<i>In situ</i> Measurement of the Electrical Potential Across the Lysosomal Membrane Using FRET
Bibliographic record
Abstract
The progressive acidification of the endocytic pathway is generated by H(+) pumping of electrogenic vacuolar-type ATPases (V-ATPases) on the endosomal/lysosomal membrane. The determinants of pH during endosome maturation are not completely understood, but the permeability to ions that neutralize the electrogenic effect of the V-ATPase has been proposed to play a central role. If counter-ion conductance becomes limiting, the generation of a large membrane potential would dominate the proton-motive force (pmf), diminishing the pH gradient proportionally. Validation of this notion requires direct measurement of the electrical potential that develops across the endosomal/lysosomal membrane. To date, the measurement of lysosomal membrane potential (ψ(φ) ) in situ has been hampered by the inability to access endosomes by electrophysiological means and the fact that individual organelles cannot be discerned when using potentiometric fluorescent dyes. Here, we describe a noninvasive procedure to estimate ψ(φ) in intact cells, based on fluorescence resonance energy transfer (FRET). At steady state, ψ(φ) averaged 19 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A (CcA). ψ(φ) was considerably increased by alkalinization of the lysosome lumen by NH(4) Cl, implying that at steady state the V-ATPase operates at submaximal rates and that the contribution of ψ(φ) to pmf is relatively small. Our method should enable systematic studies of endosomal/lysosomal potential.
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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 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 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".