LOVTRAP: A Versatile Method to Control Protein Function with Light

Hui Wang1, Klaus M. Hahn1

1 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 21.10
DOI:  10.1002/cpcb.12
Online Posting Date:  December, 2016
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Abstract

We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered. Light‐induced protein release occurs in less than a second, and the half‐life of return can be adjusted using LOV point mutations (∼2 to 500 sec). © 2016 by John Wiley & Sons, Inc.

Keywords: optogenetics; LOVTRAP; Zdk; signaling; localization; dissociation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Using LOVTRAP to Control Protein Activity with Light
  • Support Protocol 1: Use of FRAP Equipment
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Using LOVTRAP to Control Protein Activity with Light

  Materials
  • DNA encoding residues 1‐35 of TOM20 (NCBI access code: NP_055580) for mitochondrial localization (Kanaji et al., ): if the POI undesirably interacts with proteins at the mitochondria, it can be sequestered at other loci such as the ER or Golgi apparatus (DNA encoding the desired localization signal should be used)
  • DNAs encoding residues 404‐546 of the Avena sativa phototropin1 LOV2 domain (Genbank access code: AAC05083), the Zdk binding protein (Genbank access code: KX429612 for Zdk1, KX429613 for Zdk2, and KX429614 for Zdk3) (Addgene IDs: 81009, 81010, 8101,1 81012, 81041, 81057), appropriate fluorescent proteins (e.g., mVenus, mCherry, etc.), and the protein of interest
  • Plasmid vectors for mammalian cell expression: e.g., pTriEx (Novagen) or pcDNA (Invitrogen)
  • Mammalian cells lines to be studied (see annotation to step 2, below)
  • Cell culture medium (see recipe)
  • FuGENE 6 (Promega) or other transfection reagent
  • Live cell imaging medium (see recipe)
  • Coverslips appropriate for the studies to be performed (e.g., coated with fibronectin for cell adhesion in the example here)
  • Live cell imaging culture chambers (e.g. Attofluor Cell Chamber, Thermo Fisher Scientific, cat. no. A78167)
  • Inverted fluorescence microscope driven by MetaMorph 7.5, equipped with:
    • Ludl Electronics Products MAC5000 controller and programmable filter wheels for microscope automation
    • 100‐W mercury arc lamp
    • Band pass filters: ET430/24X (activation), ET572/35X (RFP excitation), and HQ620/60 M (RFP emission) from Chroma Technology Corporation; FF‐520/15 (YFP excitation) and FF‐565/24 (YFP emission) from Semrock Inc.
    • Dichroic mirrors: 440/500/580 dichroic mirror (for mCherry) and T545LP dichroic mirror (for mVenus) from Chroma Technology Corporation
    • For the spectra of the filters and dichroic mirrors, see Figure
    • Neutral density filters with 1% and 3% transmittance
    • 40×, 1.3 N.A. oil‐immersion objective
  • Additional reagents and equipment for general molecular biology techniques ( appendix 3A), preparation of fibronectin‐coated coverslips (unit 14.1; Hodgson et al., ), and cell culture (Chapter 1)

Support Protocol 1: Use of FRAP Equipment

  Materials
  • Cells transfected with LOVTRAP plasmids ( protocol 1Basic Protocol)
  • Live cell imaging medium (see recipe)
  • FRAP‐3D imaging system from MAG Biosystems equipped with:
    • Inverted fluorescence microscope driven by MetaMorph 7.5.
    • Ludl Electronics Products MAC5000 controller and programmable filter wheels for microscope automation
    • Galvo mirror‐based FRAP head
    • Laser launch module with 473 nm laser line
    • 100‐W mercury arc lamp
    • Band pass filters: ET430/24X (activation), ET572/35X (RFP excitation) and HQ620/60 M (RFP emission) from Chroma Technology Corporation, FF‐520/15 (YFP excitation) and FF‐565/24 (YFP emission) from Semrock Inc.
    • Dichroic mirrors: 440/500/580 dichroic mirror (for mCherry) and T545LP dichroic mirror (for mVenus) from Chroma Technology Corporation
    • For the spectra of the filters and dichroic mirrors, see Figure
    • Neutral density filters with 1% and 3% transmittance
    • 40×, 1.3 N.A. oil‐immersion objective
  • Additional reagents and equipment for cell culture (Chapter 1)
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.tsienlab.ucsd.edu/Documents/REF%20‐%20Fluorophore%20Spectra.xls
  Dr. Roger Y. Tsien's laboratory Web site contains excitation and emission spectra of many fluorescent proteins in this Excel spreadsheet.
  http://www.chroma.com/products/fluorochrome
  Web site of Chroma Technology Corp which contains spectra of many fluorochromes and optical filters.
  http://www.hahnlab.com
  The Hahn lab Web site contains updates regarding improvements to LOVTRAP and related imaging techniques, and discussion of frequently asked questions.
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