bottom dishes, P35G‐1.5‐14)
  • Fetal bovine serum (FBS)
  • HEPES imaging buffer (see recipe)
  • Tissue culture incubator, 5% CO 2 and 37°C
  • Aluminum foil
  • Confocal or epifluorescence microscope equipped for GFP fluorescence imaging
  • Tissue culture hood with red LED safelight (bicycle tail lights with wavelengths ∼620 nm work well)
NOTE: As an alternative to 35‐mm live cell imaging dishes, coverslips in 12‐well plates can also be used. For routine use, own inexpensive imaging dishes can be made following the protocol outlined here: http://biofrontiers.colorado.edu/core‐facilities/microscopy‐core/user‐resources‐1/making‐imaging‐dishes.

Basic Protocol 3: Controlling Transcription in Yeast Using Light

  Materials
  • YPD medium (see recipe) or synthetic dropout medium (SC −Trp/−Leu/−Ura; see recipe)
  • Laboratory yeast strain such as W303‐1A (Trp, Leu, Ura auxotroph)
  • 0.1 and 1 M LiAc
  • Dimethyl sulfoxide (DMSO)
  • 2 mg/ml ssDNA (see recipe)
  • DNA from yeast expression plasmid containing protein of interest downstream of LexA operator sites (e.g., pSH18‐34, Life Technologies, Ura+ marker)
  • LexA‐CRY2PHR or LexA‐CRY2 DNA binding domain construct (Trp+ auxotrophic marker)
  • pRH‐VP16‐CIBN or pRH‐VP16‐CIB1 activation domain fusion construct (Leu+ auxotrophic marker)
  • 50% (w/v) PEG (see recipe)
  • Synthetic dropout medium and plates (SC −Trp/−Leu/−Ura; see recipes)
  • Glass or plastic test tubes or flasks for growing yeast
  • 30°C shaking and non‐shaking incubators
  • Aluminum foil
  • Light source for CRY2 excitation (e.g., red LED; see protocol 4Support Protocol)

Support Protocol 1: Construction of a Programmable LED Device

  Materials
  • Arduino Uno ATMEGA328P microcontroller
  • LCD Keypad Shield for Arduino (e.g., SainSmart or DFRobot LCD Keypad Shield)
  • Soldering iron and solder
  • Standard 22 AWG hook up wire
  • 2100‐mA BuckBlock Luxdrive LED Driver (LEDdynamics)
  • 12‐20 V DC, 1.5‐A power supply
  • Three TIP120 Darlington transistors
  • Three 330‐Ω resistors
  • 130 × 70−mm rectangular heat sink with hex pin fin rated to 1.5‐3°C/W (fits over top of a 12‐well plate)
  • Thermal epoxy or thermal tape
  • 470‐nm Rebel LEDs, mounted on a 10 mm base (Luxeon Star LEDs)
  • Three pairs of male/female interconnects
  • 24 AWG two‐stranded flexible wires
  • Arduino software (http://www.arduino.cc)
  • LED controller program (http://pharmacology.ucdenver.edu/tucker/reagents)
  • USB A‐to‐B cable
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Figures

Videos

Literature Cited

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