A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling
Bibliographic record
Abstract
Scramblases are physiologically important proteins that translocate phospholipids bidirectionally across cell membranes. For example, scrambling facilitated by dimers of the voltage-dependent anion channel 1 (VDAC1) enables endoplasmic reticulum-derived phospholipids to cross the outer membrane to enter mitochondria. Here we describe a protocol to obtain lipid translocation rates at a single-protein level, allowing for mechanistic understanding of scramblases. We reconstituted vesicles with fluorescent phospholipids and VDAC1 dimers and use high-throughput imaging to quantify their size and dimer content. We measure scrambling in each vesicle using a new assay and find that individual human VDAC1 dimers scramble lipids at rates ranging from under 100 to over 10,000 per second. This kinetic heterogeneity, masked in ensemble measurements, revealed that rapid scrambling is facilitated by specific VDAC1 dimers. Extending our analyses to bovine opsin, a monomeric G-protein-coupled receptor scramblase, we demonstrate the versatility of our platform for quantifying lipid scrambling and exploring its regulation.
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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) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 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".