Sirk Daniel P, Zhu Ziping, Wadia Jehangir S, Mills Linda R
Cellular and Molecular Division, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, M5T 2S8 Canada.
Cytometry A. 2003 Nov;56(1):15-22. doi: 10.1002/cyto.a.10084.
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.
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.
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.
This novel GFP-based flow cytometry assay is a rapid and sensitive technique for quantifying protein import to mitochondria in live neuronal cells.
线粒体蛋白导入通常通过向分离的线粒体中添加放射性标记的前体蛋白来测量。我们开发了一种新颖的高通量方法，使用四环素（Tet）调控的、核编码的、靶向线粒体的绿色荧光蛋白（GFP）融合蛋白和流式细胞术来测量分化的活PC12细胞中的蛋白导入。
我们构建了一个稳定转染了可诱导的靶向线粒体的GFP融合蛋白（GFPmt）的PC12细胞系。在存在Tet的情况下，用神经生长因子（NGF）处理GFPmt PC12细胞一周以诱导神经元分化，从而沉默GFP表达。在第7天，通过去除Tet诱导GFPmt表达，这些“GFP开启”细胞暴露于亚致死水平的羰基氰-间氯苯腙（CCCP，2微摩尔）中24小时。在24小时时，将细胞收获于无钙磷酸盐缓冲盐水中，并使用流式细胞术测量活的完整细胞中的GFPmt信号。由于GFPmt在导入线粒体之前不发荧光，因此GFPmt信号仅反映导入线粒体的GFPmt。碘化丙啶（PI）用于排除死细胞的影响。还评估了GFPmt在线粒体中的周转情况；在这种情况下，向“GFP开启”细胞中添加Tet以阻止GFPmt表达，然后通过流式细胞术测量在没有任何新的GFP合成的情况下荧光信号的后续下降情况。
与对照相比，暴露于2微摩尔CCCP 24小时导致GFPmt荧光下降61%±0.4%。这种下降对应于通过线粒体组分的蛋白质印迹法测量的GFPmt蛋白水平下降30%±7%，以及在添加2微摩尔CCCP 24小时后1小时内通过放射自显影法测量的新合成的GFPmt导入线粒体的量下降72%±5%。CCCP使线粒体部分去极化，但在长达五天内并非致命。
这种基于GFP的新型流式细胞术检测方法是一种快速且灵敏的技术，用于定量活神经元细胞中线粒体的蛋白导入。
