In eukaryotic cells, endosomes constitute a large network of cytoplasmic vesicles that are formed through the fusion of smaller internalized vesicles arising from receptor-mediated endocytosis. Often, the endosomal vesicles ultimately transfer their contents to the lysosomes for processing, but this is not always the case. 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 human RhoB GTPase, an enzyme that is localized in early endosomes, recycling endosomes, and multivesicular bodies. Upon transcription and translation of the plasmid in transfected mammalian hosts, the fused RhoB domain is responsible for transport and distribution of the fluorescent protein chimera throughout the cellular endosomal network.

The human RhoB GTPase enzyme fused to an enhanced yellow fluorescent protein domain can be efficiently incorporated into the endosomes from a variety of mammalian cell lines (as illustrated in Figure 1). This chimera is entirely localized to both early and late endosomes and does not display a significant effect on endosomal trafficking. The RhoB GTPase-EYFP vector also contains a c-Myc epitope that enables identification of the fusion protein using immunofluorescence techniques in fixed cells. Intracellular endosomal networks labeled with resident macromolecules 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-Endosome 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 human RhoB GTPase, as discussed above. Simian virus 40 (SV40) polyadenylation signals inserted downstream from the EYFP-Endosome 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 endosomal 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.