Spatio-temporal protein interaction analysis using bimolecular fluorescence complementation in <i>C. elegans</i>
Bibliographic record
Abstract
Abstract Dynamic protein-protein interactions (PPIs) shape all aspects of cellular biology. Thus, significant efforts have been made to develop assays testing binary PPIs. The transparency of C. elegans makes it a great model organism for fluorescence-based PPI detection in vivo . However, to date, there is currently a lack of quantitative PPI assays that also provide information on the subcellular location of protein interactions in C. elegans. Here, we have made several modifications to the original bimolecular fluorescence complementation (BiFC) assay used in C. elegans to make it more quantitative and spatio-temporally controlled. First, transgenes are expressed at single copy, reducing the variability associated with multi-copy expression. Second, we have added bicistronic reference fluorescent proteins to each transgene, allowing for the normalization and quantification of the PPI. Finally, we have incorporated the auxin-inducible degradation system, allowing for small-molecule inducible control of the PPI signal. We demonstrate the utility of our modified BiFC assay by testing several model PPIs. Thus, we anticipate that our updated BiFC approach will expand the available tools for studying PPIs in C. elegans , but similar logic could be applied to other model organisms amenable to transgenesis and in vivo fluorescent imaging. Article Summary Protein-protein interactions (PPIs) play a central role in all facets of cellular biology. Here, we developed an improved assay to study PPIs in C. elegans , based on bimolecular fluorescence complementation (BiFC), where two halves of split-YFP can be reconstituted in an interaction-dependent manner. Our modifications include making the readout of the assay less variable and more quantitative, while also enabling signal to accumulate in an inducible manner. We envision that our updated BiFC approach will serve as a useful tool for C. elegans researchers interested in characterizing PPIs of interest in vivo .
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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 | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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".