From EosFP to mIrisFP: structure-based development of advanced photoactivatable marker proteins of the GFP-family

Fluorescent proteins from the GFP family have become indispensable imaging tools in life sciences research. In recent years, a wide variety of these proteins were discovered in non-bioluminescent anthozoa. Some of them feature exciting new properties, including the possibility to change their fluorescence quantum yield and/or color by irradiating with light of specific wavelengths. These photoactivatable fluorescent proteins enable many interesting applications including pulse-chase experiments and super-resolution imaging. In this review, we discuss the development of advanced variants, using a structure-function based, molecular biophysics approach, of the photoactivatable fluorescent protein EosFP, which can be photoconverted from green to red fluorescence by similar to 400 nm light. A variety of applications are presented that demonstrate the versatility of these marker proteins in live-cell imaging. [GRAPHICS] The photoactivatable fluorescent protein mIrisFP combines two forms of photoactivation, reversible on-off photoswitching and irreversible green-to-red photo-conversion, which enables pulse-chase experiments with superresolution. (Top) Light-induced structural changes upon on-off switching of the red fluorescent form, (bottom) photoactivation localization microscopy (PALM) reconstruction images of the formation of focal adhesions using the paxillin-mIrisFP fusion protein.