DOUBLE-LABELING OF SUBCELLULAR STRUCTURES WITH ORGANELLE-TARGETED GFPMUTANTS IN-VIVO

Background: The green fluorescent protein (GFP) of Aequorea victoria i s emerging as a unique tool for monitoring complex phenomena such as g ene expression and organelle structure and dynamics in living cells. T he recent description of GFP mutants with modified spectral properties opens numerous new applications in cell biology. However, the express ion and the characteristics of these GFP mutants in living eukaryotic cells have not been verified yet. Results: Here, we demonstrate the us efulness of the GFP mutants for cell biology studies in vivo, by the u se of wild-type GFP, a'bright' GFP mutant (S65T) and a mutant with blu e-shifted excitation and emission spectra (Y66H/Y145F). We have constr ucted two GFP chimeras targeted to mitochondria, mtGFP(S65T) and mtGFP (Y66H/Y145F), with the same strategy used previously for mtGFP. In add ition, two GFP chimeras targeted to the nucleus, nuGFP and nUGFP(S65T) , were constructed by fusing the wild-type GFP or the S65T mutant to t he rat glucocorticoid receptor. By co-transfecting mtGFP(Y66HN/Y145F) and nuGFP, the nucleus and the mitochondria were visualized simultaneo usly in living cells. Similarly, mtGFP and mtGFP(Y66HN/145F) were tran sfected into different populations of cells, and the events of cellula r fusion, and mitochondrial intermixing and/or fusion, were directly m onitored. Conclusions: The successful expression of organelle-targeted GPP mutants in live eukaryotes expands the uses of this fluorescent p rotein in cell biology, allowing direct access to key biological issue s, such as the study of the interactions of different organelles in vi vo. These results also open the way to other exciting applications, su ch as the direct study of protein redistribution and protein-protein i nteractions in living cells.