ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Summary

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

Abstract

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest¹, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association ^{2, 3}. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells ². The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions ^4-7. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease ⁷. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

Protocol

1. Preparation of cells for labeling with ReAsH/FlAsH

1. Using standard cell culture methods for your cell line of interest, prepare a culture of adherent cells directly in a live cell imaging slide ready for transfection.
2. Transfect your plasmid containing TC-tagged gene of interest according to your transfection method of choice.

Note it is important to use positive and negative controls to assess the extent of specific binding to the TC tags and to assess for bleedthrough of fluorescence between the channels when collecting confocal micrographs. Hence, for two colors (eg FlAsH/Cherry or ReAsH/CFP or ReAsH/GFP co....

Discussion

The approach to label protein localization with a fluorescent protein and conformational properties with a second dye offers much potential for mapping where different conformations of proteins accrue in cells and events that change the dynamics of protein conformation. ReAsH/FlAsH was first used as an in-cell sensor for protein folding of the mammalian cellular retinoic acid-binding protein I ⁴. In this example, the FlAsH bound to a TC tag engineered into cellular retinoic acid-binding protein I had redu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
8-well μ-slides	Ibidi	80826	We find these chamber slides to be particularly useful for culturing cells for imaging.
TC-FlAsH II In-cell Tetracysteine Tag Detection Kit *green fluorescence* *for live-cell imaging	Invitrogen	T34561 (FlAsH) or T34562 (ReAsH)
Hanks’ Balanced Salt Solution	Invitrogen	14175-103
2,3-Dimercapto-1-propanol	Sigma-Aldrich	D1129-5ML
1,2-Ethanedithiol	Sigma-Aldrich	02390-25ML