Chapter 5 - Fluorescent Protein Applications in Microscopy

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Abstract

The use of fluorescent proteins (FPs) in modern cell biology and microscopy has had an extraordinary impact on our ability to investigate dynamic processes in living cells. FPs are unique in that fluorescence is encoded solely by the primary amino acid sequence of the FP and does not require enzymatic modification or cofactors. This genetically encoded fluorescence enables the expression of FPs in diverse cells and organisms and the detection of that fluorescence in living systems. This chapter focuses on microscopy-based applications of FP detection to monitor protein localization, dynamics, interaction, and the cellular environment.

Section snippets

The Identification of Green Fluorescent Protein

The isolation of green fluorescent protein (GFP) was first described by Shimomura, Johnson, & Saiga, 1962 as a footnote to their studies about the aequorin protein from the jellyfish Aequorea aequorea. A. aequorea normally emits a greenish luminescence from the light organs around the rim of the jellyfish. During the isolation of the luminescence system of Aequorea, Shimomura and colleagues noted that the luminescence from aequorin was blue rather than the green luminescence of the intact

Formation of the GFP Chromophore

The primary amino acid sequence of GFP is sufficient to direct the formation of the functional chromophore. Heim, Prasher & Tsien, 1994 first proposed a reaction scheme to explain how GFP might spontaneously mature into a fluorescent protein (FP; Fig. 5.2). Four key steps were proposed to control chromophore formation: (1) the folding of the GFP protein, (2) the cyclization and dehydration of the peptide backbone between the amide nitrogen of Gly67 and the carbonyl of Ser65, (3) the oxidation

The Structure of GFP

The crystal structure of GFP revealed the importance of the entire GFP protein to the fluorescence of the chromophore. GFP is formed by an 11-stranded β-barrel that folds into a structure that has been termed as β-can (Fig. 5.3; Ormo et al., 1996, Yang et al., 1996). The chromophore is completely buried in the center of the β-can structure, protected from solvent quenching effects. Folding of the GFP protein into the β-can structure prevents access of other proteins to the chromophore region;

Mutagenesis to Alter the Properties of GFP

The thermosensitivity, slow maturation rate, and complex absorbance spectrum of Aequorea GFP were some of the factors that prompted multiple research groups to begin mutagenizing GFP to alter its functional properties. Initial mutagenesis studies on GFP were directed at improving its folding and expression at 37 °C and at altering its excitation and emission spectrum (Cormack et al., 1996, Crameri et al., 1996, Delagrave et al., 1995, Heim et al., 1995, Heim et al., 1994, Siemering et al., 1996

Imaging FPs

There are several simple factors to consider and control when planning an imaging experiment that will dramatically improve the ability to detect FPs in fixed or live specimens. Most of these factors are just as applicable to imaging conventional fluorophores as they are to FPs and thus can be considered as general guidelines for fluorescence imaging. The challenge with any live cell experiment is to image the specimen over a long enough time frame to extract meaningful data. This is almost

Applications of FP Imaging

The development of FP variants that possess diverse properties such as spectral differences, sensitivity to environmental factors, and photoswitchable responses to light has opened many new avenues in live cell imaging. Here, we discuss a few examples of microscopy-based FP applications to investigate cell biological questions.

Conclusion

The examples described earlier represent just a few of the many applications of FP imaging used to understand the dynamics of proteins in cells and to monitor the intercellular environment. Additional applications whose descriptions are outside the scope of this chapter include FP-based sensors of cell cycle state (Sakaue-Sawano et al., 2008), optical control of protein activity (Zhou, Chung, Lam & Lin, 2012), all-optical writing with a photoswitchable GFP (Grotjohann et al., 2011), and many

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