Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

Amanda Siegel,Richard Day,Michael Davidson,Michelle Baird

doi:10.3390/ijms141020340

Abstract

The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy.

Highlights

Fluorescent proteins (FPs) are valuable tools for analyzing cellular processes in living cells
The goal of the current study is to develop a methodology that enables a careful scrutiny of newly developed red FPs for spectroscopic approaches by utilizing criteria important for these applications, and to determine their ideal conditions for use in live cell fluorescence correlation spectroscopy (FCS)
Where G(τ) represents the normalized variation in intensity from average intensity measured at t + τ for all values of t, N represents the number of molecules in the confocal volume, Q represents a shape-fitting parameter and τCHAR is the characteristic diffusion time for the species, assuming it is freely diffusing

Summary

Introduction

Fluorescent proteins (FPs) are valuable tools for analyzing cellular processes in living cells. The first of the commercially available red FPs was mined from the sea anemone Discosoma striata and is commonly known as DsRed [2]. MCherry, derived by directed evolution of DsRed and first introduced nearly a decade ago [3], has been the most widely used red FP for FCS and other single molecule applications. While newer red FPs displaying increased brightness, photostability, monomeric quality, and speed of maturation have been engineered from DsRed and other anthozoa, most have not yet been evaluated for FCS or other single molecule applications. The goal of the current study is to develop a methodology that enables a careful scrutiny of newly developed red FPs for spectroscopic approaches by utilizing criteria important for these applications, and to determine their ideal conditions for use in live cell FCS

Objectives

Results

Conclusion