Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells.

Dominik Niopek,Julia Roensch,Barbara Di Ventura,Roland Eils,Thomas Draebing,Dirk Benzinger,Pierre Wehler

doi:10.1038/ncomms5404

Abstract

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

Highlights

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution
When introduced into the sequence of the C-terminal Ja helix of the AsLOV2 domain, the nuclear localization signals (NLSs) is concealed from the nuclear import machinery in the dark state (Fig. 1a)
We have shown LINuS function in yeast and mammalian cells, but given that this method relies solely on the interaction between the photocaged NLS and the endogenous import machinery, it is likely to be functional in other eukaryotic systems

Summary

Introduction

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The second method, based on phytochrome B and PIF6, is fast and can be made reversible using 750 nm light This requires a sophisticated microscopy setup when aiming at precise spatial control, an externally supplied chromophore and tagging the protein of interest with a large photosensory domain. Both systems are based on two individual components that need to be expressed at the right level to ensure precise and predictable control of nuclear translocation, which may be difficult to achieve. We report the development of LINuS, a light-inducible, fully reversible and genetically encoded nuclear localization signal (NLS) based on a single, small tag, which can be fused to proteins of different sizes, origin and function, and which is independent of externally supplied chromophores. To prove the usefulness of LINuS for cell biological applications, we created a system to trigger entry into mitosis of illuminated cells and engineered a TF that activates reporter gene expression after blue light illumination

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Conclusion