How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

Summary

Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies.

Abstract

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells and protein ensembles. Thus far, no method has been available to quantify in bulk and in live cells how many of the PA-FPs expressed are photoactivated to fluoresce.

Here, we present a protocol involving internal rulers, i.e., genetically coupled spectrally distinct (photoactivatable) fluorescent proteins, to ratiometrically quantify the fraction of all PA-FPs expressed in a cell that are switched on to be fluorescent. Using this protocol, we show that different modes of photoactivation yielded different photoactivation efficiencies. Short high-power photoactivation with a confocal laser scanning microscope (CLSM) resulted in up to four times lower photoactivation efficiency than hundreds of low-level exposures applied by CLSM or a short pulse applied by widefield illumination. While the protocol has been exemplified here for (PA-)GFP and (PA-)Cherry, it can in principle be applied to any spectrally distinct photoactivatable or photoconvertible fluorescent protein pair and any experimental set-up.

Introduction

In 2002, the first broadly applicable photoactivatable (PA-GFP¹) and photoconvertible (Kaede²) fluorescent proteins were described. These optical highlighter fluorescent proteins change their spectral properties upon irradiation with UV-light, i.e., they become bright (photoactivatable fluorescent proteins, i.e., PA-FPs), or change their color (photoconvertible FPs). To date, several reversible and irreversible photoactivatable and photoconvertible fluorescent proteins have been developed^3,4. In ensemble or bulk studies, optical highlighters hav....

Protocol

1. Plasmid Construction

1. Generate two-color fusion probes. Use a mammalian cell expression vector (see Table of Materials) in which mCherry1¹² and PA-mCherry1¹³ have been inserted with the restriction sites AgeI and BsrGI.
2. Order custom oligo-nucleotides to amplify the monomeric variants of eGFP and PA-eGFP containing the A206K mutation, i.e., mEGFP and PA-mEGFP¹⁴ without a stop codon as.......

Discussion

So far, no method existed to determine in bulk the fraction of PA-FPs expressed in live cells that is photoactivated to be fluorescent. The presented protocol can be used for any spectrally distinct fluorescent protein pair. While exemplified here for the irreversible PA-FPs PA-GFP and PA-Cherry, this approach is in principle applicable to photoconvertible proteins as well. The spectrally distinct fluorescent protein, however, must be selected carefully to minimize spectral overlap given that photoconvertible fluorescent.......

Materials

Name	Company	Catalog Number	Comments
pEGFP-N1 mammalian cell expression vector	Clontech
DMEM w/o phenol red	Thermo Fisher Scientific	11054020
Trypsin w/o phenol red	Thermo Fisher Scientific	15400054
L-Glutamine (200 mM)	Thermo Fisher Scientific	25030081
HEPES	Thermo Fisher Scientific	15630080
LabTek 8-well chambers #1.0	Thermo Fisher Scientific	12565470
Fugene 6	Promega	E2691