Flow cytometry and GFP: a novel assay for measuring the import and turnover of nuclear-encoded mitochondrial proteins in live PC12 cells.
BACKGROUND
Mitochondrial protein import is typically measured by adding radiolabeled precursor proteins to isolated mitochondria. We have developed a novel, high-throughput method for measuring protein import in live differentiated PC12 cells using a tetracycline (Tet) regulated, nuclear encoded, mitochondrially-targeted GFP fusion protein and flow cytometry.


METHODS
We generated a PC12 cell line stably transfected with an inducible GFP fusion protein (GFPmt) targeted to mitochondria. GFPmt PC12 cells were treated with NGF for one week to induce neuronal differentiation in the presence of Tet to silence GFP expression. On day seven GFPmt expression was induced by removal of Tet and these "GFP-on" cells were exposed to sublethal levels of CCCP (2 microM) for 24 h. At 24 h, the cells were harvested in Ca(++)-free PBS and the GFPmt signal in live intact cells was measured using flow cytometry. Since GFPmt is not fluorescent prior to being imported into mitochondria, the GFPmt signal reflected only GFPmt imported to mitochondria. PI was used to gate out contributions from dead cells. Turnover of GFPmt in mitochondria was also assessed; in this case, Tet was added to arrest GFPmt expression in GFP-on cells, and the subsequent decline of the fluorescent signal, in the absence of any new GFP synthesis, was measured by flow cytometry.


RESULTS
Exposure to 2 microM CCCP for 24 h caused a 61% +/- 0.4 decline in GFPmt fluorescence compared to controls. This decline corresponded to a 30% +/- 7 decrease in GFPmt protein levels measured by Western blot of mitochondrial fractions, and a 72% +/- 5 decline in the import of newly synthesized GFPmt to mitochondria over a 1 h period 24-h after addition of 2 microM CCCP measured by autoradiography. CCCP partially depolarized mitochondria but was not lethal for up to five days.


CONCLUSIONS
This novel GFP-based flow cytometry assay is a rapid and sensitive technique for quantifying protein import to mitochondria in live neuronal cells.
Sirk
Daniel P
DP
Cellular and Molecular Division, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, M5T 2S8 Canada.
Zhu
Ziping
Z
Wadia
Jehangir S
JS
Mills
Linda R
LR
D000818:Animals; D015153:Blotting, Western; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D016716:PC12 Cells; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D021381:Protein Transport; D051381:Rats; D013347:Subcellular Fractions; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D045304:Cytochromes c; D013752:Tetracycline