Paul Gavin# and Mark Prescott#, Ph.D
Daren J. Fyfe, Ph.D*
#Department of Biochemistry and Molecular Biology, Monash University, Clayton campus Victoria 3800, Australia
* Technical assistance: Varian Australia Pty Ltd, Mulgrave, Victoria 3170, Australia Email: firstname.lastname@example.org
Since first isolated in 1994 by Chalfie and colleagues, green fluorescent protein
(GFP) has proven to be an immensely popular and powerful tool for the non-invasive
monitoring of cellular events in vivo
Upon excitation with light of 395 nm (peak excitation) or 475 nm, the GFP fluorophore emits bright green fluorescence at 509 nm. This can be detected using a variety of instruments such as fluorescence microscopes, fluorescence-activated cell sorters and fluorometers.
GFP is an extremely attractive tool for todays life scientist, mainly because the protein matures without the need for any additional substrates or co-factors. This allows expression of GFP in an array of living organisms and cell systems, resulting in autocatalytic maturation and a fluorescent signal. In other words, GFP allows cells to produce their own fluorescent marker. This omits the need for conventional fluorescence imaging procedures, which often damage or kill cells, and are not generally feasible for in vivo studies. GFP has allowed the real time study of cellular events in live, intact cells and organisms, allowing a better understanding of biological mechanisms in physiological models
Fused to a protein of interest, one of the first applications of GFP was as a reporter of gene expression and protein localisation. It was used to illuminate cellular organelles, the cytoskeleton, and the secretory pathway (reviewed by Gerdes and Kaether2). Since then, a rapidly increasing variety of applications for GFP and its derivatives have been developed. Mutational studies of the chromophore and surrounding amino acids have produced new GFPs with altered excitation and emission spectra. These include blue (BFP), cyan (CFP), and yellow (YFP) fluorescent proteins, all with enhanced maturation rates and fluorescent intensities3. The color spectrum was recently extended by Matz and co-workers4, who isolated a red fluorescent protein from the Discosoma coral (DsRed) with 30% sequence homology to Aequoria GFP. This wide variety of spectrally distinct fluorescent proteins provides distinguishable markers for the simultaneous study of multiple cellular events, as well as allowing more advanced applications such as the study of protein-protein interactions using the phenomena of fluorescence resonance energy transfer (FRET).
The fluorescent properties of the GFP family vary in terms of spectral profiles, intensity and susceptibility to photobleaching. Therefore it is important to optimise detection and measurement protocols for GFP and its derivatives prior to experimental analysis in each experimental model. The present study aimed to detect and characterise GFPs expressed in the cytosol of live yeast cells (Saccharomyces cerevisiae) using the Varian Cary Eclipse fluorescence spectrophotometer.
Materials and Methods
(For part numbers see reference 5)
Varian Cary Eclipse fluorescence spectrophotometer
Peltier-thermostatted multicell holder (with electromagnetic stirring)
Magnetic stirrer bars
Quartz cuvettes (4)
YRD15 (MATa, his3, ura3, leu2, p+) of the yeast S. cerevisiae was the parental strain used in this study. Genes encoding the GFP derivatives (green, blue, cyan, yellow) and red fluorescent protein (DsRED) were cloned into the yeast expression plasmid pAS1N and transformed into the yeast strain YRD15 as previously described6. Transformants were plated out on yeast minimal medium (0.75% yeast minimal medium w/o amino acids, 2% glucose, 1.5% agar) with selective markers as required and grown at 28C for 35 days.
Yeast cells were washed twice in 1ml MilliQ water to remove contaminating media before being resuspended in MilliQ to a final optical density of 0.55 Abs at 650 nm. Cell suspensions (2 ml) were placed in disposable fluorescence cuvettes (Sarstedt) in the multicell holder, which was positioned in the sample compartment of the Cary Eclipse fluorescence spectrophotometer. The temperature within the cuvettes was set to 25C. Using the Scan application, cell suspensions were excited with light of the appropriate wavelength for each specific fluorescent protein (See the Clontech Web Site3 for a comprehensive list of excitation/emission maxima of fluorescent proteins.) Emission scans were recorded for GFP, BFP, CFP, YFP and DsRed. Operating parameters for scans of DsRed are given in Figure 2.
Emission spectra for each of the fluorescent proteins are shown in Figure 3.
Spectra from all fluorescent proteins expressed in the cytosol of the yeast cells were easily recorded and correspond to expected characteristics. Noise produced by cellular autofluorescence and scatter was negligible, mainly due to internal filters fitted to both excitation and emission monochromators on the Varian Cary Eclipse. Reducing autofluorescence (from either sample of interest or media) is of paramount importance when using fluorescent proteins that have either low fluorescent intensity, or are excited by high energy UV light (such as BFP). UV light initiates a strong fluorescent response from many components of the cellular environment and must be minimized to prevent masking of emission from target fluorescent proteins, especially when subtle protein interactions are being monitored7.
The Varian Cary Eclipse with multicell and temperature control accessories provides a simple, rapid and reproducible assembly with which to measure and characterise different fluorescent proteins expressed in whole cells. The opportunity now exists to specifically tag proteins of interest within cells using different GFPs and monitor interactions simultaneously using multiwavelength readings which allow automated excitation and reading of emission at up to six different fluorescent probes in vivo.