Flow cytometry and GFP: A novel assay for measuring the import and turnover of nuclear‐encoded mitochondrial proteins in live PC12 cells
Bibliographic record
Abstract
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.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.001 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.003 | 0.002 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.002 | 0.002 |
| Open science | 0.003 | 0.001 |
| Research integrity | 0.003 | 0.004 |
| Insufficient payload (model declined to judge) | 0.004 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".