Characterization of a CFTR construct with a C‐terminal tetracysteine sequence and its use in the visualization of trafficking pathways
Bibliographic record
Abstract
Membrane permeable bi‐arsenical dyes, such as fluorescein arsenical hairpin (FlAsH), bind tetracysteine‐tagged‐proteins emitting fluorescence in‐vivo . This fluorescence method (developed by R. Tsien) provides a tool to evaluate trafficking pathways in living cells. We generated a tetracysteine‐tagged Cystic Fibrosis Transmembrane Regulator (CFTR) protein and studied its expression with the long‐term goal of using this protein to study its trafficking during endocytosis. The tetracysteine‐tagged CFTR (CFTR‐TC) was predominantly expressed in both BHK and in Cos‐7 cells as the immature core‐glycosylated form (band B) rather than the mature glycosylated band C. Iodide efflux assays of CFTR‐TC's function as a chloride channel at the cell surface revealed an insignificant response after cAMP stimulation, likely reflecting its low band C expression. Interestingly, 1 hour FlAsH pre‐treatment evoked a mean response difference of −16.0 mV versus untreated (one‐way ANOVA p<0.05), suggesting increased band C expression. Our results suggest that FlAsH rescues the surface expression of CFTR‐TC and studies are ongoing to visualize its localization and the regulation of its trafficking by proteins involved in the endocytic pathway. The work is supported by the Natural Sciences and Engineering Research Council (postgraduate scholarship) and the Canadian Cystic Fibrosis Foundation BREATHE Program.
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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 | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".