Optogenetic Control of Nuclear Protein Import in Living Cells Using Light‐Inducible Nuclear Localization Signals (LINuS)

Pierre Wehler1, Dominik Niopek2, Roland Eils2, Barbara Di Ventura1

1 Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Heidelberg, Germany, 2 Department of Theoretical Bioinformatics, German Cancer Research Center, Heidelberg, Germany
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.4
Online Posting Date:  June, 2016
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Abstract

Many biological processes are regulated by the timely import of specific proteins into the nucleus. The ability to spatiotemporally control the nuclear import of proteins of interest therefore allows study of their role in a given biological process as well as controlling this process in space and time. The light‐inducible nuclear localization signal (LINuS) was developed based on a natural plant photoreceptor that reversibly triggers the import of proteins of interest into the nucleus with blue light. Each LINuS is a small, genetically encoded domain that is fused to the protein of interest at the N or C terminus. These protocols describe how to carry out initial microscopy‐based screening to assess which LINuS variant works best with a protein of interest. © 2016 by John Wiley & Sons, Inc.

Keywords: LOV2; nuclear import; optogenetics; protein engineering; synthetic biology

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Quantification of Blue‐Light‐Dependent Nuclear Accumulation of LINuS Fusion Proteins in a Population of Cells Using Epifluorescence Microscopy
  • Alternate Protocol 1: Quantification of Blue‐Light Dependent Nuclear Accumulation of LINuS Fusion Proteins in Individual cells Using Confocal Microscopy
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Quantification of Blue‐Light‐Dependent Nuclear Accumulation of LINuS Fusion Proteins in a Population of Cells Using Epifluorescence Microscopy

  Materials
  • Adherent cells (e.g., HEK 293T) grown in a glass‐bottom microscope dish
  • Dulbecco's modified Eagle medium (DMEM, phenol‐red free) with 10% (v/v) fetal bovine serum (FBS)
  • LINuS fusion construct (see Strategic Planning)
  • Stuffer plasmid (e.g., pBlueScriptIIS/K)
  • Transfection reagent (jetPRIME, Polyplus Transfection) and buffer
  • 35‐mm glass‐bottom microscope dishes (e.g., Greiner One, 627871)
  • Tissue culture incubator, 5% CO 2 and 37°C
  • Aluminum foil and/or light‐protected box
  • Red LED safelight
  • Epifluorescence microscope equipped with GFP (or CFP, FITC) and mCherry filter sets
  • Dark incubation chamber, 5% CO 2 and 37°C
  • Computer with appropriate image analysis software (e.g., ImageJ)

Alternate Protocol 1: Quantification of Blue‐Light Dependent Nuclear Accumulation of LINuS Fusion Proteins in Individual cells Using Confocal Microscopy

  Additional Materials (also see protocol 1Basic Protocol)
  • Confocal laser scanning microscope equipped with 458‐, 476‐ or 488‐nm laser line (for LINuS activation) and 561‐ or 594‐nm laser line (for mCherry excitation)
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Figures

Videos

Literature Cited

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Key Reference
  Niopek et al. (2014). See above.
  Primary publication describing how LINuS was engineered and how nuclear protein import is quantitatively regulated by choosing appropriate illumination conditions, LINuS variants, and/or LOV2 mutants. This publication also showcases the utility of LINuS for cell biological applications by controlling entry into mitosis and gene expression with blue light.
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