Two - Color GFP Expression System for

We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported “W7” form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described “10C” variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community. INTRODUCTION The ability to observe a population of molecules in living cells using the green fluorescent protein (GFP) has led to major advances in our understanding of many different biological questions (1,3,11,14,19). Recently, the utility of this technology has been enhanced by the development of GFP variants with altered fluorescence spectra. These different colored GFPs should prove to be very useful for marking distinct protein populations or cellular organelles for doubleor multiple-labeling experiments (7,16,20). For a multiple-labeling system to be generally useful in fluorescence microscopy, a number of criteria must be fulfilled. First, the different GFP forms must be sufficiently bright to be visible under standard epi-fluorescence illumination. Second, the fluorescence signal must be sufficiently stable to allow observation and documentation over extended times (several seconds to several minutes). Third, the spectra of the different GFP forms must be sufficiently different that they can be unambiguously distinguished using barrier filters. Although a blue-shifted variant of GFP (BFP) can be spectrally resolved from GFP, it has a low quantum yield and is not photostable, which limits its usefulness (4,20). In contrast, more recently developed GFP mutants with cyan-shifted (CFP) and yellow-shifted (YFP) emission spectra are brighter and more stable than BFP (4,7,16). In addition, CFP and YFP exhibit well-separated emission and excitation spectra. Here, we describe expression vectors and sets of barrier filters that can be used for CFP and YFP double-labeling experiments in Caenorhabditis elegans. Two recent publications have demonstrated the utility of CFP and YFP for dual-labeling in the wide-field microscope (4,21). In these studies, CFP and YFP are selectively excited through discrete band-pass filters, but common dichroic and emission barrier filters are used to capture both signals. This configuration avoids the registration shift that occurs with the use of separate dichroics and is therefore optimal for certain applications such as time-lapse imaging or high-resolution co-localization studies. This arrangement, however, effectively excludes most of the CFP emission spectrum. In addition, the use of a common emission filter produces spectrally overlapping signals from CFP and YFP, which therefore must be collected in separate channels and pseudo-colored before merging. We have used separate dichroic and emission filters to capture a substantially greater fraction of the CFP signal, with negligible cross talk between CFP and YFP, and also to produce direct two-color images that can be easily discerned by eye. Thus, the filter sets that we describe provide a useful alternative for cases in which registration shift is either not a limiting factor or can be easily corrected. MATERIALS AND METHODS Fluorescence Spectra For in vitro analysis, CFP (Y66W, N146I, M153T and V163A) and YFP (S65G, V68A, S72A and T203Y) genes were subcloned into the pRSETA vector (6×His tag). Bacterially expressed CFP and YFP proteins were purified over a Ni-NTA Spin Column 914 BioTechniques Vol. 26, No. 5 (1999) Two-Color GFP Expression System