Enhanced Yellow Fluorescent Protein (EYFP) Nuclear Localization
Localization of specific proteins and complexes to the nucleus in mammalian cells is generally accomplished through the use of peptide signals that mediate transport to the organelle. 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 multiple copies of nuclear localization signal peptides. Upon transcription and translation of the plasmid in transfected mammalian hosts, the nuclear localization signals are responsible for transport of the fluorescent protein chimera to the cell nucleus. The nucleus can be subsequently visualized using fluorescence microscopy, as illustrated for a variety of adherent cell lines in Figure 1. The single bandpass emission filter featured by the Nikon YFP HYQ optical block, which was employed to capture these images, produces sharp contrast with little interference from autofluorescence or other fluorescent species.
Figure 1 - EYFP-Nucleus Subcellular Localization in Transfected Cells
Plasmid pEYFP-Nucleus 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 three tandem repeats of the nuclear localization signal (NLS) from simian virus 40 (SV40) large T-antigen. Reiteration of the NLS sequence significantly enhances the efficiency of EYFP translocation to the nucleus in mammalian cells. 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.
The collection of specimens illustrated in Figure 1 demonstrates the effectiveness of the Nikon YFP HYQ filter combination for imaging a series of cell lines containing the chimeric EYFP plasmid vector that expresses a fluorescent fusion protein targeted at the cell nucleus. The fusion protein is transported into the nucleus to enable visualization of the subcellular structure in living and fixed cells. 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 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.
Additional EYFP-Nucleus Images with the YFP HYQ Filter Combination
Rat Thoracic Aorta Myoblast Cell Nuclei
Fluorescence emission intensity from a culture of embryonic rat thoracic aorta medial layer (A-10 line) myoblast cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector.
Transformed African Green Monkey Kidney Cell Nuclei
Fluorescence emission intensity from a culture of simian virus 40 (SV40) transformed African green monkey kidney fibroblast (COS-7 line) cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector.
Bovine Pulmonary Artery Endothelial Cell Nuclei
Fluorescence emission intensity from a culture of bovine pulmonary artery endothelial (BPAE line) cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector.
Human Brain Mixed Glioblastoma and Astrocytoma Cell Nuclei
Fluorescence emission intensity from a culture of mixed human glioblastoma and astrocytoma (U-118 line) cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector.
Human Bone Osteosarcoma Cell Nuclei
Fluorescence emission intensity from a culture of human bone osteosarcoma (U2OS line) cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector.
Indian Muntjac Deer Skin Cell Nuclei
Fluorescence emission intensity from a culture of Indian Muntjac deer skin fibroblast cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector.
Normal African Green Monkey Kidney Cell Nuclei
Examine the fluorescence emission in a culture of normal African green monkey kidney fibroblast (CV-1 line) cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector. Note the relatively constant level of green fluorescence defining the chromatin and nuclear geometry, as well as the bright regions that correspond to nucleoli and Cajal bodies.
Albino Swiss Mouse Embryo Cell Nuclei
A culture of albino Swiss mouse embryo (3T3 line) fibroblast cells was transfected with the pEYFP-Nucleus plasmid subcellular localization vector. The cells were transiently transfected and cultured in nutrient medium for a minimum of 24 hours before recording images.
Rat Kangaroo Kidney Cell Nuclei
Observe fluorescence intensity from a culture of rat kangaroo kidney epithelial (PtK2 line) cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector. The enhanced yellow fluorescent protein gene used in these studies contains several important amino acid substitutions that shift the emission maximum of green fluorescent protein (GFP) by approximately 18 nanometers, from 509 to 527 nanometers.
Human Fetal Lung Cell Nuclei
Normal human fetal lung (MRC-5 line) fibroblast cells were transfected with a pEYFP-Nucleus plasmid subcellular localization vector, and then the cells were cultured in nutrient medium for a minimum of 24 hours before recording images.
Normal Rat Kidney Cell Nuclei
Fluorescence emission intensity from a culture of normal rat kidney (NRK line) epithelial cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector is presented in this section. Similar to other images in the nucleus localization series, the rat cell nuclei display a relatively constant level of green fluorescence defining the chromatin and nuclear geometry, as well as the bright regions that correspond to nucleoli and Cajal bodies.
Human Cervical Carcinoma Cell Nuclei
Examine human cervical adenocarcinoma (HeLa line) epithelial cells that were transfected with a pEYFP-Nucleus plasmid subcellular localization vector. The enhanced yellow fluorescent protein gene used in these studies contains several important amino acid substitutions that shift the emission maximum from 509 to 527 nanometers. However, the fluorescence emission still appears green when observed in the microscope eyepieces.
Canine Kidney Cell Nuclei
The Madin-Darby canine kidney (MDCK) cell line was derived in 1958 from an apparently normal adult female cocker spaniel. The cells are positive or keratin by immunoperoxidase staining and have been extensively employed to study processing of beta-amyloid precursor protein and sorting of the resulting proteolytic products. The nuclei of transfected cells appear similar to others in this section.
Normal African Green Monkey Kidney Cell Nuclei
The Vero cell line, derived from apparently normal kidney tissue of the African green monkey (Cercopithecus aethiops), was initiated in 1962 at the Chiba University in Japan. These cells are efficacious in the detection of virus in ground beef, as transfection hosts (they display nuclear EYFP distributions similar to other cell lines), and for the detection of verotoxin (hence the cell line designation).
Contributing Authors
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
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