Spectrally resolved microscopy of GFP trafficking.
Folding and chromophore cyclization-oxidation processes of green and cyan fluorescent fusion proteins (GFP and CFP) in subcellular microenvironments of transfected C6 glioma cells were studied by multipixel spectrally resolved microscopy (SRM). Discrete time-dependent spectral transitions were characterized during protein folding and chromophore maturation in the cytosol, nucleus, mitochondria, endoplasmic reticulum (ER), and Golgi. Spectral similarity mapping of fluorophore transition phases demarcated spatio-temporal fluorescence correlation at a subcellular level. Folding stages were characterized by a transition from red-shifted spectral populations in the time interval of 7-10 hr after transfection to a fully matured fluorophore emitting typical GFP or CFP fluorescence after 10-15 hr. The nascent protein revealed an initial focal accumulation in cytosol emitting in the range of 580-680 nm. After 10 hr, mixed pixel population spectra were measured and at 15 hr GFP was visualized in the cytoplasm by its specific spectral fingerprints with maxima at 545 nm. For nucleus- and mitochondrion-targeted CFPs, the mature conformer was discovered only in its final destination, whereas intermediate steps of fluorophore synthesis (at 10 hr) were found in the cytoplasm. Enhanced fluorescence maturation was manifested only by the ER-Golgi-targeted CFP after 10 hr post transfection by spectral imaging. Moreover, only remnants of initial intermediate fluorescent pixels were localized externally to the Golgi framework at 15 hr. SRM assessed the competence of ER-Golgi to maintain efficient CFP folding in comparison to the rest of the cellular compartments.
Microscopy Unit, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Lior
L
Greenbaum
Debby
D
Schwartz
Zvi
Z
Malik
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000235:genetics; D005583:Fourier Analysis; D005700:Galactosyltransferases / Q000235:genetics; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D017510:Protein Folding; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D003576:Electron Transport Complex IV; D005700:Galactosyltransferases