Photoconvertible Fluorescent Protein EosFP: Biophysical Properties and Cell Biology Applications

Abstract

EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30 degrees C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37 degrees C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.

Dual Color Photoactivation Localization Microscopy of Cardiomyopathy-associated Desmin Mutants

“…Mutant and wildtype desmin were fused either with a thermostable variant of the green to red convertible fluorescent protein mEosFP termed mEosFPthermo (31)(32)(33) or with the green photoswitchable mIrisGFP1, a variant of IrisFP (24,34,35). After co-transfection with desmin expression constructs under the same constitutive promoter, filament formation was ana- lyzed in living cells by dual color PALM.…”

Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression

“…However, neither up to 400 mM DETA/NO nor 400 mM DETA showed any effect on cellular AR protein levels. To investigate, whether NO affects nuclear AR translocation, DU-145 prostate cancer cells were transfected with a reporter construct coding for a fusion of the AR and the green fluorescent protein (GFP)-like AR-t1EosFP (Wiedenmann et al, 2004;Nienhaus et al, 2005). In the absence of hormone, fluorescence activity was predominantly found in the cytoplasm (Figure 4a), whereas 10 nM R1881 induced maximal nuclear translocation of the AR-t1EosFP construct after 6 h ( Figure 4b).…”

“…Subsequently cells were transfected with 1 mg/well of green fluorescent AR-t1EosFP reporter construct (Wiedenmann et al, 2004;Nienhaus et al, 2005) for 4 h in RPMI 1640 and allowed to grow for another 24 h in RPMI 1640/5% cFBS. Cells were then cultured in RPMI 1640/2.5% cFBS in the presence or absence of 10 nM R1881 and DETA/NO or DETA for 6 h. Subsequently, localization of AR-t1EosFP was determined in 50-100 fluorescent cells.…”

Optical imaging of nanoscale cellular structures

“…For these experiments, photoactivatable fluorescent proteins (PAFPs) have been shown to be very convenient, as they can be used as fusion markers for a particular protein under study. EosFP is a popular photoactivatable fluorescent protein, which changes its fluorescence irreversibly from green to red upon illumination with ∼400 nm light (Wiedenmann et al 2004;Nienhaus et al 2006;Wiedenmann and Nienhaus 2006). By appropriate adjustment of the 400-nm activating laser intensity, only a few molecules are converted to red emitters and registered in the red color channel during acquisition of a CCD image for 30-100 ms.…”