The Golgi apparatus (often referred to as the Golgi complex) consists of stacked and flattened vesicles in eukaryotic cells, which are often located near the nucleus, but can also be distributed widely throughout the cytoplasmic matrix. Golgi vesicles are important in the synthesis of complex carbohydrates, and in the processing and packaging of secretory proteins produced by the endoplasmic reticulum. Recombinant plasmids have been constructed that contain a fusion protein consisting of the yellow-green variant (referred to as enhanced yellow fluorescent protein; EYFP) of the Aequorea victoria green fluorescent protein (GFP) coupled to a sequence encoding the N-terminal 81 amino acids of human beta-1,4-galactosyltransferase. This region of the glycoprotein contains a membrane-anchoring signal peptide that targets the plasmid fusion protein to the trans-medial region of the Golgi apparatus. Upon transcription and translation of the plasmid in transfected mammalian hosts, the fused glycoprotein domain is responsible for transport and distribution of the fluorescent protein chimera throughout the cellular Golgi network.
The human beta-1,4-galactosyltransferase N-terminal sequence fused to an enhanced yellow fluorescent protein domain can be efficiently incorporated into the Golgi apparatus from a variety of mammalian cell lines (as illustrated in Figure 1). Intracellular Golgi networks labeled with resident macromolecules or localization peptides containing fluorescent protein domains, such as GFP or EYFP, can be readily visualized using fluorescence microscopy, as illustrated for a variety of established adherent cell lines in Figure 1. The single bandpass emission filter featured by the Nikon YFP HYQ optical block, which was employed to capture all of these images, produces sharp contrast with little interference from autofluorescence or other fluorescent species.
Plasmid pEYFP-Golgi vector gene product expression in various cell types (from both transiently and stably transfected clones; see Figure 1) occurs due to the efficient intracellular translation of a fusion nucleotide sequence combining the enhanced yellow fluorescent protein domain with the Golgi targeting peptide sequence, as discussed above. Simian virus 40 (SV40) polyadenylation signals inserted downstream from the EYFP-Golgi fusion sequence direct proper processing of the 3' end of the transcribed messenger RNA. The fluorescence excitation maximum of EYFP is 513 nanometers and the corresponding emission maximum occurs at 527 nanometers, with a relatively high (approximately 0.60) fluorescence quantum yield. In addition to the four chromophore mutations that shift the fluorescence emission maximum, the nucleotide coding sequence of the EYFP gene contains over 190 silent base alterations, which correspond to human codon-usage preferences that are designed to increase translational efficiency.
The collection of specimens illustrated in Figure 1 demonstrates the effectiveness of the Nikon YFP HYQ filter combination for imaging a variety of cell lines that express gene products from the EYFP plasmid vector sequence for a fluorescent fusion protein chimera targeted at the intracellular Golgi network. Susceptible adherent cell cultures were transfected with the appropriate vector using proprietary lipophilic reagents, and were then cultured for a period of at least 24 hours in nutrient medium supplemented with fetal bovine serum to allow high expression levels of the fluorescent fusion protein.
The enhanced yellow fluorescent protein gene used in these studies contains four amino acid substitutions that shift the emission maximum of green fluorescent protein (GFP) by 18 nanometers, from approximately 509 to 527 nanometers. The gene is optimized with human codons (as described above) and features a consensus Kozak translation initiation signal to achieve higher expression levels in mammalian cell cultures. In general, vectors targeted at specific subcellular organelles contain a fusion gene segment, which couples the EYFP gene to a peptide sequence or complete protein that is localized to a region of interest in living cells.
Anna Scordato and Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
Nathan S. Claxton, John D. Griffin, Matthew J. Parry-Hill, Thomas J. Fellers