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Furthermore, high-resolution imaging techniques include the yeast-enhanced TetCys motif, which is compatible with diaminobenzidine photo-oxidation used for protein localization by electron microscopy, and mEos2, which is ideal for super-resolution microscopy.
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This series of modules, complete with six different selection markers, provides the optimal flexibility during live-cell imaging and multicolour labelling in vivo.
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Thus, we have constructed a series of cassettes containing codon-optimized epitope tags, fluorescent protein variants that cover the full spectrum of visible light, a TetCys motif used for fluorescein arsenical hairpin (FlAsH)-based localization, and the first evaluation in yeast of a photoswitchable variant, mEos2, to monitor discrete subpopulations of proteins via confocal microscopy. Several approaches have been developed to identify, characterize and monitor the spatial localization of proteins and complexes at the suborganelle level, yet many of these techniques have not been applied to yeast. Although fluorescent protein variants are ubiquitously used to monitor protein dynamics, localization and abundance fluorescent light microscopy techniques often lack the resolution to explore protein heterogeneity and cellular ultrastructure.
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cerevisiae, techniques such as fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) enables the evaluation of protein dynamics and kinetics (Luedeke, et al., 2005 Mc Intyre, et al., 2007 Muller, et al., 2005 Raicu, et al., 2005) fluorescence resonance energy transfer (FRET)-based technology facilitates studies of protein-protein interactions (Hailey, et al., 2002 Malinska, et al., 2004 Qiu, et al., 2008 Sprouse, et al., 2008) and the application of fluorescent proteins with different spectral characteristics permits simultaneous imaging of multiple proteins at the sub-cellular level (Melloy, et al., 2007 Reinke, et al., 2004 Szymanski, et al., 2007 Takeda and Nakano, 2008).ĭuring the past decade, it has become clear that protein function and regulation are highly dependent upon intracellular localization. Engineered fluorescent proteins (FPs) provide a diverse array of tools for biological imaging and in vivo studies (Lippincott-Schwartz and Patterson, 2003 Lippincott-Schwartz and Patterson, 2009 Lukyanov, et al., 2005 Shaner, et al., 2005). Future improvements in the spectral properties of CFP and YFP will increase the general applicability of FRET to study a broad range of protein–protein interactions in yeast. In instances where interactions give robust FRET signals, FRET is a valuable tool used to study the dynamic spatial and temporal behavior of protein–protein interactions in living cells. However, with careful controls, FRET is a powerful indication of protein–protein interaction. Dynamic intracellular conditions-such as changes in pH or protein concentrations-can complicate the interpretation of experimental data. The detection of a FRET signal is limited by background cellular autofluorescence and the relatively weak fluorescent signal intensities. The extreme sensitivity of the current YFP to bleaching constrains image acquisition. Spectral overlap between excitation and emission spectra complicates the analysis. Live cell FRET detection in yeast is in its infancy, and there are significant complications with the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) pair. This chapter discusses the fluorescence resonance energy transfer (FRET) using color variants of green fluorescent protein (GFP).