Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells.
BACKGROUND
It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic (Escherichia coli) and eukaryotic (Caenorhabditis elegans and Drosophila melanogaster) living cells; it can therefore be used as a powerful marker of gene expression in vivo. The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles.


RESULTS
In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol. Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide). When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria. We also describe two of the many possible applications of this recombinant GFP in physiological studies. The targeted chimera allows the visualization of mitochondrial movement in living cells. Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential. Moreover, when GFP is cotransfected with a membrane receptor, such as the alpha 1-adrenergic receptor, the fluorescence of the GFP in intact cells can be used in recognizing the transfected cells. Thus, specific changes in intracellular Ca2+ concentration that occur in cells expressing the recombinant receptor can be identified using a classical fluorescent Ca2+ indicator.


CONCLUSIONS
GFP is an invaluable new tool for studies of molecular biology and cell physiology. As a marker of transfection in vivo, it provides a simple means of identifying genetically modified cells to be used in physiological studies. More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters.
Rizzuto
R
R
Department of Biomedical Sciences, University of Padova, Italy.
Brini
M
M
Pizzo
P
P
Murgia
M
M
Pozzan
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015415:Biomarkers; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism / Q000648:ultrastructure*; D005456:Fluorescent Dyes / Q000032:analysis / Q000528:radiation effects; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000737:chemistry / Q000648:ultrastructure; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D008027:Light; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000528:radiation effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D015388:Organelles / Q000648:ultrastructure*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism / Q000528:radiation effects; D020112:Rhodamine 123; D012235:Rhodamines / Q000032:analysis; D037401:Scyphozoa / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D014466:Ultraviolet Rays
D015415:Biomarkers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D049452:Green Fluorescent Proteins; D020112:Rhodamine 123; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D006632:Histamine