Shine on … proteins

The use of fluorescent proteins and live-cell imaging has greatly improved our understanding of cell biology in recent years (Patterson et al., 2001; Tsien, 1998; Zimmer, 2002). The discovery in different organisms and development of fluorescent proteins are revolutionizing the study of cell behavior by providing convenient in vivo markers for gene expression and protein targeting in intact cells and organisms. Visible fluorescent proteins (VFPs) can be attached, by genetically programmed encoding, to virtually any protein of interest and still assemble into a fluorescent molecule. The resulting chimaeras can be used to localize previously uncharacterized proteins or to visualize and track known proteins to further understand cellular events. So far, protein trafficking and relationships between different organelles can be elucidated in a noninvasive manner (Chudakov et al., 2004; Miyawaki, 2003; Voss et al., 2005). VFPs coupling to three-dimensional fluorescence imaging methods like confocal and twophoton excitation microscopy (Diaspro, 2002) is very effective in live-cell imaging. Moreover, the need to understand the fluorescence mechanism under different conditions has inspired a renaissance in single molecule methods (Blum et al., 2004; Garcia-Parajo et al., 2000). The use of multiple distinct fluorochromes makes possible the observation of interactions between organelles and between proteins, making this one of the fastest-developing and exciting fields of study. The origin of the visible fluorescent proteins can be found in the seminal work by Shimomura et al. (1962). The story of the cnidarian Aequorea victoria, the green fluorescent protein (GFP) and other VFPs, has involved several biological and technical steps. In the early 1990s, GFP was recognized as being of potential use to cell biologists and the revolution in fluorescent microscopy began, as reported in the paper by Hanton and Brandizzi (pages 152–159 in this issue). In this paper, the authors discuss the multitude of fluorescent markers that have been generated to study the plant secretory pathway. Although these markers have been used to solve many mysteries in this field, some areas still require further investigation. This special issue was organized to contribute to some of those remaining investigations. Anna-Katerina Hadjantonakis and her group have performed excellent work using live imaging of fluorescent proteins in chordate embryos (pages 160–167). Because embryonic development takes place in three-dimensional space over time, to fully understand the events embryo morphogenesis in multiple dimensions in situ must be visually investigated. Since most fluorescent protein probes and modes of data acquisition are similar in different species, the authors have chosen the mouse and the ascidian, as two model organisms at opposite ends of the chordate clade, to review the use of some of the current genetically encoded fluorescent proteins and their visualization in living embryos for the generation of imaging data. In the next study, presented by Visser and colleagues (pages 168–174), the spectral properties of autofluorescent Dictyostelium have been characterized using fluorescence microscopy in a scenario where whole cell autofluorescence potentially interferes with measurements of GFP fusion proteins. In this case, the cellular autofluorescence is a factor of seven times less bright than the molecular emission from eGFP (enhanced GFP). The autofluorescence is localized in discrete regions in the cell, and the localization pattern and intensity does not change with long incubations in buffer. The influence of autofluorescence (Harms et al., 2001) FFS (fluorescence fluctuation spectroscopy) (Skakun et al., 2005; Chen et al., 2002) measurements can be minimized by incubating cells in buffer during the measurements, prebleaching, and making use of lowexcitation intensities. The results of this study offer guidelines for the design of future fluorescence studies of Dictyostelium and outlines experimental methods useful for a wide range of studies. To obtain a quantitative understanding and to more effectively design VFPbased experiments, one needs a good knowledge of the fluorescent molecules behavior starting with an isolated single molecule and then understanding molecular crowding effects that are present in all live-cell imaging studies (van de Berg et al., 2000; Zimmermann and Minton, 1993) in addition to complementary photophysical parameters. In the complete study by Jung and Zumbusch, the photophysical behavior of eight mutants of green fluorescent protein (GFP) has been evaluated at the single molecule level. Fluorescence correlation spectroscopy has been used in a comparative analysis of the effective brightness of the GFP mutants. The authors propose to use the mutant T203Y/E222Q in imaging studies whenever the expression yield is not a limiting factor. One important factor is related to the probability of exceeding the limitations given by out-of-focus bleaching in live-cell microscopy (pages 175–185). To characterize the photophysical properties of the VFPs, a very useful and important parameter is the determination of their fluorescent lifetime (Gadella et al., 1993). The fluorescent lifetime gives information about the VFP’s environment and conformational changes. Becker et al. (pages 186–195) report about a multidimensional approach for lifetime measurements combined with a confocal, two-photon laser scanning microscope as well as a pulsed laser, reveals a near-ideal counting efficiency, and the capability to resolve multiexponential decay functions related to different protein arrangements. In addition, FRET (Forster resonance energy transfer) interactions can usefully be exploited to study protein–protein interactions and to design environmental switchers (Selvin, 2000). Wallrabe and colleagues of the Keck Center for Cellular Imaging at University of Virginia, in a collaboration with the University of Albany in NY, have contributed a paper (pages 196– 206) on the methodological issues in FRET quantitative imaging, such as spectral bleed-through and back-